CFGparadigms - User contributions [en]

Candida glabrata EPA7

2011-09-15T18:55:43Z

Carole Weaver: /* Knockout mouse lines */

'''Fungal adhesins with lectin properties''' 
Cell adhesion proteins on fungal cell surfaces mediate interactions both with other cells of the same type and with the external environment<ref>Douglas, L.M., Li, L., Yang, Y. and Dranginis, A.M. 2007. Expression and characterization of the flocculin Flo11/Muc1, a Saccharomyces cerevisiae mannoprotein with homotypic properties of adhesion. Eukaryot Cell, 6, 2214-2221.</ref><ref>Dranginis, A.M., Rauceo, J.M., Coronado, J.E. and Lipke, P.N. 2007. A biochemical guide to yeast adhesins: glycoproteins for social and antisocial occasions. Microbiol Mol Biol Rev, 71, 282-294.</ref>. These interactions impact critical processes including mating, pathogenesis, and biofilm formation. Fungal adhesins are typically GPI-anchored proteins that have been covalently linked to the cell wall, such that their N-terminal ligand binding domains extend from the cell surface. They frequently occur as families of related proteins<ref>Tronchin, G., Pihet, M., Lopes-Bezerra, L.M. and Bouchara, J.P. 2008. Adherence mechanisms in human pathogenic fungi. Med Mycol, 46, 749-772. </ref>. Members of two such groups, the flocculation/agglutination genes of the model yeast ''Saccharomyces cerevisiae''<ref>Kobayashi, O., Hayashi, N., Kuroki, R. and Sone, H. 1998. Region of FLO1 proteins responsible for sugar recognition. J Bacteriol, 180, 6503-6510.</ref> and the related EPA genes<ref>Kaur, R., Domergue, R., Zupancic, M.L. and Cormack, B.P. 2005. A yeast by any other name: Candida glabrata and its interaction with the host. Curr Opin Microbiol, 8, 378-384.</ref> of the pathogenic fungus ''Candida glabrata'', are lectins. Several of the 23 identified EPA genes have been functionally shown to mediate binding of ''C. glabrata'' to host cells<ref name="Castano 2005">Castano, I., Pan, S.J., Zupancic, M., Hennequin, C., Dujon, B. and Cormack, B.P. 2005. Telomere length control and transcriptional regulation of subtelomeric adhesins in Candida glabrata. Mol Microbiol, 55, 1246-1258.</ref><ref name="Domergue 2005">Domergue, R., Castano, I., de las Penas, A., Zupancic, M., Lockatell, V., Hebel, J.R. et al. 2005. Nicotinic acid limiation regulates silencing of Candida albicans adhesins during UTI. Science, 308, 866-870.</ref>, an essential step in infection and virulence. Defining the specificity of these proteins and their biological roles will elucidate the interactions between host and pathogen, and potentially indicate ways in which to inhibit them for the benefit of the host.

'''''Candida glabrata'' EPA7''' 
The EPA family was chosen as a paradigm because of its relevance to a fungal pathogen that affects human health that can be studied in mouse models of infection. EPA7 was chosen to represent this group because it has been demonstrated to function as an adhesin<ref name="Castano 2005"/> and is one of the EPA proteins that has been studied in the most detail. The N-terminal binding domain of this protein, expressed on the surface of S''. cerevisiae'', has been analyzed on the CFG glycan array. These studies demonstrated EPA7 binding specificity for β1,3- and β1,4-linked galactosides
<ref name=" Zupancic, M.L., 2008">
Zupancic, M.L., Frieman, M., Smith, D., Alvarez, R.A., Cummings, R.D. and Cormack, B.P. 2008. Glycan microarray analysis of Candida glabrata adhesin ligand specificity. Mol Microbiol, 58, 547-559.</ref><ref>de Groot, P.W.J. and Klis, F.M. 2008. The conserved PA14 domain of cell wall-associated fungal adhesins governs their glycan-binding specificity. Mol Microbiol, 68, 535-537. </ref>. This work represents a significant step forward in the area of lectin-like fungal adhesins; in general the specificity of these important proteins remains unexplored.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) who have contributed to studies of this paradigmatic protein include: Brendan Cormack, Rick Cummings
* PIs using CFG resources to study related ''S. cerevisiae'' proteins include: Lars-Oliver Essen (several flocculins), Peter Lipke (alpha agglutinin and ''Candida albicans'' Als adhesins)

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

Carbohydrate ligands of Epa7 have been examined by glycan array analysis in work from the Cormack group <ref name=" Zupancic, M.L., 2008"></ref>and found to bind almost exclusively to non-reducing terminal β1-4 or β1-3-linked galactose residues. The Epa1 family member has similar specificity while Epa6 binds to most ligands with terminal galactose residues. The related ''S. cerevisiae'' flocculins bind α-mannose or α-glucose residues.

=== Cellular expression of GBP and ligands ===

EPA7 is expressed by the pathogenic fungus ''Candida glabrata''. The EPA gene family is subject to an interesting mechanism of regulation mediated by telomeric silencing. This silencing is relieved in low niacin, leading to increased protein expression. Because urine provides a low niacin growth environment, these adhesins are upregulated in precisely the niche where ''C. glabrata'' must adhere to cause urinary tract infections. 
Consistent with the site of ''C. glabrata'' infection, Epa6 and Epa7 mediate binding to uroepithelial cells in vitro <ref name="Castano 2005"></ref><ref name="Domergue 2005">Domergue, R., Castano, I., de las Penas, A., Zupancic, M., Lockatell, V., Hebel, J.R. et al. 2005. Nicotinic acid limiation regulates silencing of Candida albicans adhesins during UTI. Science, 308, 866-870.</ref>. It has also been noted <ref name=" Zupancic, M.L., 2008"></ref> that "Epa1, Epa6, and Epa7 recognize the T antigen Gal-β1-3GalNAc-α-R, one of the major mucin-type O-glycans found in the colonic epithelium", another site colonized by ''C. glabrata''. 

=== Biosynthesis of ligands ===
 

=== Structure ===

Epa proteins are C-type lectins, but no detailed structural information for these proteins is yet available.

=== Biological roles of GBP-ligand interaction ===

Epa7 and other Epa family members, as summarized above, participate in adhesion of the pathogenic yeast ''Candida glabrata'' cells to host cells, leading to infection. Related proteins participate in cell:cell interactions between yeast that can be important for critical processes such as mating or biofilm formation.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=EPA7&maxresults=20 CFG database search results for "EPA7"].

=== Glycan profiling ===

Glycan profiling of host cell glycans has not been used in connection with this paradigm.

=== Glycogene microarray ===
Mammalian glycogene profiling has not been performed for this paradigm of a fungal protein. Although yeast binding to mammalian cells could conceivably trigger changes in glycogene expression, the critical issue for this adhesin is which glycans are present on the mammalian cell surface upon initial contact. These structures will mediate the cell:cell interactions that are important for establishment of infection.

=== Knockout mouse lines ===

CFG knockout mouse lines have not been used for studies pertaining to this paradigm. Examining ''C. glabrata'' infection using wild type and ''epa'' mutant strains in mice with defects in terminal galactosylation could potentially be of interest.

=== Glycan array ===
The specificity of Epa7 and related proteins was determined through CFG glycan array analysis<ref name=" Zupancic, M.L., 2008"></ref> (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1056 here] to see results). To see glycan array results for other EPA family members, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=EPA&cat=coreh here]. Flocculins from ''S. cerevisiae'' have also been examined by CFG glycan array analysis (click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=flocculin&cat=coreh here]), and glycan array studies of flocculins from ''P. Pastoris'' and of additional ''Candida'' Epa domains have been approved.

== Related GBPs ==
* 22 additional EPA family members in ''C. glabrata''
* Related proteins in ''S. cerevisiae''
* EPA7-like glycan-binding domain also occurs in predicted proteins of ''Ashbya gossypii'' and ''Kluyveromyces lactis''.
(Click here for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=EPA&maxresults=20 CFG data] on EPA family members)

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Richard Cummings, Tamara Doering, Peter Lipke

Candida glabrata EPA7

2011-09-15T18:55:33Z

Carole Weaver: /* Glycogene microarray */

'''Fungal adhesins with lectin properties''' 
Cell adhesion proteins on fungal cell surfaces mediate interactions both with other cells of the same type and with the external environment<ref>Douglas, L.M., Li, L., Yang, Y. and Dranginis, A.M. 2007. Expression and characterization of the flocculin Flo11/Muc1, a Saccharomyces cerevisiae mannoprotein with homotypic properties of adhesion. Eukaryot Cell, 6, 2214-2221.</ref><ref>Dranginis, A.M., Rauceo, J.M., Coronado, J.E. and Lipke, P.N. 2007. A biochemical guide to yeast adhesins: glycoproteins for social and antisocial occasions. Microbiol Mol Biol Rev, 71, 282-294.</ref>. These interactions impact critical processes including mating, pathogenesis, and biofilm formation. Fungal adhesins are typically GPI-anchored proteins that have been covalently linked to the cell wall, such that their N-terminal ligand binding domains extend from the cell surface. They frequently occur as families of related proteins<ref>Tronchin, G., Pihet, M., Lopes-Bezerra, L.M. and Bouchara, J.P. 2008. Adherence mechanisms in human pathogenic fungi. Med Mycol, 46, 749-772. </ref>. Members of two such groups, the flocculation/agglutination genes of the model yeast ''Saccharomyces cerevisiae''<ref>Kobayashi, O., Hayashi, N., Kuroki, R. and Sone, H. 1998. Region of FLO1 proteins responsible for sugar recognition. J Bacteriol, 180, 6503-6510.</ref> and the related EPA genes<ref>Kaur, R., Domergue, R., Zupancic, M.L. and Cormack, B.P. 2005. A yeast by any other name: Candida glabrata and its interaction with the host. Curr Opin Microbiol, 8, 378-384.</ref> of the pathogenic fungus ''Candida glabrata'', are lectins. Several of the 23 identified EPA genes have been functionally shown to mediate binding of ''C. glabrata'' to host cells<ref name="Castano 2005">Castano, I., Pan, S.J., Zupancic, M., Hennequin, C., Dujon, B. and Cormack, B.P. 2005. Telomere length control and transcriptional regulation of subtelomeric adhesins in Candida glabrata. Mol Microbiol, 55, 1246-1258.</ref><ref name="Domergue 2005">Domergue, R., Castano, I., de las Penas, A., Zupancic, M., Lockatell, V., Hebel, J.R. et al. 2005. Nicotinic acid limiation regulates silencing of Candida albicans adhesins during UTI. Science, 308, 866-870.</ref>, an essential step in infection and virulence. Defining the specificity of these proteins and their biological roles will elucidate the interactions between host and pathogen, and potentially indicate ways in which to inhibit them for the benefit of the host.

'''''Candida glabrata'' EPA7''' 
The EPA family was chosen as a paradigm because of its relevance to a fungal pathogen that affects human health that can be studied in mouse models of infection. EPA7 was chosen to represent this group because it has been demonstrated to function as an adhesin<ref name="Castano 2005"/> and is one of the EPA proteins that has been studied in the most detail. The N-terminal binding domain of this protein, expressed on the surface of S''. cerevisiae'', has been analyzed on the CFG glycan array. These studies demonstrated EPA7 binding specificity for β1,3- and β1,4-linked galactosides
<ref name=" Zupancic, M.L., 2008">
Zupancic, M.L., Frieman, M., Smith, D., Alvarez, R.A., Cummings, R.D. and Cormack, B.P. 2008. Glycan microarray analysis of Candida glabrata adhesin ligand specificity. Mol Microbiol, 58, 547-559.</ref><ref>de Groot, P.W.J. and Klis, F.M. 2008. The conserved PA14 domain of cell wall-associated fungal adhesins governs their glycan-binding specificity. Mol Microbiol, 68, 535-537. </ref>. This work represents a significant step forward in the area of lectin-like fungal adhesins; in general the specificity of these important proteins remains unexplored.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) who have contributed to studies of this paradigmatic protein include: Brendan Cormack, Rick Cummings
* PIs using CFG resources to study related ''S. cerevisiae'' proteins include: Lars-Oliver Essen (several flocculins), Peter Lipke (alpha agglutinin and ''Candida albicans'' Als adhesins)

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

Carbohydrate ligands of Epa7 have been examined by glycan array analysis in work from the Cormack group <ref name=" Zupancic, M.L., 2008"></ref>and found to bind almost exclusively to non-reducing terminal β1-4 or β1-3-linked galactose residues. The Epa1 family member has similar specificity while Epa6 binds to most ligands with terminal galactose residues. The related ''S. cerevisiae'' flocculins bind α-mannose or α-glucose residues.

=== Cellular expression of GBP and ligands ===

EPA7 is expressed by the pathogenic fungus ''Candida glabrata''. The EPA gene family is subject to an interesting mechanism of regulation mediated by telomeric silencing. This silencing is relieved in low niacin, leading to increased protein expression. Because urine provides a low niacin growth environment, these adhesins are upregulated in precisely the niche where ''C. glabrata'' must adhere to cause urinary tract infections. 
Consistent with the site of ''C. glabrata'' infection, Epa6 and Epa7 mediate binding to uroepithelial cells in vitro <ref name="Castano 2005"></ref><ref name="Domergue 2005">Domergue, R., Castano, I., de las Penas, A., Zupancic, M., Lockatell, V., Hebel, J.R. et al. 2005. Nicotinic acid limiation regulates silencing of Candida albicans adhesins during UTI. Science, 308, 866-870.</ref>. It has also been noted <ref name=" Zupancic, M.L., 2008"></ref> that "Epa1, Epa6, and Epa7 recognize the T antigen Gal-β1-3GalNAc-α-R, one of the major mucin-type O-glycans found in the colonic epithelium", another site colonized by ''C. glabrata''. 

=== Biosynthesis of ligands ===
 

=== Structure ===

Epa proteins are C-type lectins, but no detailed structural information for these proteins is yet available.

=== Biological roles of GBP-ligand interaction ===

Epa7 and other Epa family members, as summarized above, participate in adhesion of the pathogenic yeast ''Candida glabrata'' cells to host cells, leading to infection. Related proteins participate in cell:cell interactions between yeast that can be important for critical processes such as mating or biofilm formation.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=EPA7&maxresults=20 CFG database search results for "EPA7"].

=== Glycan profiling ===

Glycan profiling of host cell glycans has not been used in connection with this paradigm.

=== Glycogene microarray ===
Mammalian glycogene profiling has not been performed for this paradigm of a fungal protein. Although yeast binding to mammalian cells could conceivably trigger changes in glycogene expression, the critical issue for this adhesin is which glycans are present on the mammalian cell surface upon initial contact. These structures will mediate the cell:cell interactions that are important for establishment of infection.

=== Knockout mouse lines ===

 CFG knockout mouse lines have not been used for studies pertaining to this paradigm. Examining ''C. glabrata'' infection using wild type and ''epa'' mutant strains in mice with defects in terminal galactosylation could potentially be of interest.

=== Glycan array ===
The specificity of Epa7 and related proteins was determined through CFG glycan array analysis<ref name=" Zupancic, M.L., 2008"></ref> (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1056 here] to see results). To see glycan array results for other EPA family members, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=EPA&cat=coreh here]. Flocculins from ''S. cerevisiae'' have also been examined by CFG glycan array analysis (click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=flocculin&cat=coreh here]), and glycan array studies of flocculins from ''P. Pastoris'' and of additional ''Candida'' Epa domains have been approved.

== Related GBPs ==
* 22 additional EPA family members in ''C. glabrata''
* Related proteins in ''S. cerevisiae''
* EPA7-like glycan-binding domain also occurs in predicted proteins of ''Ashbya gossypii'' and ''Kluyveromyces lactis''.
(Click here for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=EPA&maxresults=20 CFG data] on EPA family members)

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Richard Cummings, Tamara Doering, Peter Lipke

Reovirus hemagglutinin (sigma 1)

2011-09-15T17:21:32Z

Carole Weaver: /* Glycan profiling */

'''Mammalian orthoreoviruses (reoviruses)''' are useful models for studies of viral receptor recognition and the pathogenesis of viral disease. Reovirus also efficiently lyses tumor cells in experimental animals<ref>Duncan, M.R., Stanish, S.M., and Cox, D.C. Differential sensitivity of normal and transformed human cells to reovirus infection. J. Virol. 28:444-449, 1978.</ref><ref> Coffey, M.C., Strong, J.E., Forsyth, P.A., and Lee, P.W. Reovirus therapy of tumors with activated Ras pathway. Science 282:1332-1334, 1998.</ref> and has shown efficacy in clinical trials for aggressive and refractory human tumors<ref>Stoeckel, J., and Hay, J.G. Drug evaluation: Reolysin--wild-type reovirus as a cancer therapeutic. Curr. Opin. Mol. Ther. 8:249-260, 2006.</ref><ref>Twigger, K., Vidal, L., White, C.L., De Bono, J.S., Bhide, S., Coffey, M., Thompson, B., Vile, R.G., Heinemann, L., Pandha, H.S., et al. Enhanced in vitro and in vivo cytotoxicity of combined reovirus and radiotherapy. Clin. Cancer Res. 14:912-923, 2008. </ref>. Reovirus forms double-shelled particles<ref>Dryden, K.A., Wang, G., Yeager, M., Nibert, M.L., Coombs, K.M., Furlong, D.B., Fields, B.N., and Baker, T.S. Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction. J. Cell Biol. 122:1023-1041, 1993.</ref> that contain a segmented dsRNA genome. The reovirus sigma 1 protein is a long, fiber-like molecule that extends from the virion surface<ref>Furlong, D.B., Nibert, M.L., and Fields, B.N. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles. J. Virol. 62:246-256, 1988.</ref> and mediates viral attachment<ref>Weiner, H.L., Ault, K.A., and Fields, B.N. Interaction of reovirus with cell surface receptors. I. Murine and human lymphocytes have a receptor for the hemagglutinin of reovirus type 3. J. Immunol. 124:2143-2148, 1980.</ref><ref>Lee, P.W.K., Hayes, E.C., and Joklik, W.K. Protein σ1 is the reovirus cell attachment protein. Virology 108:156-163, 1981.</ref>. The three human serotypes (T1, T2, and T3) differ in cell tropism, which correlates directly with receptor-binding properties of sigma 1. Sialic acid serves as an essential receptor for T3 reovirus on murine erythroleukemia (MEL) cells<ref name="Rubin1992">Rubin, D.H., Wetzel, J.D., Williams, W.V., Cohen, J.A., Dworkin, C., and Dermody, T.S. Binding of type 3 reovirus by a domain of the σ1 protein important for hemagglutination leads to infection of murine erythroleukemia cells. J. Clin. Invest. 90:2536-2542, 1992.</ref>, and it functions as a coreceptor on murine L929 (L) cells<ref name="Gentsch1985">Gentsch, J.R., and Pacitti, A.F. Effect of neuraminidase treatment of cells and effect of soluble glycoproteins on type 3 reovirus attachment to murine L cells. J. Virol. 56:356-364, 1985.</ref><ref name="Pacitti1987">Pacitti, A., and Gentsch, J.R. Inhibition of reovirus type 3 binding to host cells by sialylated glycoproteins is mediated through the viral attachment protein. J. Virol. 61:1407-1415, 1987. </ref><ref name="name4">.</ref><ref name="Rubin1992"></ref><ref name="Nibert1995">Nibert, M.L., Chappell, J.D., and Dermody, T.S. Infectious subvirion particles of reovirus type 3 Dearing exhibit a loss in infectivity and contain a cleaved σ1 protein. J. Virol. 69:5057-5067, 1995.</ref>. Residues involved in sialic acid-binding map to the center of the long fiber, close to the midpoint of the molecule<ref name="Chappell2002">Chappell, J.D., Prota, A., Dermody, T.S., and Stehle, T. Crystal structure of reovirus attachment protein σ1 reveals evolutionary relationship to adenovirus fiber. EMBO J. 21:1-11, 2002.</ref>, in a repetitive structural region known as the triple β-spiral. The T1 sigma 1 protein binds to cell-surface glycans of unknown structure.

The triple β-spiral of '''sigma 1''' functions as a trimerization domain and defines a novel carbohydrate-recognition motif. Other carbohydrate-recognition domains, such as those of the C-type lectin superfamily<ref>Weis, W.I., Taylor, M.E., and Drickamer, K. The C-type lectin superfamily in the immune system. Immunol. Rev. 163:19-34, 1998.</ref> or the sialic acid-binding domains in the Siglec family of adhesion proteins<ref>Crocker, P.R., and Varki, A. Siglecs in the immune system. Immunology 103:137-145, 2001.</ref> (see [http://glycobank.mit.edu/glycoWiki/Main_Page Siglec paradigms]), have been described, but none are formed by a repetitive, fiber-like structure such as the one present in sigma 1. In fact, the domain in sigma 1 that binds sialic acid constitutes a carbohydrate-binding “cassette” that could be endowed with altered ligand-binding properties or grafted onto other trimeric structures and used to create avidity for carbohydrates. For example, the adenovirus fiber shaft could be licensed with sialic acid-binding capacity using this approach. These properties render the sigma 1 protein unique among the structurally known glycan-binding moieties.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of sigma 1 include: Terence Dermody, Thilo Stehle

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
Reovirus strains of all three serotypes are capable of binding to carbohydrates, which is a property mediated by viral attachment protein sigma 1<ref name="Chappell2000">Chappell, J.D., Duong, J.L., Wright, B.W., and Dermody, T.S. Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses. J. Virol. 74:8472-8479, 2000.</ref>. Substantial evidence indicates that serotype 3 reoviruses bind to sialic acid, whereas the identity of carbohydrates bound by serotype 1 and 2 reoviruses is less clear. Serotype 3 reoviruses bind to alpha-linked sialic acid in either alpha2,3 or alpha2,6 linkages on a variety of cell types<ref name="Chappell2000">.</ref><ref name="Gentsch1985">.</ref><ref name="name4">Paul, R.W., Choi, A.H., and Lee, P.W.K. The a-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus. Virology 172:382-385, 1989.</ref><ref>Dermody, T.S., Nibert, M.L., Bassel-Duby, R., and Fields, B.N. Sequence diversity in S1 genes and S1 translation products of 11 serotype 3 reovirus strains. J. Virol. 64:4842-4850, 1990.</ref><ref name="Chappell1997">Chappell, J.D., Gunn, V.L., Wetzel, J.D., Baer, G.S., and Dermody, T.S. Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein s1. J. Virol. 71:1834-1841, 1997.</ref>. Hemagglutination by serotype 3 reoviruses is mediated by interactions with alpha-linked sialic acid on several glycosylated erythrocyte proteins such as glycophorin A<ref>Gentsch, J.R., and Pacitti, A.F. Differential interaction of reovirus type 3 with sialylated receptor components on animal cells. Virology 161:245-248, 1987.</ref><ref>Paul, R.W., and Lee, P.W.K. Glycophorin is the reovirus receptor on human erythrocytes. Virology 159:94-101, 1987.</ref>. Reovirus strain T3D binds to sialoglycophorin, but not to asialoglycophorin, with an avidity of ~ 5 x 10e-9 M<ref name="Barton2001a">Barton, E.S., Connolly, J.L., Forrest, J.C., Chappell, J.D., and Dermody, T.S. Utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening. J. Biol. Chem. 276:2200-2211, 2001.</ref>.

Serotype 1 reoviruses also appear to bind to sialic acid in some contexts. Reovirus strain T1L, but not strain T3D, binds to the apical surface of microfold (M) cells, but not to enterocytes, in tissue sections of rabbit Peyer’s patches<ref name="name10">Helander, A., Silvey, K.J., Mantis, N.J., Hutchings, A.B., Chandran, K., Lucas, W.T., Nibert, M.L., and Neutra, M.R. The viral s1 protein and glycoconjugates containing a2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces. J. Virol. 77:7964-7977, 2003.</ref>. Binding is inhibited by pre-incubation of the tissue sections with neuraminidase or with lectins that specifically recognize alpha2-3-linked sialic acid.

=== Cellular expression of GBP and ligands ===
Sialic acid serves as an essential receptor for type 3 reoviruses on murine erythroleukemia (MEL) cells<ref name="Chappell1997">.</ref><ref name="Rubin1992"></ref>. Sialic acid also functions as a coreceptor on murine L929 cells<ref name="Gentsch1985">.</ref><ref name="name4">.</ref><ref name="Rubin1992">.</ref><ref name="Pacitti1987">.</ref><ref name="Nibert1995">.</ref> and human HeLa cells<ref name="Barton2001a">.</ref>. Although not all serotype 3 strains are capable of binding to sialic acid, the majority bind to this carbohydrate.

Serotype 1 reoviruses are incapable of infecting MEL cells, which support infection only by sialic-acid-binding strains<ref name="Rubin1992">.</ref>. Serotype 1 reoviruses also are insensitive to the growth-inhibitory effects of neuraminidase treatment of L929 cells<ref name="Nibert1995">.</ref>. However, binding of serotype 1 reoviruses to intestinal M cells is diminished by neuraminidase treatment<ref name="name10">.</ref>. The explanation for this discrepancy is not known.

Reovirus employs a multi-step mechanism of viral attachment in which a low-affinity interaction with sialic acid serves to tether the virion to target cells and precedes a high-affinity interaction with JAM-A<ref name="Barton2001a">.</ref>, an immunoglobulin superfamily protein engaged by reovirus<ref name="Barton2001b">Barton, E.S., Forrest, J.C., Connolly, J.L., Chappell, J.D., Liu, Y., Schnell, F., Nusrat, A., Parkos, C.A., and Dermody, T.S. Junction adhesion molecule is a receptor for reovirus. Cell 104:441-451, 2001.</ref><ref>Prota, A.E., Campbell, J.A., Schelling, P., Forrest, J.C., Peters, T.R., Watson, M.J., Aurrand-Lions, M., Imhof, B., Dermody, T.S., and Stehle, T. Crystal structure of human junctional adhesion molecule 1: implications for reovirus binding. Proc. Natl. Acad. Sci. U. S. A. 100:5366-5371, 2003.</ref><ref>Campbell, J.A., Shelling, P., Wetzel, J.D., Johnson, E.M., Wilson, G.A.R., Forrest, J.C., Aurrand-Lions, M., Imhof, B., Stehle, T., and Dermody, T.S. Junctional adhesion molecule-A serves as a receptor for prototype and field-isolate strains of mammalian reovirus. J. Virol. 79:7967-7978, 2005.</ref>. By virtue of its rapid association rate, virus binding to sialic acid adheres the virion to the cell surface, thereby enabling it to diffuse laterally until it encounters JAM-A. Such lateral diffusion has been reported for influenza virus<ref>Sagik, B., Puck, T., and Levine, S. Quantitative aspects of the spontaneous elution of influenza virus from red cells. J. Exp. Med. 99:251-260, 1954.</ref> and phage T4<ref>Wilson, J.H., Luftig, R.B., and Wood, W.B. Interaction of bacteriophage T4 tail fiber components with a lipopolysaccharide fraction from Escherichia coli. J. Mol. Biol. 51:423-434, 1970.</ref>. After attachment, reovirus is internalized by receptor-mediated endocytosis<ref>Borsa, J., Morash, B.D., Sargent, M.D., Copps, T.P., Lievaart, P.A., and Szekely, J.G. Two modes of entry of reovirus particles into L cells. J. Gen. Virol. 45:161-170, 1979.</ref><ref>Borsa, J., Sargent, M.D., Lievaart, P.A., and Copps, T.P. Reovirus: evidence for a second step in the intracellular uncoating and transcriptase activation process. Virology 111:191-200, 1981.</ref><ref>Ehrlich, M., Boll, W., Van Oijen, A., Hariharan, R., Chandran, K., Nibert, M.L., and Kirchhausen, T. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118:591-605, 2004.</ref><ref name="Maginnis2006">Maginnis, M.S., Forrest, J.C., Kopecky-Bromberg, S.A., Dickeson, S.K., Santoro, S.A., Zutter, M.M., Nemerow, G.R., Bergelson, J.M., and Dermody, T.S. b1 integrin mediates internalization of mammalian reovirus. J. Virol. 80:2760-2770, 2006.</ref><ref>Sturzenbecker, L.J., Nibert, M.L., Furlong, D.B., and Fields, B.N. Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle. J. Virol. 61:2351-2361, 1987.</ref><ref name="Maginnis2008">Maginnis, M.S., Mainou, B.A., Derdowski, A.M., Johnson, E.M., Zent, R., and Dermody, T.S. NPXY motifs in the b1 integrin cytoplasmic tail are required for functional reovirus entry. J. Virol. 82:3181-3191, 2008.</ref> using a mechanism dependent on beta 1 integrin<ref name="Maginnis2006">.</ref><ref name="Maginnis2008">.</ref>.
=== Biosynthesis of ligands ===
Serotype 3 reovirus can bind to glycans that terminate with sialic acid in α2-3, α2-6, or α2-8 linkage. Thus, any of the known sialyl transferases ([http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/gtdb.jsp?species=Homo+sapiens&classification=SialylT&linkage_attaching=%3F&linkage_anomeric=%3F&linkage_position=%3F&linkage_base=%3F&pgname=&from=multiple&title=Multiple+Criteria+Search+Results&slideNumber=multipleQuery Human sialyltransferases]) are potentially involved in biosynthesis of target ligands.
 

=== Structure ===
Structural analysis of the C-terminal half of reovirus attachment protein sigma 1 from strain T3D (residues 246-455) reveals a trimeric structure, in which each monomer is composed of a slender tail and a compact head<ref name="Chappell2002">.</ref>. The C-terminal residues that form the head domain (310-455) consist of two Greek-key motifs that fold into a beta-barrel. The sigma 1 head domain binds to JAM-A<ref name="Barton2001b">.</ref><ref>Kirchner, E., Guglielmi, K.M., Strauss, H.M., Dermody, T.S., and Stehle, T. Structure of reovirus s1 in complex with its receptor junctional adhesion molecule-A. PLoS Path. 4:e1000235, 2008.</ref>. N-terminal residues in the crystallized fragment form a portion of the tail, residues 246-309, which consists of three beta-spiral repeats. Each repeat is composed of two short beta-strands connected by a four-residue beta-turn that has either a proline or a glycine residue at its third position<ref name="Chappell2002">.</ref>. A surface-exposed, variable loop links successive repeats, and trimerization generates an unusual triple beta-spiral motif that also is observed in the adenovirus fiber<ref>van Raaij, M.J., Mitraki, A., Lavigne, G., and Cusack, S. A triple b-spiral in the adenovirus fibre shaft reveals a new structural motif for a fibrous protein. Nature 401:935-938, 1999.</ref>, bacteriophage PRD1 P5 protein<ref>Merckel, M.C., Huiskonen, J.T., Bamford, D.H., Goldman, A., and Tuma, R. The structure of the bacteriophage PRD1 spike sheds light on the evolution of viral capsid architecture. Mol. Cell 18:161-170, 2005.</ref>, and avian reovirus attachment protein sigma C<ref>Guardado, C.P., Fox, G.C., Hermo Parrado, X.L., Llamas-Saiz, A.L., Costas, C., Martinez-Costas, J., Benavente, J., and van Raaij, M.J. Structure of the carboxy-terminal receptor-binding domain of avian reovirus fibre sigmaC. J. Mol. Biol. 354:137-149, 2005.</ref>.

In addition to the three beta-spiral repeats observed in the crystallized sigma 1 fragment, sequence analysis suggests that residues 167-249 in the T3D sigma 1 tail form an additional five N-terminal beta-spiral repeats<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">Guglielmi, K.M., Johnson, E.M., Stehle, T., and Dermody, T.S. Attachment and cell entry of mammalian orthoreovirus. Curr. Top. Microbiol. Immunol. 309:1-38, 2006.</ref>. Alternatively, these residues may form a combination of beta-spiral repeats and alpha-helical coiled-coil, as suggested by sequence analysis<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">.</ref><ref name="Nibert1990">Nibert, M.L., Dermody, T.S., and Fields, B.N. Structure of the reovirus cell-attachment protein: a model for the domain organization of s1. J. Virol. 64:2976-2989, 1990.</ref> and an observed narrowing in this region in a composite negative-stain electron micrograph<ref>Fraser, R.D.B., Furlong, D.B., Trus, B.L., Nibert, M.L., Fields, B.N., and Steven, A.C. Molecular structure of the cell-attachment protein of reovirus: correlation of computer-processed electron micrographs with sequence-based predictions. J. Virol. 64:2990-3000, 1990.</ref>. The structure of N-terminal residues 1-160 of sigma 1 is unknown. However, a repeating heptad sequence motif is predictive of an amphipathic alpha-helix, which likely assembles into an alpha-helical coiled-coil in the trimer<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">.</ref><ref name="Nibert1990">.</ref>.

Although the structure of sigma 1 in complex with sialic acid is not yet available, studies using expressed proteins indicate that the region of T3D sigma 1 required for sialic binding resides near the midpoint of the tail, whereas a region just N-terminal to the head domain of T1L sigma 1 binds to carbohydrate<ref name="Chappell2000">.</ref>. In both T1L and T3D sigma 1, interactions with carbohydrate are mediated by a region of predicted beta-spiral<ref name="Chappell2002">.</ref>. Adaptation of non-sialic-acid-binding reoviruses to growth in MEL cells results in amino acid substitutions at residues 198, 202, and 204 of sigma 1 that confer sialic-acid-binding capacity on the resultant viruses<ref name="Chappell1997">.</ref>. Molecular modeling of the sigma 1 tail, based on available structure and sequence data, suggests that these residues are surface-exposed and proximal to one another in the predicted beta-spiral region<ref name="Chappell2002">.</ref>. Thus, residues 198, 202, and 204 are likely to contribute to a sialic-acid-binding site in T3D sigma 1.

=== Biological roles of GBP-ligand interaction ===
Sialic acid binding serves an important role in reovirus tropism and pathogenesis in vivo<ref>Barton, E.S., Youree, B.E., Ebert, D.H., Forrest, J.C., Connolly, J.L., Valyi-Nagy, T., Washington, K., Wetzel, J.D., and Dermody, T.S. Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease. J. Clin. Invest. 111:1823-1833, 2003.</ref>. Strain T3SA+, which binds to sialic acid, and strain T3SA-, which does not bind to this carbohydrate, produce equivalent titers in the intestine of newborn mice following peroral inoculation. However, T3SA+ spreads more rapidly from the intestine to sites of secondary replication and produces higher titers in the spleen, liver, and brain. Mice infected with T3SA+, but not T3SA-, develop clinical evidence of bile duct obstruction including jaundice and steatorrhea. Liver tissue from mice infected with T3SA+ show intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in human infants, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ binds 100-fold more efficiently than T3SA- to human cholangiocarcinoma cells. Thus, binding to sialic acid targets reovirus to bile duct epithelium and produces a disease reminiscent of infantile biliary atresia.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=reovirus&maxresults=20 CFG database search results for "reovirus"].

=== Glycan profiling ===
Not applicable.

=== Glycogene microarray ===
Sigma 1 is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.

=== Knockout mouse lines ===
Not applicable.

=== Glycan array ===
Experiments in progress. No definitive results have been obtained thus far.

== Related GBPs ==
The attachment protein of adenovirus, fiber, is a structural homolog of sigma 1. At least one adenovirus serotype (Ad37; [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=ad37&maxresults=20 CFG data]) is known to bind glycan receptors via residues in the fiber protein<ref>Burmeister WP, Guilligay D, Cusack S, Wadell G, Arnberg N: Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites. Journal of Virology 78:7727-7736, 2004.</ref>. The actual binding site is not homologous. However, information about reovirus glycan binding could also be used to engineer adenovirus fiber proteins (or other trimeric fiber-like proteins) that possess novel glycan-binding properties.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Terence Dermody, Mavis McKenna, Thilo Stehle.

C. difficile toxin A (TcdA)

2011-09-13T22:29:03Z

Carole Weaver:

'''The large clostridial cytotoxins (LCT)''' are a family of structurally and functionally related exotoxins from ''Clostridium difficile'' (toxins A and B [also termed TcdA and TcdB, respectively), ''C. sordellii'' (lethal and hemorrhagic toxin) and ''C. novyi'' (alpha-toxin). The LCTs are major virulence factors, which in addition to their in vivo effects, are cytotoxic to cultured cell lines, causing reorganization of the cytoskeleton accompanied by morphological changes<ref name="Just 1">Just. I., Gerhard, R. (2004). Large clostridial cytotoxins. Rev. Physiol. Biochem. Pharmacol. 152:23-47.</ref>. The LCTs are single-chain protein toxins, comprising three domains: receptor-binding, translocation and catalysis, which mediate cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and enzymatic cytotoxic activity, respectively. Enzymatic activity involves transfer of a glucosyl moiety from UDP-glucose (or the N-acetyl-glucosaminyl moiety from UDP-GlcNAc in the case of alpha toxin) to a conserved threonine within the effector regions of the intracellular Rho and Ras GTPases<ref name="Just 1"/>. The C-terminal receptor-binding domain comprises up to one third of the LCT molecule, and contains repetitive peptide elements called combined repetitive oligopetides (CROPs). The repeating units binding regions of certain streptococcal glycosyltransferases<ref name="Just 1"/>.

'''TcdA''' is the better characterized of the two LCTs produced by ''C. difficile.'' Collectively, TcdA and TcdB are directly responsible for the increasingly common and serious human gastrointestinal disease caused when antibiotic treatment enables ''C. difficile'' to out-compete commensal gut microflora. Recently, the crystal structure of a C-terminal fragment of TcdA has been solved, revealing a solenoid-like structure, which consists of 32 short repeats with 15–21 residues and seven long repeats with 30 residues<ref name="Jank 2007">Thomas Jank, Torsten Giesemann and Klaus Aktories. (2007). Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function. Glycobiology. 17: 15R–22R.
</ref>. Solenoid structures are often found in bacterial surface proteins, and their extended surface allows protein–protein or protein–carbohydrate interactions. TcdA is known to bind to Galα1-3Galβ1-4GlcNAc- glycans, although this structure is not present in humans. However, it is also known to recognize a variety of other structures with a Galβ[1→4]GlcNAcβ- backbone, including the fucosylated blood group antigens Lewisx and Lewisy, both of which are present in human intestinal epithelium<ref name="Voth 2005">Voth, D. E., and Ballard, J. D. (2005). Clostridium difficile toxins: mechanism of action and role in disease. Clin. Microbiol. Rev. 18, 247-263.</ref>. Clearly, a better understanding of the identity/structure of the human receptor(s) for these toxins will facilitate design of novel therapeutics capable of blocking toxin binding. Glycan array analysis conducted by the CFG showed that TcdA bound most strongly to glycans terminating in Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4-GlcNAc-, which mimics the blood group antigen Lewisa; other bound structures included Galα1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ-, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-, and Fucαa1-2[GalNAcαa1-3]Galβ1-4GlcNAcβ1-3Galβb1-4GlcNAcβ-.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) contributing to the understanding of TcdA include: Borden Lacy, Yashwant Mahida, Kenneth Ng
* Non-CFG PIs include: Brian Dieckgraefe

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
*Galα1-3Galβ1-4GlcNAc- glycans (not present in humans). 
*Other structures with a Galβ[1→4]GlcNAcβ- backbone, including the fucosylated blood group antigens Lewisx and Lewisy, both of which are present in human intestinal epithelium<ref name="Voth 2005"/>. 
*Strongest binding: glycans terminating in Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4-GlcNAc-, which mimics the blood group antigen Lewisa. 
*Also binds: Galα1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ-, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-, and Fucαa1-2[GalNAcαa1-3]Galβ1-4GlcNAcβ1-3Galβb1-4GlcNAcβ-. 
 
=== Cellular expression of GBP and ligands ===
TcdA is produced by ''Clostridium difficile''. Structurally and functionally related exotoxins in the LCT family are produced by ''C. sordellii'' (lethal and hemorrhagic toxin) and ''C. novyi'' (alpha-toxin).
 
TcdA binds the fucosylated blood group antigens Lewisx and Lewisy, which are present in human intestinal epithelium<ref name="Voth 2005"/> (see above).
=== Biosynthesis of ligands ===

 
=== Structure ===
Members of the LCT family are single-chain protein toxins with receptor-binding, translocation and catalysis domains, which mediate cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and enzymatic cytotoxic activity, respectively. Up to one third of the LCT molecule is comprised of the C-terminal receptor-binding domain, which contains repetitive peptide elements called combined repetitive oligopeptides (CROPs).
 
X-ray crystallography has revealed the three-dimensional structures of several recombinant fragments of TcdA and related toxins<ref name="Jank 2007"/>. The N-terminal domain consists of a GT-A family glucosyltransferase that catalyzes the transfer of glucose from UDP-glucose to specific threonine residues of small GTPases regulating the structure and dynamics of the cytoskeleton. Although the three-dimensional structure of the GT domain of TcdA has not been solved by crystallography, the structures of closely related GT domains from TcdB and the letahal toxin from ''Clostridium sordellii'' have been determined.
 
The crystallographic structure of a cysteine protease domain bound to inositol hexakisphosphate has also been determined. This domain follows the N-terminal GT domain and is responsible for the autoproteolysis of TcdA that is important for releasing the GT domain into the cytoplasm.
 
Crystallographic structures of several recombinant fragments from the highly repetitive C-terminal domain have revealed an extended beta-solenoid structure that presents seven binding sites for complex oligosaccharide receptors. The bulk of this carbohydrate-binding domain is comprised of multiple copies of short repeats that interspersed with single copies of long repeats. Carbohydrate-binding sites appear to be formed at the junctions of long repeats and the preceding and following short repeats.
 
Crystallographic structures of recombinant fragments of TcdA have been determined with oligosaccharides, including the Gal-α(1,3)-LacNAc trisaccharide shown below<ref name = "Greco 2006">Greco, A et al. (2006). Carbohydrate recognition by ''Clostridium difficile'' toxin A. Nat. Struct. Mol. Biol. 13:460-461.</ref>. This trisaccharide was determined to be a likely native receptor for TcdA in the brush border membranes of intestinal epithelial cells from hamster<ref name = "Krivan 1986">Krivan, HC et al (1986). Cell-surface binding site for ''Clostridium difficile'' enterotoxin: evidence for a glycoconjugate containing the sequence Galα1,3-Galβ1,4-GlcNAc. Infect. Imm. 53:573-581.</ref>, an organism which is susceptible to ''C. difficile'' infection (CDI) and is often used as a model for studying the disease.
 
[[File:CD_fig1.png]]

=== Biological roles of GBP-ligand interaction ===
TcdA and TcdB, the other LCT produced by ''C. difficile'', cause the serious human gastrointestinal disease that results when antibiotic treatment enables ''C. difficile'' to out-compete commensal gut microflora. The LCTs are major virulence factors, which in addition to their in vivo effects, are cytotoxic to cultured cell lines, causing reorganization of the cytoskeleton accompanied by morphological changes<ref name="Just 1"/>.
 
== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=difficile&maxresults=20 CFG database search results for "difficile"].

=== Glycan profiling ===

 
=== Glycogene microarray ===
TcdA is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG glycan array has been used to examine the [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2693 glycan-binding specificity of TcdA]. In addition, the CFG's glycan synthesis capability is being used to prepare synthetic oligosaccharides indentified in the glycan array screens (with or without biotin tags) to further probe TcdA-receptor interactions.

== Related GBPs ==
''C. difficile'' toxin B (TcdB; [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=TcdB&maxresults=20 CFG data]), ''C. sordellii'' lethal toxin and hemorrhagic toxin, ''C. novyi'' alpha-toxin.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, Kenneth Ng, James Paton

Botulinum toxin serotype A (BoNT/A)

2011-09-13T22:24:00Z

Carole Weaver: /* Structure */

'''The clostridial neurotoxins''' are the most lethal protein toxins for humans and recently have been utilized as therapeutic agents to treat numerous human neurological inflictions. The Botulinum neurotoxins produced by ''Clostridium botulinum'' and Tetanus toxin produced by ''C. tetani'' are members of the family of clostridial neurotoxins. The clostridial neurotoxins are di-chain toxins with the N-terminal catalytic domains (Light Chain, LC) possessing metalloprotease activity that is disulfide linked to the C-terminal
domain (Heavy Chain, HC). The neurological toxicity and therapeutic utility of the clostridial neurotoxins is due to the HC’s tropism for neuronal receptors and the LC’s cleavage of neuron-specific target proteins, termed SNARE proteins. SNARE proteins are responsible for the fusion of neurotransmitter vesicles with the plasma membrane. There are seven serotypes of the BoNTs that share primary and ternary structure-function properties. Each BoNT serotype utilizes dual receptors for entry into neurons and each cleaves a specific SNARE protein or a unique site on a specific SNARE protein.

'''The Botulinum neurotoxins''' elicit flaccid paralysis, while tetanus toxin elicits spastic paralysis. The differential toxicity is due to the unique trafficking of these toxins in motor neurons. Botulinum neurotoxin HC binds to dual host receptors on the surface of motor neurons to deliver the BoNT to acidified vesicles where the LC is translocated into the cytoplasm of the neuron. Upon entry into the cytoplasm, LC cleaves a SNARE protein which inhibits fusion of neurotransmitter vesicles to the plasma membrane, inhibiting the release of neurotransmitter molecules. In contrast, TeNT HC binds to dual host receptors on the surface of motor neurons to deliver the TeNT to neutral vesicles for retrograde trafficking to the central nervous system. Following this transcytosis, Tetanus toxin binds dual receptors on inhibitory neurons to deliver TeNT to acidified endosomes where the LC is translocated into the cytoplasm of the neuron. TeNT LC cleaves the SNARE proteins, VAMP-2, which inhibits the release of neurotransmitters from inhibitory motor neurons. The inability to release neurotransmitter from inhibitory motor neurons yields spastic paralysis.

'''Botulinum toxin serotype A (BoNT/A)''' was chosen as the paradigm for the clostridial neurotoxins, since the dual host receptors for BoNT/A have been determined and the basis for recognizing cleavage of the SNARES substrate SNAP25 has been characterized. In addition, BoNT/A is the most common serotype used in clinical therapies. It is anticipated that understanding BoNT/A action will provide new information relevant to the entire family of clostridial neurotoxins. These studies will enhance the understanding of the unique
properties of each BoNT serotype and Tetanus toxin to extend their utility in human inflictions. The CFG has been used to facilitate the characterization of the ganglioside binding pocket of these neurotoxins. Prior studies utilized low throughput analyses that provided limited insight into interactions between these neurotoxins and gangliosides, and but high throughput analysis of the CFG core provided a better understanding of the biochemical and structural interactions of the neurotoxins with glycans. For example, array analysis showed that the dual receptors for Tetanus toxin recognized unique components of gangliosides.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of BoNT/A include: Joseph Barbieri, Edwin Chapman, Minoru Fukuda, Raymond Stevens, Willie Vann

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

 
=== Cellular expression of GBP and ligands ===
Botulinum toxins produced by ''Clostridium botulinum'' bind to dual host receptors on the surface of motorneurons.
 
=== Biosynthesis of ligands ===

 
=== Structure ===
The crystal structure of BoNT/A has been solved both alone and bound to other molecules (see [http://pathema.jcvi.org/cgi-bin/Clostridium/shared/HtmlPage.cgi?page=bont_structures#typeA entry in Panthema] NIAID Bioinformatics Resource Center).
 

=== Biological roles of GBP-ligand interaction ===
Botulinum toxins cause neurotoxicity by cleaving SNARE proteins, which normally allow neurotransmitter-containing vesicles to fuse with the neuronal plasma membrane.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=botulinum&maxresults=20 CFG database search results for botulinum].

=== Glycan profiling ===

 
=== Glycogene microarray ===
BoNT/A is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG synthesized ganglioside derivates that have been used in co-crystallization studies with the clostridial neurotoxins. The CFG glycan array was used to identify the ganglioside binding specificity to the clostridial neurotoxins ([http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2011 BoNT/C], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1972 BoNT/D], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1681 BoNT/F]).

== Related GBPs ==

Botulinum neurotoxins serotypes B-G (CFG data: [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FB&maxresults=20 BoNT/B,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FC&maxresults=20 BoNT/C,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FD&maxresults=20 BoNT/D,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FE&maxresults=20 BoNT/E,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Botulinum+AND+neurotoxin+AND+F&maxresults=20 BoNT/F,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FG&maxresults=20 BoNT/G)], Tetanus toxin [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=tetanus+AND+toxin&maxresults=20 (CFG data)]

== References ==
<references/>
* C. Chen, Z. Fu, J-J. P. Kim, J.T. Barbieri, and M. R. Baldwin. 2009. Gangliosides as High Affinity Receptors for Tetanus Neurotoxin. J Biol Chem. 284: 26569-77. PMC2785345
* M. Dong, W. H. Tepp, H. Liu, E. A. Johnson, and E. R. Chapman. 2007. Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons. J. Cell Biol. 179: 1511-1522. PMC2373501

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton

Botulinum toxin serotype A (BoNT/A)

2011-09-13T22:22:41Z

Carole Weaver: /* Structure */

'''The clostridial neurotoxins''' are the most lethal protein toxins for humans and recently have been utilized as therapeutic agents to treat numerous human neurological inflictions. The Botulinum neurotoxins produced by ''Clostridium botulinum'' and Tetanus toxin produced by ''C. tetani'' are members of the family of clostridial neurotoxins. The clostridial neurotoxins are di-chain toxins with the N-terminal catalytic domains (Light Chain, LC) possessing metalloprotease activity that is disulfide linked to the C-terminal
domain (Heavy Chain, HC). The neurological toxicity and therapeutic utility of the clostridial neurotoxins is due to the HC’s tropism for neuronal receptors and the LC’s cleavage of neuron-specific target proteins, termed SNARE proteins. SNARE proteins are responsible for the fusion of neurotransmitter vesicles with the plasma membrane. There are seven serotypes of the BoNTs that share primary and ternary structure-function properties. Each BoNT serotype utilizes dual receptors for entry into neurons and each cleaves a specific SNARE protein or a unique site on a specific SNARE protein.

'''The Botulinum neurotoxins''' elicit flaccid paralysis, while tetanus toxin elicits spastic paralysis. The differential toxicity is due to the unique trafficking of these toxins in motor neurons. Botulinum neurotoxin HC binds to dual host receptors on the surface of motor neurons to deliver the BoNT to acidified vesicles where the LC is translocated into the cytoplasm of the neuron. Upon entry into the cytoplasm, LC cleaves a SNARE protein which inhibits fusion of neurotransmitter vesicles to the plasma membrane, inhibiting the release of neurotransmitter molecules. In contrast, TeNT HC binds to dual host receptors on the surface of motor neurons to deliver the TeNT to neutral vesicles for retrograde trafficking to the central nervous system. Following this transcytosis, Tetanus toxin binds dual receptors on inhibitory neurons to deliver TeNT to acidified endosomes where the LC is translocated into the cytoplasm of the neuron. TeNT LC cleaves the SNARE proteins, VAMP-2, which inhibits the release of neurotransmitters from inhibitory motor neurons. The inability to release neurotransmitter from inhibitory motor neurons yields spastic paralysis.

'''Botulinum toxin serotype A (BoNT/A)''' was chosen as the paradigm for the clostridial neurotoxins, since the dual host receptors for BoNT/A have been determined and the basis for recognizing cleavage of the SNARES substrate SNAP25 has been characterized. In addition, BoNT/A is the most common serotype used in clinical therapies. It is anticipated that understanding BoNT/A action will provide new information relevant to the entire family of clostridial neurotoxins. These studies will enhance the understanding of the unique
properties of each BoNT serotype and Tetanus toxin to extend their utility in human inflictions. The CFG has been used to facilitate the characterization of the ganglioside binding pocket of these neurotoxins. Prior studies utilized low throughput analyses that provided limited insight into interactions between these neurotoxins and gangliosides, and but high throughput analysis of the CFG core provided a better understanding of the biochemical and structural interactions of the neurotoxins with glycans. For example, array analysis showed that the dual receptors for Tetanus toxin recognized unique components of gangliosides.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of BoNT/A include: Joseph Barbieri, Edwin Chapman, Minoru Fukuda, Raymond Stevens, Willie Vann

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

 
=== Cellular expression of GBP and ligands ===
Botulinum toxins produced by ''Clostridium botulinum'' bind to dual host receptors on the surface of motorneurons.
 
=== Biosynthesis of ligands ===

 
=== Structure ===
The crystal structure of BoNT/A has been solved both alone and bound to other molecules (see its [http://pathema.jcvi.org/cgi-bin/Clostridium/shared/HtmlPage.cgi?page=bont_structures#typeA entry in Panthema] NIAID Bioinformatics Resource Center).
 

=== Biological roles of GBP-ligand interaction ===
Botulinum toxins cause neurotoxicity by cleaving SNARE proteins, which normally allow neurotransmitter-containing vesicles to fuse with the neuronal plasma membrane.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=botulinum&maxresults=20 CFG database search results for botulinum].

=== Glycan profiling ===

 
=== Glycogene microarray ===
BoNT/A is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG synthesized ganglioside derivates that have been used in co-crystallization studies with the clostridial neurotoxins. The CFG glycan array was used to identify the ganglioside binding specificity to the clostridial neurotoxins ([http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2011 BoNT/C], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1972 BoNT/D], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1681 BoNT/F]).

== Related GBPs ==

Botulinum neurotoxins serotypes B-G (CFG data: [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FB&maxresults=20 BoNT/B,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FC&maxresults=20 BoNT/C,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FD&maxresults=20 BoNT/D,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FE&maxresults=20 BoNT/E,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Botulinum+AND+neurotoxin+AND+F&maxresults=20 BoNT/F,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FG&maxresults=20 BoNT/G)], Tetanus toxin [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=tetanus+AND+toxin&maxresults=20 (CFG data)]

== References ==
<references/>
* C. Chen, Z. Fu, J-J. P. Kim, J.T. Barbieri, and M. R. Baldwin. 2009. Gangliosides as High Affinity Receptors for Tetanus Neurotoxin. J Biol Chem. 284: 26569-77. PMC2785345
* M. Dong, W. H. Tepp, H. Liu, E. A. Johnson, and E. R. Chapman. 2007. Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons. J. Cell Biol. 179: 1511-1522. PMC2373501

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton

CBM47

2011-09-13T21:11:23Z

Carole Weaver:

Enzymes that degrade host glycans are increasingly being found as virulence factors in pathogenic bacteria<ref>Shelburne, S. A., Davenport, M. T., Keith, D. B. & Musser, J. M. (2008). The role of complex carbohydrate catabolism in the pathogenesis of invasive streptococci. Trends Microbiol 16, 318-25.</ref><ref name="Hava 16">Hava, D. L. & Camilli, A. (2002). Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol Microbiol 45, 1389-406.</ref>. A common property of extracellular glycan degrading enzymes found in such bacteria is multi-modularity; these enzymes often comprise a large number of modules with a variety of functions. The most common class of ancillary module are carbohydrate-binding modules (CBMs)<ref>Boraston, A. B., Bolam, D. N., Gilbert, H. J. & Davies, G. J. (2004). Carbohydrate-binding modules: fine tuning polysaccharide recognition. Biochem J 382, 769-782.</ref>, which are alternatively referred to as lectin-domains. These modules are responsible for targeting carbohydrate-degrading enzymes to a glycan substrate or, when the enzymes are attached to the bacterial cell-surface, likely also function to adhere the bacterium to a glycan<ref>Ficko-Blean, E., Gregg, K. J., Adams, J. J., Hehemann, J. H., Czjzek, M., Smith, S. P. & Boraston, A. B. (2009). Portrait of an enzyme, a complete structural analysis of a multimodular {beta}-N-acetylglucosaminidase from Clostridium perfringens. J Biol Chem 284, 9876-84.</ref>. The presence of these lectin-domains in multi-modular proteins and their contribution of glycan binding function to catalytically active proteins distinguishes these modules from other bacterial glycan-binding proteins (GBPs). The CBM47 modules from the ''Streptococcus pneumoniae'' enzyme SpGH98 (or "fucolectin-related protein") are specific to the Lewisy antigen<ref name ="Boraston 2006">Boraston, A. B., Wang, D. & Burke, R. D. (2006). Blood group antigen recognition by a Streptococcus pneumoniae virulence factor. J Biol Chem 281, 35263-35271.</ref>, which is quite rare among all GBPs, and function to target this enzyme to this antigen when present on epithelial cells<ref name="Higgins 2009">Higgins, M. A., Whitworth, G. E., El Warry, N., Randriantsoa, M., Samain, E., Burke, R. D., Vocadlo, D. J. & Boraston, A. B. (2009). Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases. J Biol Chem 284, 26161-73.</ref>. Recognition and destruction of this antigen appears to be a critical process in pneumococcal virulence<ref name="Hava 16"/><ref name="Embry 2007">Embry, A., Hinojosa, E. & Orihuela, C. J. (2007). Regions of Diversity 8, 9 and 13 contribute to Streptococcus pneumoniae virulence. BMC Microbiol 7, 80.</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
This is a very new area of investigation. CFG Participating Investigators (PIs) that have screened other CBMs or proteins containing CBMs include: Alisdair Boraston, Garry Taylor, Warren Wakarchuck

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about CBM47, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===
CBM47 is a fucose specific binding module that is related to ''Anguilla anguilla'' fucolectin. The high affinity ligand for CBM47 has been determined from glycan microarray screening on the CFG microarray[http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_34_08192004#]to be Fucα1-2Galβ1-4(Fucα1-3)GlcNAc [Lewis y]<ref name="Boraston 2006"/>.

[[File:Lewisy.jpg]]
 

=== Cellular expression of GBP and ligands ===
The CBM47 modules from the ''Streptococcus pneumoniae'' enzyme SpGH98 (or "fucolectin-related protein") are specific to the Lewisy antigen<ref name="Boraston 2006"/>, which is quite rare among all GBPs, and function to target this enzyme to this antigen when present on epithelial cells<ref name="Higgins 2009"/>.
 
=== Biosynthesis of ligands ===

 
=== Structure ===

CBM47 originates from a multimodular ''S. pneumoniae'' that has an N-terminal, Lewisy degrading catalytic module. Indeed, three CBM47 modules are found in tandem.

[[File:modular.jpg]]

The high resolution X-ray structures of the N-terminal and C-terminal CBM47 modules have been determined and the N-terminal module in complex with the Lewisy tetrasaccharide<ref name="Boraston 2006"/>.

[[File:CBM47.jpg]]

 

=== Biological roles of GBP-ligand interaction ===

The primary role of CBM47, and indeed other CBMs found in carbohydrate-active enzymes, is to direct the entire enzyme to its glycan substrate. However, CBMs found in carbohydrate-active enzymes attached to microbial cell surfaces may also play a role in the adhesion of the bacterium to host glycan-bearing tissues.
 
Recognition and destruction of the Lewisy antigen by CBM47 appears to be a critical process in pneumococcal virulence<ref name="Hava 16"/><ref name="Embry 2007"/>.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CBM&maxresults=20 CFG database search results for CBM].

=== Glycan profiling ===

 
=== Glycogene microarray ===
CBM47 is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The specificity of several CBMs have been investigated by CFG glycan array analysis (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1831 here] for example). Isolated glycans for structural and quantitative binding studies have also been obtained from the CFG.

== Related GBPs ==

There are presently over 55 families of CBMs that are defined on the basis of amino acid sequence similarity; however, the majority of these CBMs families contain members specific for plant cell wall polysaccharides. CFG resources have been instrumental in studying the subset of CBMs that recognize complex glycans. In addition to CBM47 there are a number of CBMs belonging to family 32 and 51 that share the property of binding complex glycans. Though unrelated at the amino acid sequence level CBMs in families 32, 47, and 51 are structurally related and functionally related. It is important to note, however, that the diversity of complex glycan binding among these family members is considerable and only coming to light through glycan array screening. The availability of purfied glycans is facilitating structural and quantitative studies of glycan binding by CBMs in families 32, 47, and 51.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Alisdair Boraston, Anne Imberty

Siglec-8

2011-09-12T22:18:49Z

Carole Weaver: /* Structure */

Siglec-8 is a human siglec expressed predominantly on eosinophils and mast cells, and is a paradigm for the rapidly evolving sub-family of CD33-related siglecs that are expressed on various white blood cells<ref>Crocker, P. R., Paulson, J. C. & Varki, A. Siglecs and their roles in the immune system. Nat Rev Immunol 7, 255-266 (2007).</ref>
<ref name=" Kikly 2009">
Kikly, K.K., Bochner, B.S., et al. [http://www.ncbi.nlm.nih.gov/pubmed/10856141 Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils.] J Allergy Clin Immunol 105, 1093-100 (2000)</ref>
<ref name="Bochner 2009">Bochner, B.S. [http://www.ncbi.nlm.nih.gov/pubmed/19178537 Siglec-8 on human eosinophils and mast cells, and Siglec-F on murine eosinophils, are functionally related inhibitory receptors.] Clin Exp Allergy 39, 317-324 (2009).</ref>
<ref name=" Floyd H 2000"></ref>. A characteristic feature of Siglec-8 and most other CD33-related siglecs is a cytoplasmic domain with a single immunoreceptor tyrosine inhibitory motif (ITIM) and a single ITIM-like motif that participate in siglec-mediated regulation of cell signaling and endocytosis. While there is no clear ortholog in mice, Siglec-F has been documented as a functional paralog that has a similar expression pattern on murine leukocytes and similar ligand specificity<ref name="Bochner 2009"></ref><ref name=" Tateno 1125">Tateno, H., Crocker, P. R. & Paulson, J. C. Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6'-sulfo-sialyl Lewis X as a preferred glycan ligand. Glycobiology 15, 1125-1135 (2005).</ref>
<ref name=" Zhang 2007"></ref>. Siglec-8, and its murine paralog Siglec-F, recognize a ligand containing both sialic acid and sulfate (NeuAcα2-3[6S]Galβ1-4G[Fucα1-3]GlcNAc-), a specificity that is distinct from all other siglecs. Ligation of Siglec-8 (or Siglec-F) with antibodies or polymeric ligands induces apoptosis of eosinophils, suggesting a therapeutic approach for treating eosinophil (or mast cell) mediated disease by targeting Siglec-8<ref>O'Reilly, M. K. & Paulson, J. C. Siglecs as targets for therapy in immune-cell-mediated disease. Trends Pharmacol Sci 30, 240-248 (2009).</ref><ref>Zimmermann, N. et al. Siglec-F antibody administration to mice selectively reduces blood and tissue eosinophils. Allergy 63, 1156-1163 (2008).</ref>
<ref name=" Bochner 4307 ">Bochner, B. S. et al. Glycan array screening reveals a candidate ligand for Siglec-8. J Biol Chem 280, 4307-4312 (2005).</ref>
<ref name=" Nutku, E 2003">
Nutku, E., Aizawa, H., Hudson, S. A. & Bochner, B. S. Ligation of Siglec-8: a selective mechanism for induction of human eosinophil apoptosis. Blood 101, 5014-5020 (2003).</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
Participating Investigators (PIs) of the CFG have made major contributions to the understanding of the biology of Siglec-8 and its murine paralog, Siglec-F. These include: Bruce Bochner, Nicolai Bovin, Paul Crocker, James Paulson, Ronald Schnaar, Ajit Varki

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about Siglec-8, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information about Siglec-8 can be found in its [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Itlect_00146&sideMenu=no GBP Molecule Page] in the CFG database.
=== Carbohydrate ligands ===
The high affinity ligand for Siglec-8 has been deduced from glycan microarray screening on the CFG microarray (click [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_49_09152004 here] and [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v2_19_02202004 here]) to be NeuAcα2-3(6-SO3)Galβ1-4(Fucα1-3)GlcNAc [6'Su-SLeX] <ref name=" Bochner 4307 ">
</ref><ref name=" Tateno 1125"></ref>

[[File:6pso3slex.jpg]]

For Siglec-F, histologic studies suggest the presence of an α2,3-linked sialylated glycoprotein ligand expressed by airway epithelium. Its constitutive expression requires the enzyme St3Gal3.<ref>Guo JP, Brummet ME, Myers AC, Na HJ, Rowland E, Schnaar RL, Zheng T, Zhu Z, Bochner BS. Characterization of expression of glycan ligands for Siglec-F in normal mouse lungs. Am J Respir Cell and Molec Biol 2010 Apr 15 [Epub ahead of print] 2010 and </ref> Levels of this ligand are increased during allergic pulmonary inflammation.
<ref name=" Zhang 2007">Zhang M, Angata T, Cho JY, Miller M, Broide DH, Varki A. Defining the in vivo function of Siglec-F, a CD33-related Siglec expressed on mouse eosinophils. Blood 2007; 109:4280-7</ref>
 

=== Cellular expression of GBP and ligands ===
Siglec-8 is expressed in human eosinophils and mast cells, and weakly in basophils. <ref name=" Kikly 2009"></ref>
<ref name=" Floyd H 2000">Floyd H, Ni J, Cornish AL, Zeng Z, Liu D, Carter KC, Steel J, Crocker PR. Siglec-8: a novel eosinophil-specific member of the immunoglobulin superfamily. J Biol Chem 2000; 275:861-6.</ref><ref>Yokoi H, Myers A, Matsumoto K, Crocker PR, Saito H, Bochner BS. Alteration and acquisition of Siglecs during in vitro maturation of CD34+ progenitors into human mast cells. Allergy 2006; 61:769-76</ref>
 
=== Biosynthesis of ligands ===
Constitutive expression in airway epithelium of the putative α2,3-linked sialylated glycoprotein ligand for Siglec-F requires the enzyme St3Gal3 which has been characterized in
[http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_hum_624&sideMenu=true&pageType=general Human] and
[http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_mou_644&sideMenu=true&pageType=general Mouse].
 

=== Structure ===
[[File:Siglec8 SiglecF.jpg]]
 
Siglec-8 and most other CD33-related siglecs have a characteristic cytoplasmic domain with a single immunoreceptor tyrosine inhibitory motif (ITIM) and a single ITIM-like motif that participate in siglec-mediated regulation of cell signaling and endocytosis.

=== Biological roles of GBP-ligand interaction ===
'''In vitro'''
Eosinophil apoptosis. <ref name=" Nutku, E 2003"></ref><ref>Nutku E, Hudson SA, Bochner BS. Mechanism of Siglec-8-induced human eosinophil apoptosis: role of caspases and mitochondrial injury. Biochem Biophys Res Commun 2005; 336:918-24</ref><ref>1Nutku-Bilir E, Hudson SA, Bochner BS. Interleukin-5 priming of human eosinophils alters Siglec-8 mediated apoptosis pathways. Am J Respir Cell Mol Biol 2008; 38:121-4</ref>
Inhibition of mast cell mediator release.<ref>Yokoi H, Choi OH, Hubbard W, Lee H-S, Canning BJ, Lee HH, Ryu S-D, Bickel CA, Hudson SA, MacGlashan DW, Jr., Bochner BS. Inhibition of FcεRI-dependent mediator release and calcium flux from human mast cells by Siglec-8 engagement. J Allergy Clin Immunol 2008; 121:499-505</ref>

'''In vivo (for Siglec-F)'''
Antibody administration to mice causes selective depletion of eosinophils in blood and gastrointestinal tissues via apoptosis.<ref>Zimmermann N, McBride ML, Yamada Y, Hudson SA, Jones C, Cromie KD, Crocker PR, Rothenberg ME, Bochner BS. Siglec-F antibody administration to mice selectively reduces blood and tissue eosinophils. Allergy 2008; 63:1156-63</ref> They are also effective in reversing some sequelae of mouse models of eosinophilic gastroenteritis and asthma.<ref>Song DJ, Cho JY, Miller M, Strangman W, Zhang M, Varki A, Broide DH. Anti-Siglec-F antibody inhibits oral egg allergen induced intestinal eosinophilic inflammation in a mouse model. Clin Immunol 2009; 131:157-69</ref><ref>Song DJ, Cho JY, Lee SY, Miller M, Rosenthal P, Soroosh P, Croft M, Zhang M, Varki A, Broide DH. Anti-Siglec-F antibody reduces allergen-induced eosinophilic inflammation and airway remodeling. J Immunol 2009; 183:5333-41</ref>
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Siglec-8&maxresults=20 CFG database search results for Siglec-8].

=== Glycan profiling ===
Glycan structure analysis has been conducted by the CFG for [http://www.functionalglycomics.org/glycomics/common/jsp/samples/searchSample.jsp?5=Human&15=&10=&9=Eosinophils+&operation=refine&templateKey=2&12=CellType&submit=submit human ] and [http://www.functionalglycomics.org/glycomics/common/jsp/samples/searchSample.jsp?5=Mouse&15=&10=&9=Eosinophils+&operation=refine&templateKey=2&12=CellType&submit=submit mouse] eosinophils.

=== Glycogene microarray ===
Analysis has been conducted on glycosyltransferase expression using the glycogene microarray for [http://www.functionalglycomics.org/static/coreE/flash/search.swf?maExpKey=100393 murine eosinophils] that relates to the enzymes required for expression of cis ligands of Siglec-F on these cells. Probes for human Siglec-8 and mouse Siglec-F have been included on all four versions of the CFG glycogene microarray.

=== Knockout mouse lines ===
Mice deficient in Siglec-F have normal blood and bone marrow eosinophils at baseline, but develop exaggerated bone marrow, blood and lung eosinophilia after allergen sensitization and challenge. <ref name=" Zhang 2007"></ref>
 

=== Glycan array ===
The discovery of the [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_49_09152004 ligand for Siglec-8] [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v2_19_02202004] and its murine paralog, [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_47_10132004 Siglec-F ] [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v2_58_07262004], was made by investigator-initiated resource requests for glycan array analysis and carbohydrate compounds. To see all glycan array results for Siglec-8, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=siglec-8&cat=coreh here].

== Related GBPs ==
hSiglec-3 (CD33), Siglec-5, Siglec-6 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-6&maxresults=20 (CFG data)], Siglec-7 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-7&maxresults=20 (CFG data)], Siglec-9 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-9&maxresults=20 (CFG data)], Siglec-10 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-10&maxresults=20 (CFG data)], Siglec-11, Siglec-F [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-F&maxresults=20 (CFG data)], Siglec-E [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-E&maxresults=20 (CFG data)], Siglec-G [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-G&maxresults=20 (CFG data)].

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Bruce Bochner, Paul Crocker, James Paulson, Ron Schnaar

P-Selectin

2011-04-01T19:02:47Z

Carole Weaver: /* Knockout mouse lines */

The three selectins (P-selectin, L-selectin, and E-selectin) have related and sometimes overlapping functions in cell adhesion and mediate some of the best characterized glycan-dependent cell adhesion events. Of the ligands for these three C-type lectins, the target ligand of P-selectin, P-selectin glycoprotein ligand 1 (PSGL-1), is the best understood. Thus, P-selectin is used here to represent all three selectins.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
Selectin research was already at a relatively mature stage when the CFG began. Early PI work included structural studies, extensive analysis of leukocyte adhesion to endothelia ''in vivo'', and characterization of knockout mice to demonstrate physiological function. In addition to submitting samples for glycan array analysis, PIs have been involved in analyzing selectin expression under different conditions, including in knockout mice lacking enzymes for making target ligands.
* PIs working on P-selectin include: Hans-Peter Altevogt, Bruce Bochner, Pi-Wan Cheng, Richard Cummings, Robert Fuhlbrigge, Minoru Fukuda, Geoff Kansas, Klaus Ley, John Lowe, Rodger McEver, Steve Rosen, Ron Schnaar, Karen Snapp, Lloyd Stoolman, Martin Wild, Hermann Ziltener
* Non-PIs who have used CFG resources to study P-selectin include: Roland Contreras, Leonard Seymour

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about P-selectin, its carbohydrate ligands, and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Pages for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Ctlect_354&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_mou_Ctlect_284&sideMenu=no mouse] P-selectin in the CFG database.
=== Carbohydrate ligands ===

P-selectin binds cooperatively to tyrosine sulfates, other amino acids, and a core 2 O-glycan capped with sialyl Lewis x, all positioned near the N terminus of PSGL-1<ref>Leppanen A, White SP, Helin J, McEver RP, Cummings RD (2000) Binding of glycosulfopeptides to P-selectin requires stereospecific contributions of individual tyrosine sulfate and sugar residues. J Biol Chem 275, 39569-39578.</ref>

=== Cellular expression of GBP and ligands ===

P-selectin is expressed on platelets, endothelial cells, and some macrophages
<ref name=" McEver RP 581">McEver RP (2002) Selectins: lectins that initiate cell adhesion under flow. Curr Opin Cell Biol 14, 581-588.</ref><ref>Tchernychev B, Furie B, Furie BC (2003) Peritoneal macrophages express both P-selectin and PSGL-1. J Cell Biol 163, 1145-1155</ref>. PSGL-1 is expressed on leukocytes and some endothelial cells<ref name=" McEver RP 581"></ref><ref>Rivera-Nieves J, Burcin TL, Olson TS, Morris MA, McDuffie M, Cominelli F, Ley K (2006) Critical role of endothelial P-selectin glycoprotein ligand 1 in chronic murine ileitis. J Clin Invest 203, 907-919</ref>.

=== Biosynthesis of ligands ===
 
=== Structure ===
[[image:P-selectin.jpg]] 
The structures of a fragment of P-selectin containing the CRD and EGF domains in complex with a sialyl Lewisx-containing oligosaccharide as well as a glycopeptide from PSGL-1 that contains sulfated tyrosine residues have been determined.<ref name="Somers2000">Somers W. S., Tang J., Shaw G. D,. Camphausen R. T. (2000) Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLex and PSGL-1. Cell 103, 467-479</ref> The fucose residue of the sialyl Lewisx tetrasaccharide fits in the primary binding site and interacts with a Ca2+ bound to the protein, with secondary contacts to the galactose residue and electrostatic interactions with the carboxyl group of sialic acid. The EGF domain is located on the opposite side of the CRD from the glycan-binding site.
 

=== Biological roles of GBP-ligand interaction ===

Interactions between P-selectin and PSGL-1 initiate rolling of leukocytes on activated platelets and endothelial cells as one of the earliest responses to tissue injury and infection<ref>McEver, R. P. (2001) Adhesive interactions of leukocytes, platelets, and the vessel wall during hemostasis and inflammation. Thromb Haemost 86, 746-756.</ref><ref name=" McEver RP 581"></ref><ref>McEver, R. P. and Zhu, C. (2010) Rolling cell adhesion. Annu Rev Cell Dev Biol 26, in press.</ref>. Engagement of PSGL-1 on neutrophils transduces signals that activate integrin LFA-1 to slow rolling on ICAM-1, thus augmenting neutrophil recruitment to inflammatory sites.<ref>Zarbock, A., Lowell, C. A., Ley, K. (2007) Spleen tyrosine kinase Syk is necessary for E-selectin-induced αLβ2 integrin-mediated rolling on intercellular adhesion molecule-1. Immunity 26, 773-783.</ref><ref>Zarbock, A., Abram, C. L., Hundt, M., Altman, A., Lowell, C. A., Ley, K. (2008) PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcR gamma to induce slow leukocyte rolling. J Exp Med 205, 2339-2347.</ref><ref>Miner, J. J., Xia, L., Yago, T., Kappelmayer, J., Liu, Z., Klopocki, A. G., Shao, B., McDaniel, J. M., Setiadi, H., Schmidtke, D. W., McEver, R. P. (2008) Separable requirements for cytoplasmic domain of PSGL-1 in leukocyte rolling and signaling under flow. Blood 112, 2035-2045.</ref><ref>Yago, T., Shao, B., Miner, J. J., Yao, L., Klopocki, A. G., Maeda, K., Coggeshall, K. M., McEver, R. P. (2010) E-selectin engages PSGL-1 and CD44 through a common signaling pathway to induce integrin αLβ2-mediated slow leukocyte rolling. Blood March 18 [Epub ahead of print].</ref><ref>Mueller, H., Stadtmann, A., Van Aken, H., Hirsch, E., Wang, D., Ley, K., Zarbock, A. (2010) Tyrosine kinase Btk regulates E-selectin-mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) gamma2 and PI3Kgamma pathways. Blood 115, 3118-27.</ref>
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=P-selectin&maxresults=20 CFG database search results for P-selectin].

=== Glycan profiling ===
The glycans on the main target for P-selectin, P-selectin glycoprotein ligand 1 (PSGL-1), were analyzed.<ref name="Kawar2008">Kawar ZS, Johnson TK, Natunen S, Lowe JB, Cummings RD (2008) PSGL-1 from the murine leukocytic cell line WEHI-3 is enriched for core 2-based O-glycans with sialyl Lewis x antigen. Glycobiology 18, 441-446</ref>

=== Glycogene microarray ===
Probes for all three human and mouse selectins have been included in all versions of the CFG glycogene chip. Regulation of P-selectin expression was analyzed under multiple conditions; see all results [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=p-selectin&cat=coree here].

=== Knockout mouse lines ===
The [https://www.functionalglycomics.org/glycomics/publicdata/phenotyping.jsp phenotype] of PSGL-1 knockout mice was analyzed by the CFG. The CFG did not generate mice deficient in the P-selectin gene, as these mice were published in 1993<ref name="Mayadas 1993">Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD. Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell. 1993 Aug 13;74(3):541-54. PubMed PMID: 7688665.</ref> and have since been extensively studied. P-selectin knockout mice exhibit defects in leukocyte behavior, including elevated numbers of circulating neutrophils, loss of leukocyte rolling in mesenteric venules, and delayed neutrophil recruitment to the peritoneal cavity after induction of inflammation<ref name="Mayadas 1993"/>. They also show attentuated polymorphonuclear leukocyte accumulation and myocardial injury following brief ischaemia-reperfusion of the myocardium (i.e., P-selectin deficiency is cardioprotective; reviewed in <ref>Kakkar AK, Lefer DJ. Leukocyte and endothelial adhesion molecule studies in knockout mice. Curr Opin Pharmacol. 2004 Apr;4(2):154-8. Review. PubMed PMID:15063359.</ref>.)

=== Glycan array ===
The comparative binding specificities of [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1659 human] and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1095 mouse] P-selectins have been analyzed. See all glycan array results for P-selectin [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=P-selectin&cat=coreh here].

== Related GBPs ==
L-selectin [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=L-selectin&maxresults=20 (CFG data)]and E-selectin [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=E-selectin&maxresults=20 (CFG data)].

== References ==
<references/>
* Mitoma J, Miyazaki T, Sutton-Smith M, Suzuki M, Saito H, Yeh JC, Kawano T, Hindsgaul O, Seeberger PH, Panico M, Haslam SM, Morris HR, Cummings RD, Dell A, Fukuda M (2009) The N-glycolyl form of mouse sialyl Lewis X is recognized by selectins but not by HECA-452 and FH6 antibodies that were raised against human cells. Glycoconj J 26, 511-523.
* Kawar ZS, Johnson TK, Natunen S, Lowe JB, Cummings RD (2008) PSGL-1 from the murine leukocytic cell line WEHI-3 is enriched for core 2-based O-glycans with sialyl Lewis x antigen. Glycobiology 18, 441-446.
* Mitoma J, Bao X, Petryanik B, Schaerli P, Gauguet JM, Yu SY, Kawashima H, Saito H, Ohtsubo K, Marth JD, Khoo KH, von Andrian UH, Lowe JB, Fukuda M (2007) Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment. Nat Immunol 8, 409-418.
* Veerman KM, Williams MJ, Uchimura K, Singer MS, Merzaban JS, Naus S, Carlow DA, Owen P, Rivera-Nieves J, Rosen SD, Ziltener HJ (2007) Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs. Nat Immunol 8, 532-539.
* Chen S, Kawashima H, Lowe JB, Lanier LL, Fukuda M (2005) Suppression of tumor formation in lymph nodes by L-selectin-mediated natural killer cell recruitment. J Exp Med 202, 1679-1689.
* Kawashima H, Petryniak B, Hiraoka N, Mitoma J, Huckaby V, Nakayama J, Uchimura K, Kadomatsu K, Muramatsu T, Lowe JB, Fukuda M (2005) N-acetylglucosamine-6-O-sulfotransferases 1 and 2 cooperatively control lymphocyte homing through L-selectin ligand biosynthesis in high endothelial venules. Nat Immunol 6, 1096-1104.
* Piccio L, Rossi B, Colantonio L, Grenningloh R, Gho A, Ottoboni L, Homeister JW, Scarpini E, Martinello M, Laudanna C, DAmbrosio D, Lowe JB, (2005) Constantin G Efficient recruitment of lymphocytes in inflamed brain venules requires expression of cutaneous lymphocyte antigen and fucosyltransferase-VII. J Immunol 174, 5805-5813.
* Homeister JW, Daugherty A, Lowe JB (2004) α(1,3)Fucosyltransferases FucT-IV and FucT-VII control susceptibility to atherosclerosis in apolipoprotein E -/- mice. Arterioscler Thromb Vasc Biol 24, 1897-1903.
* Lowe JB (2003) Glycan-dependent leukocyte adhesion and recruitment in inflammation Curr Opin Cell Biol 15, 531-538.
* Smith PL, Myers JT, Rogers CE, Zhou L, Petryniak B, Becher DJ, Homeister JW, Lowe JB (2002) Conditional control of selectin ligand expression and global fucosylation events in mice with a targeted mutation at the FX locus. J Cell Biol 158, 801-815.

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Kurt Drickamer, Rodger McEver, Yvette van Kooyk

Cation-dependent Mannose-6-phosphate receptor

2011-03-31T22:18:57Z

Carole Weaver: /* Knockout mouse lines */

The cation-dependent mannose 6-phosphate receptor is one of two transmembrane receptors that bind mannose-6-phosphate on lysosomal proteins in the Golgi apparatus and direct their trafficking to the lysosome.<ref>Sahagian, G. G. and Neufeld, E. F. Biosynthesis and turnover of the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. J Biol Chem 258, 7121-7128 (1983)</ref><ref>Hoflack, B. and Kornfeld, S. Purification and characterization of a cation-dependent mannose 6-phosphate receptor from murine P388D1 macrophages and bovine liver. J Biol Chem 260, 12008-12014 (1985)</ref><ref>Dahms, N. M. and Kornfeld, S. The cation-dependent mannose 6-phosphate receptor. Structural requirements for mannose 6-phosphate binding and oligomerization. J Biol Chem 264, 11458-11467 (1989)</ref><ref>Nair, P., Schaub, B. E. and Rohrer, J. Characterization of the endosomal sorting signal of the cation-dependent mannose 6-phosphate receptor. J Biol Chem 278, 24753-24758 (2003)</ref><ref>Sun, G., Zhao, H., Kalyanaraman, B. and Dahms, N. M. Identification of residues essential for carbohydrate recognition and cation dependence of the 46-kDa mannose 6-phosphate receptor. Glycobiology 15, 1136-1149 (2005)</ref><ref name="Kim 2009">Kim, J. J., Olson, L. J. and Dahms, N. M. Carbohydrate recognition by the mannose-6-phosphate receptors. Curr Opin Struct Biol 19, 534-542 (2009)</ref><ref name="Olson 2010">Olson, L. J., Sun, G., Bohnsack, R. N., Peterson, F. C., Dahms, N. M. and Kim, J. J. Intermonomer interactions are essential for lysosomal enzyme binding by the cation-dependent mannose 6-phosphate receptor. Biochemistry 49, 236-246 (2010)</ref>. The other receptor is termed the cation-independent mannose-6-phosphate receptorand is also the receptor for Insulin-like growth factor II.<ref>Tong, P. Y., Tollefsen, S. E. and Kornfeld, S. The cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II. J Biol Chem 263, 2585-2588 (1988)</ref><ref>Lobel, P., Dahms, N. M. and Kornfeld, S. Cloning and sequence analysis of the cation-independent mannose 6-phosphate receptor. J Biol Chem 263, 2563-2570 (1988)</ref><ref>Tong, P. Y. and Kornfeld, S. Ligand interactions of the cation-dependent mannose 6-phosphate receptor. Comparison with the cation-independent mannose 6-phosphate receptor. J Biol Chem 264, 7970-7975 (1989) </ref><ref>Hancock, M. K., Yammani, R. D. and Dahms, N. M. Localization of the carbohydrate recognition sites of the insulin-like growth factor II/mannose 6-phosphate receptor to domains 3 and 9 of the extracytoplasmic region. J Biol Chem 277, 47205-47212 (2002)</ref><ref>Bohnsack, R. N., Song, X., Olson, L. J., Kudo, M., Gotschall, R. R., Canfield, W. M., Cummings, R. D., Smith, D. F. and Dahms, N. M. Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites. J Biol Chem 284, 35215-35226 (2009)</ref><ref>Brown, J., Jones, E. Y. and Forbes, B. E. Keeping IGF-II under control: lessons from the IGF-II-IGF2R crystal structure. Trends Biochem Sci 34, 612-619 (2009)</ref><ref>Laube, F. Mannose-6-phosphate/insulin-like growth factor-II receptor in human melanoma cells: effect of ligands and antibodies on the receptor expression. Anticancer Res 29, 1383-1388 (2009)</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) with interest in mannose 6-phosphate receptors include: Ajit Varki
* Non-PIs using CFG resources to study mannose 6-phosphate receptors include: Nancy Dahms

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about the cation-dependent mannose 6-phosphate receptor, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===
The preferred ligands for the cation-dependent mannose 6-phosphate receptor are N-linked glycans that bear two terminal mannose 6-phosphate residues.<ref name=”Song2009”>Song, X, Lasanajak, Y, Olson, LJ, Boonen, M, Dahms, NM, Kornfeld, S, Cummings, RD and Smith, DF (2009) Glycan microarray analysis of P-type lectins reveals distinct phospho-mannose glycan recognition J. Biol. Chem. 284, 35201-35214</ref>
 

=== Cellular expression of GBP and ligands ===
The cation-dependent mannose 6-phosphate receptor is expressed in most cell types in mammals.
 

=== Biosynthesis of ligands ===
The signal for binding to the mannose 6-phosphate receptor is generated in several steps. GlcNAc phosphate is first added to the reducing end mannose in terminal Manα1-2Man disaccharides in N-linked glycans by N-acetylglucosaminephosphotransferase to generate phosphodiesters. Removal of the GlcNAc moieties by an N-acetylglucosaminidase, known as an uncapping enzymes, which generates phosphomonoesters, which are required for binding to the cation-dependent mannose 6-phosphate receptor. The mannose residues at the non-reducing termini can then be removed. There are typically two phosphomonoesters per N-linked glycan. <ref name”Dahms2008”>Dahms, NM, Olson, LJ and Kim, J-JP (2008) Strategies for carbohydrate recognition by the mannose 6-phosphate receptors. Glycobiology 18, 664–678</ref>
 

=== Structure ===
The cation-dependent mannose 6-phosphate receptor has a luminal domain consisting of a single carbohydrate-recognition domain, and forms dimers in which these two domains typically interact with two mannose 6-phosphate residues on a single glycan. The crystal structure of the carbohydrate-recognition domain shows that the sugar moiety of mannose 6-phosphate makes hydrogen bonds to the hydroxyl groups at positions 2, 3 and 4, the orientations of which identify the sugar as mannose. The phosphate moiety makes hydrogen bonds with backbone atoms and interacts with a Mn2+ cation in the binding site.<ref name=”Roberts1998”>Roberts, DL, Weix, DJ, Dahms, NM and Kim, J-JP (1998) Molecular Basis of Lysosomal Enzyme Recognition: Three-Dimensional Structure of the Cation-Dependent Mannose 6-Phosphate Receptor Cell 93, 639-648</ref>
 

=== Biological roles of GBP-ligand interaction ===
The CD-MPR is found in all eukaryotes and is known to play a highly conserved role in recognition and targeting of lysosomal enzymes. Both CD-MPRs and CI-MPRs are glycan-binding proteins that bind their M6P-tagged cargo in the lumen of the Golgi apparatus <ref name="Kim 2009"/>. Once bound to their cargo, the MPRs are recognized by the GGA family of clathrin adaptor proteins and accumulate in forming clathrin-coated vesicles. Upon arriving at the early endosome, the low pH environment of the endosome induces the MPRs release their cargo. The MPRs are recycled back to the Golgi, again by way of interaction with GGAs and vesicles. The cargo proteins are then trafficked to the lysosome via the late endosome in a process independent of the MPRs <ref name="Kim 2009"/>. Cargo molecules undergo extensive processing terminating in terminal mannose-6 phosphate on one or more arms of the oligosaccharide.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Mannose-6-phosphate&maxresults=20 CFG database search results for Mannose-6-phosphate].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Probes for the cation-dependent mannose 6-phosphate receptor have been included on all versions of the glycogene micro-array.
 

=== Knockout mouse lines ===
The CFG did not generate mice deficient in the cation-dependent mannose-6-phosphate receptor gene, as these mice were created and published in 1993 <ref>Ludwig T, Ovitt CE, Bauer U, Hollinshead M, Remmler J, Lobel P, Rüther U,
Hoflack B. Targeted disruption of the mouse cation-dependent mannose 6-phosphate receptor results in partial missorting of multiple lysosomal enzymes. EMBO J. 1993 Dec 15;12(13):5225-35. PubMed PMID: 8262065; PubMed Central PMCID: PMC413788.</ref>. CD-M6PR-deficient mice are viable with no obvious developmental abnormalities. They exhibit high levels of phosphorylated lysosomal enzymes in serum and urine, indicating that these enzymes are being mis-sorted, a conclusion that was supported by studies in cultured cells from these mice.
 

=== Glycan array ===
In 2006, soluble forms of MPR were screened on the CFG glycan array (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_852 here]), but results were inconclusive.

== Related GBPs ==
The cation-dependent mannose 6-phosphate receptor has a single carbohydrate-recognition domain in the MRH family, while the cation-independent mannose 6-phosphate receptor has 15 such repeats, at least three of which bind mannose 6-phosphate.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Kurt Drickamer, John Hanover

Ficolins/Mannose-binding protein

2011-03-31T22:08:39Z

Carole Weaver: /* Knockout mouse lines */

The ficolins share a common organization and function with the collectins: serum mannose-binding and the pulmonary surfactant proteins A and D. All of these proteins are soluble mediators of innate immunity and consist of globular sugar-binding domains attached to collagenous stalks, which can invoke innate immune responses either through complement fixation or interaction with receptors on the surfaces of macrophages. Amongst these proteins, the ficolins have been most extensively investigated with CFG resources, while mannose-binding protein is the best characterized. The ficolins have fibrinogen-like sugar-binding domains, rather than C-type carbohydrate-recognition domains, but conceptually fall within the same group.

See also: paradigm page for [[Ficolin M (Ficolin 1)]]
== CFG Participating Investigators contributing to the understanding of this paradigm ==
Participating Investigators have generated and characterized knockout mice, defined the sugar-binding properties and undertaken structural analysis for members of this glycan-binding protein (GBP) group.
* PIs working on ficolins include: Raymond Dwek, Daniel Mitchell, Nicole Thielens
* PIs investigating other paradigms in this GBP group include: Kurt Drickamer, Ten Feizi, Toshisuke Kawasaki, Laura Kiessling, Reiko Lee, Yuan Lee, Jamie Marth, Kenneth Ng, Michel Nussenzweig, Pauline Rudd, Maureen Taylor, Bill Weis
* Non-PIs with who have used CFG resources to study ficolins include: David Stephens

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about ficolins and mannose-binding protein, their carbohydrate ligands, and how they interact with ligands to mediate cell communication. Further information can be found in the GBP Molecule Pages for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Ctlect_227&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_mou_Ctlect_169&sideMenu=no mouse] mannose-binding protein in the CFG database.
=== Carbohydrate ligands ===

L-ficolin preferentially recognizes disulfated LacNAc and tri- and tetrasaccharides containing a terminal LacNAc or GlcNAc unit, provided that the linkage with the following carbohydrate is not of the β1-3 type<ref>Gout E, Garlatti V, Smith DF, Lacroix M M, Dumestre-Perard C, Lunardi T, Martin L, Cesbron JY, Arlaud GJ, Gaboriaud C, Thielens NM (2010) Carbohydrate recognition properties of human ficolins: Glycan array screening reveals the sialic acid binding specificity of M-ficolin. J Biol Chem 285:6612-22</ref><ref>Krarup A, Mitchell DA, Sim RB (2008) Recognition of acetylated oligosaccharides by human L-ficolin. Immunol Lett 118:152-6</ref>. H-ficolin does not bind to any of the glycans.

Mannose-binding protein, also known as mannan-binding lectin (MBL), binds to terminal mannose, fucose and GlcNAc residues on the outer surfaces of bacterial and fungal cell walls. MBL belongs to a family of soluble immune proteins known as the collectins that consist of N-terminal collagen tail regions and C-terminal C-type lectin domains. Other family members include lung surfactant protein A (SP-A) that preferentially binds to galactose, mannose and fucose residues on microbial glycolipids <ref>Childs RA, Wright JR, Ross GF, Yuen CT, Lawson AM, Chai W, Drickamer K, Feizi T (1992) Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids. J Biol Chem 267:9972-9</ref>, and lung surfactant protein D (SP-D) that has been shown to interact with mannoside and glucoside moieties. <ref>Shrive AK, Martin C, Burns I, Paterson JM, Martin JD, Townsend JP, Waters P, Clark HW, Kishore U, Reid KB, Greenhough TJ (2009) Structural characterisation of ligand-binding determinants in human lung surfactant protein D: influence of Asp325. J Mol Biol 394:776-88.</ref>

Ficolins and mannose-binding protein share the ability to associate with mannan-binding lectin-associated serine protease-2.

=== Cellular expression of GBP and ligands ===

Mannose-binding protein is produced mostly by hepatocytes and secreted into the circulation. SP-A and SP-D are produced mostly by alveolar cells and secreted to the pulmonary surfactant that lines the lung.
 L- and H-ficolins are serum proteins that are essentially synthesized in the liver. H-ficolin is also synthesized by bile duct epithelial cells, by lung ciliated bronchial and type II alveolar epithelial cells, and by glioma cells <ref> Akaiwa M, Yae Y, Sugimoto R, Suzuki SO, Iwaki T, Izuhara K, Hamasaki N (1999) Hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile. Histochem Cytochem 47:777-86</ref><ref> Kuraya M, Matsushita M, Endo Y, Thiel S, Fujita T (2003) Expression of H-ficolin/Hakata antigen, mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 by human glioma cell line T98G. Int Immunol 2003:15:109-17</ref>.

L-ficolin recognizes ligands on several strains of opportunistic capsulated bacteria and ''Salmonella typhimurium'' whereas H-ficolin specifically recognizes ''Aerococcus viridans''.

=== Biosynthesis of ligands ===
Glycans on viruses 
High mannose oligosaccharides on viral envelope proteins that are ligands for mannose-binding protein result from incomplete processing of glycans in the pathway for biosynthesis of complex N-linked glycans ([http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/geMolecule.jsp?slideNumber=default GT Database]).
 
Glycans on bacteria 
The biosynthesis pathways for the bacterial lipopolysaccharides have been extensively studied and the gene families responsible for the expression of different glycan sequences have been characterized.<ref name”Raetz2002”>Raetz CR and Whitfield C (2002) Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635-700</ref>
 
Glycans on fungi 
The polysaccharide beta(1,3)-D-glucan is a component of the cell wall of many fungi. The linear polymer is synthesized from UDP-glucose (UDGG) by the multisubunit enzyme UDP-glucose beta(1,3)-D-glucan beta(3)-D-glucosyltransferase. This enzymatic complex contains two catalytic and one regulatory subunits that were first identified in "S. cerevisiae".<ref>Douglas CM, Foor F, Marrinan JA, Morin N, Nielsen JB, Dahl AM, Mazur P, Baginsky W, Li W, el-Sherbeini M, Clemas JA, Mandala SM, Frommer BR, Kurz MB (1994) The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase. Proc Natl Acad Sci U S A 91:12907-11</ref><ref>Mazur P, Morin N, Baginsky W, el-Sherbeini M, Clemas JA, Nielsen JB, Foor F. (1995) Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase. Mol Cell Biol. 15:5671-81</ref><ref>Qadota H, Python CP, Inoue SB, Arisawa M, Anraku Y, Zheng Y, Watanabe T, Levin DE, Ohya Y (1996) Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-glucan synthase. Science 272:279-81</ref>

=== Structure ===

The 3-D structures of the trimeric fibrinogen-like recognition domains of L- and H-ficolins have been solved by X-ray crystallography, revealing similar three-lobed clover-like assemblies, whereas different recognition mechanisms have been deciphered from the structure of complexes with various ligands<ref> Garlatti V, Belloy N, Martin L, Lacroix M, Matsushita M, Endo Y, Fujita T, Fontecilla-Camps JC, Arlaud GJ, Thielens NM, Gaboriaud C (2007) Structural insights into the innate immune recognition specificities of L- and H-ficolins. EMBO J 26:623-33</ref>. An external ligand binding site able to accommodate neutral carbohydrates such as galactose and D-fucose has been identified for H-ficolin. In contrast, L-ficolin exhibited three additional binding sites which define a continuous recognition surface able to bind acetylated and neutral carbohydrates in the context of extended polysaccharides such as 1,3-β-D-glucan.

=== Biological roles of GBP-ligand interaction ===

Ficolins share with mannan-binding lectin the ability to associate with mannan-binding lectin-
associated serine protease-2, thus triggering activation of the lectin complement pathway upon binding to suitable targets and enhancing their phagocytosis. L-ficolin recognizes several strains of opportunistic capsulated bacteria and ''Salmonella typhimurium'' whereas H-ficolin specifically recognizes ''Aerococcus viridans''. In addition to pathogenic microorganisms, L- ficolin binds specifically to apoptotic HL60, U937 and Jurkat T cells, whereas binding of H-ficolin is restricted to apoptotic Jurkat T cells <ref>Kuraya M, Ming Z, Liu X, Matsushita M, Fujita T (2005) Specific binding of L-ficolin and H-ficolin to apoptotic cells leads to complement activation. Immunobiology 209:689-97</ref><ref>Honoré C, Hummelshoj T, Hansen BE, Madsen HO, Eggleton P, Garred P(2007) The innate immune component ficolin 3 (Hakata antigen) mediates the clearance of late apoptotic cells. Arthritis Rheum 56:1598-1607</ref><ref> Jensen ML, Honoré C, Hummelshøj T, Hansen BE, Madsen HO, Garred P(2007) Ficolin-2 recognizes DNA and participates in the clearance of dying host cells. Mol Immunol 44:856-65</ref>.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=ficolin&maxresults=20 ficolin] and [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=mannose-binding&maxresults=20 mannose-binding protein].

=== Glycan profiling ===
Because L and H ficolin and mannose-binding protein bind ligands on bacteria and other micro-organisms, profiling of mammalian glycans is not relevant for these proteins.
 

=== Glycogene microarray ===
Probes for mouse and human MBP have been included in all versions of the CFG glycogene chip.
 

=== Knockout mouse lines ===
Mice, unlike humans, have two genes encoding MBP: MBP-A and MBP-C. The CFG did not generate mice deficient in these genes, as the double knockout was published in 2004 <ref>Shi L, Takahashi K, Dundee J, Shahroor-Karni S, Thiel S, Jensenius JC, Gad F,
Hamblin MR, Sastry KN, Ezekowitz RA. Mannose-binding lectin-deficient mice are
susceptible to infection with Staphylococcus aureus. J Exp Med. 2004 May
17;199(10):1379-90. PubMed PMID: 15148336; PubMed Central PMCID: PMC2211809.
</ref>. These mice display increased susceptibility to infection by certain pathogens, including ''Staphylococcus aureus'' and ''Pseudomonas aeruginosa''; a reduced inflammatory response; and resistance to ischemia/reperfusion injury <ref>Takahashi K: Lessons learned from murine models of mannose-binding lectin deficiency. Biochem Soc Transact 2008, 36:1487-1490.</ref>.
 

=== Glycan array ===
The binding specificities of several of the ficolins have been analyzed and other members of the group were screened on the CFG glycan array.
 Investigators have used CFG glycan arrays to study ligand binding specificity of human L-ficolin (see examples [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2.1_609_10172006 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2.1_611_10172006 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1604 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2121 here], and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2122 here]) and of its rat homologue ficolin A (see example [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1707 here]).
Several analyses with human H-ficolin, which has no homologue in rodents, yielded inconclusive results (see examples [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2.1_610_10172006 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1511 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1603 here],
[http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1950 here], and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2120 here]). See all glycan array results for ficolin [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=ficolin&cat=coreh here].

== Related GBPs ==
Serum mannose-binding protein ([http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=Mannose-binding%20AND%20protein&cat=coreh CFG data]; MBP, also designated mannose-binding lectin, MBL [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Mannose-binding+AND+lectin&maxresults=20 (CFG data)]); and the pulmonary surfactant proteins SP-C and SP-D

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Kurt Drickamer, Nicole Thielens, Daniel Mitchell, Yvette van Kooyk

Galectin-9

2011-03-31T21:11:40Z

Carole Weaver: /* Knockout mouse lines */

Galectin-9 is the best-studied of the tandem-repeat galectins and the crystal structure of the N-terminal carbohydrate recognition domain (CRD) is known. In addition, Galectin-9...
* uniquely binds poly-N-acetyllactosamine sequences by recognizing internal N-acetyllactosamine repeats<ref>Nagae, M. et al. Structural analysis of the recognition mechanism of poly-N-acetyllactosamine by the human galectin-9 N-terminal carbohydrate recognition domain. Glycobiology 19, 112-117 (2009). </ref>
* binds distinct ligands from [[Galectin-1]]<ref>Bi, S., Earl, L.A., Jacobs, L. & Baum, L.G. Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways. J Biol Chem 283, 12248-12258 (2008).</ref>
* has three well-characterized linker domains between the CRDs, generated by alternative splicing, that regulate cellular localization and function of the protein
* is the only tandem-repeat galectin that has been administered in animal models of disease to assess therapeutic potential<ref>Baba, M. et al. Galectin-9 inhibits glomerular hypertrophy in db/db diabetic mice via cell-cycle-dependent mechanisms. J Am Soc Nephrol 16, 3222-3234 (2005). </ref><ref name="Seki 2008">Seki, M. et al. Galectin-9 suppresses the generation of Th17, promotes the induction of regulatory T cells, and regulates experimental autoimmune arthritis. Clin Immunol 127, 78-88 (2008).
</ref><ref>Tsuchiyama, Y. et al. Efficacy of galectins in the amelioration of nephrotoxic serum nephritis in Wistar Kyoto rats. Kidney Int 58, 1941-1952 (2000). </ref>
* null mice have increased susceptibility to autoimmune disease
* binds to a unique glycoprotein ligand Tim-3 expressed in Th1 and Th17 cells<ref name="Seki 2008" /><ref>Naka, E.L., Ponciano, V.C., Cenedeze, M.A., Pacheco-Silva, A. & Camara, N.O. Detection of the Tim-3 ligand, galectin-9, inside the allograft during a rejection episode. Int Immunopharmacol 9, 658-662 (2009).
</ref><ref>Niwa, H. et al. Stable form of galectin-9, a Tim-3 ligand, inhibits contact hypersensitivity and psoriatic reactions: a potent therapeutic tool for Th1- and/or Th17-mediated skin inflammation. Clin Immunol 132, 184-194 (2009).</ref><ref>Anderson, D.E. TIM-3 as a therapeutic target in human inflammatory diseases. Expert Opin Ther Targets 11, 1005-1009 (2007). </ref>

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of Galectin-9 include: Linda Baum, Richard Cummings, Gabriel Rabinovich, Sachiko Sato

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about Galectin-9, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Page for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Stlect_00120&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_1307&sideMenu=no mouse] Galectin-9 in the CFG database.
=== Carbohydrate ligands ===

 
=== Cellular expression of GBP and ligands ===

 
=== Biosynthesis of ligands ===

 
=== Structure ===

 
=== Biological roles of GBP-ligand interaction ===

 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-9&maxresults=20 CFG database search results for Galectin-9].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Probes for human galectin-9 have been included in all versions of the CFG glycogene chip, and probes for mouse galectin-9 are included on versions 2, 3, and 4.
 

=== Knockout mouse lines ===
CFG-generated [http://www.functionalglycomics.org/static/consortium/resources/DataCoreFGJb4.shtml Galectin-9 knockout mice] have been used to study the biological functions of this paradigm GBP. [http://www.functionalglycomics.org/glycomics/publicdata/investigator.jsp?investigator=judyteale (CFG PI data)]
 

=== Glycan array ===
Investigators have used CFG carbohydrate compounds and glycan array screening to study ligand binding specificity of Galectin-9 (for example, click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2735 here]). To see all glycan array results for Galectin-9, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=galectin-9&cat=coreh here].

== Related GBPs ==
Galectin-4 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-4&maxresults=20 (CFG data)], galectin-6, galectin-8 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-8&maxresults=20 (CFG data)], and galectin-12 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-12&maxresults=20 (CFG data)].

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Linda Baum, Richard Cummings

Siglec-15

2011-03-30T23:37:46Z

Carole Weaver: /* Glycogene microarray */

Siglec-15<ref name="Angata 2007">Angata, T., Tabuchi, Y., Nakamura, K. & Nakamura, M. Siglec-15: an immune system Siglec conserved throughout vertebrate evolution. Glycobiology 17, 838-846 (2007).</ref> serves as a paradigm for several siglecs, including Siglec-14<ref>Angata, T., Hayakawa, T., Yamanaka, M., Varki, A. & Nakamura, M. Discovery of Siglec-14, a novel sialic acid receptor undergoing concerted evolution with Siglec-5 in primates. FASEB J 20, 1964-1973 (2006).</ref><ref>Yamanaka, M., Kato, Y., Angata, T. & Narimatsu, H. Deletion polymorphism of SIGLEC14 and its functional implications. Glycobiology 19, 841-846 (2009)</ref>, Siglec-16<ref>Cao, H. et al. SIGLEC16 encodes a DAP12-associated receptor expressed in macrophages that evolved from its inhibitory counterpart SIGLEC11 and has functional and non-functional alleles in humans. Eur J Immunol 38, 2303-2315 (2008).</ref> and Siglec-H<ref>Zhang, J. et al. Characterization of Siglec-H as a novel endocytic receptor expressed on murine plasmacytoid dendritic cell precursors. Blood 107, 3600-3608 (2006).</ref><ref>Blasius, A. L., Cella, M., Maldonado, J., Takai, T. & Colonna, M. Siglec-H is an IPC-specific receptor that modulates type I IFN secretion through DAP12. Blood 107, 2474-2476 (2006).</ref>, that contain a basic amino acid within the transmembrane domain. This leads to association of these siglecs with a transmembrane adaptor protein containing an immunoreceptor tyrosine based activation motif (ITAM). Siglec-15 is unusual compared to other siglecs that share this paradigm in two respects. For one, it can associate with two ITAM containing adaptors, DAP12 and DAP10, whereas Siglec-14, Siglec-16, and Siglec-H show a restricted association with DAP12. In addition, Siglec-15 is unusual in having four cysteine residues in the V-set domain predicted to result in an inter-sheet disulfide that is absent from all other known siglecs. These potentially ‘activating’ siglecs are expressed on myeloid cells and dendritic cells and may be involved in innate responses to pathogen challenge.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
As yet, no CFG Participating Investigators (PIs) have contributed to Siglec-15, but contributors to the related Siglec-H include Marco Colonna and Paul Crocker.

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about Siglec-15, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===
Human Siglec-15 has been shown to prefer the sialyl Tn (Neu5Acα2-6GalNAcα1-) structure, while mouse Siglec-15 recognizes both sialyl Tn and 3'-sialyl Lac[NAc] (Neu5Acα2-3Galβ1-4Glc[NAc]) structures<ref name="Angata 2007"/>.

=== Cellular expression of GBP and ligands ===
Human Siglec-15 is expressed on DC-SIGN positive cells in lymph nodes<ref name="Angata 2007"/>. Exact identity of these cells (i.e., whether these cells are dendritic cells or macrophages) is not yet conclusively determined.

=== Biosynthesis of ligands ===
 
=== Structure ===
Siglec-15 has two Ig-like domains, followed by a single-pass transmembrane domain and a short cytoplasmic tail. Siglec-15 is unusual compared to other siglecs in that it has four cysteine residues in the V-set domain predicted to result in an inter-sheet disulfide that is absent from all other known siglecs.
The transmembrane domain of Siglec-15 contains a basic amino acid. This leads to association with a transmembrane adaptor protein containing an immunoreceptor tyrosine based activation motif (ITAM), DAP12 and DAP10. A stretch of conserved amino acids (containing a tyrosine residue) is found in the cytoplasmic tail of human and mouse Siglec-15, although its functional importance is not yet known.

=== Biological roles of GBP-ligand interaction ===
As yet, no clear biological roles for the GBP-ligand interaction have been shown for Siglec-15. Considering the absence of reported cases of Siaα2-6GalNAcα1- structure in pathogens<ref>Angata, T. & Varki, A. Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem Rev 102, 439-469 (2002)</ref>, the ligand for Siglec-15 may be of endogenous origin.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Siglec-15&maxresults=20 CFG database search results for Siglec-15].

=== Glycan profiling ===
No data available.

=== Glycogene microarray ===
No data available. Probes for human Siglec-15 were included on version 4 of the CFG glycogene microarray.

=== Knockout mouse lines ===
The CFG has generated Siglec-15-deficient ES cells that will permit generation of a Siglec-15-deficient mouse in the future. Two [https://www.functionalglycomics.org/static/consortium/resources/DataCoreFsigH.shtml Siglec-H-deficient mouse lines] (Siglec-H-conditional knockout and Siglec-H-total knockout) were also generated and are currently under investigation.

=== Glycan array ===
No data available.

== Related GBPs ==
Siglec-14, Siglec-16, Siglec-H [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Siglec-H&maxresults=20 (CFG data)].

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Takashi Angata, Paul Crocker, James Paulson

MAG

2011-03-30T23:36:29Z

Carole Weaver: /* Glycogene microarray */

Myelin-associated glycoprotein (MAG, Siglec-4) is unique among the siglecs in that it is expressed exclusively on neuronal glial cells<ref>Crocker, P. R., Paulson, J. C. & Varki, A. Siglecs and their roles in the immune system. Nat Rev Immunol 7, 255-266 (2007).</ref><ref name="Schnaar 2009">Schnaar, R. L. Brain gangliosides in axon-myelin stability and axon regeneration. FEBS Lett (2009).</ref>. It is the most highly conserved among the siglecs in mammalian species. This siglec paradigm is unique in its activity of stabilizing axon-myelin interactions. MAG has a cytoplasmic domain that is devoid of ITIMs, but contains a tyrosine-based motif associated with binding the FYN tyrosine kinase, believed to play a role in its activity in myelin-axon interactions. MAG recognizes as ligands sialoside sequences found on gangliosides that are abundant in axonal membranes<ref name="Schnaar 2009"/>. It is one of several proteins in myelin that negatively regulate axon outgrowth following tissue injury, an activity that involves MAG-ligand interactions. Evidence suggests that inhibition of MAG-ligand interactions may enhance neurite outgrowth and repair of injured neurons<ref>Yang, L. J. et al. Sialidase enhances spinal axon outgrowth in vivo. Proc Natl Acad Sci U S A 103, 11057-11062 (2006).</ref><ref name="mountney2010">Mountney A., Zahner M.R., Lorenzini I., Oudega M., Schramm L.P., Schnaar R.L. Sialidase enhances recovery from spinal cord contusion injury. Proc Natl Acad Sci U S A 107, 11561-11566, 2010</ref><ref name="vyas2005">Vyas, A. A., Blixt, O., Paulson, J. C. & Schnaar, R. L. Potent glycan inhibitors of myelin-associated glycoprotein enhance axon outgrowth in vitro. J Biol Chem 280, 16305-16310 (2005).</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
Several CFG Participating Investigators (PIs) have contributed to identification of MAG as a siglec and to understanding the functions of MAG, including: Paul Crocker, Sørge Kelm, James Paulson, Ronald Schnaar

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about MAG, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Pages for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Itlect_271&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_mou_Itlect_196&sideMenu=no mouse] MAG (a.k.a. Siglec-4a) in the CFG database.
=== Carbohydrate ligands ===
The glycan specificity of Siglec-4 has been investigated using resialylated erythrocytes<ref>Kelm, S. et al. Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily. Curr Biol. 4, 965-972 (1994)</ref>, gangliosides<ref>Collins, B. E., Kiso, M., Hasegawa, A., Tropak, M. B., Roder, J. C., Crocker, P. R., Schnaar, R. L. Binding specificities of the sialoadhesin family of I-type lectins. Sialic acid linkage and substructure requirements for binding of myelin-associated glycoprotein, Schwann cell myelin protein, and sialoadhesin. J Biol Chem. 272, 16889-16895 (1997)</ref><ref>Collins, B. E., Yang, L. J., Mukhopadhyay, G., Filbin, M. T., Kiso, M., Hasegawa, A., Schnaar, R.L. Sialic acid specificity of myelin-associated glycoprotein binding. J Biol Chem. 272, 1248-1255 (1997)</ref>, and inhibition assays with oligosaccharides<ref name="strenge1998">Strenge, K., Schauer, R., Bovin, N., Hasegawa, A., Ishida, H., Kiso, M., Kelm, S. Glycan specificity of myelin-associated glycoprotein and sialoadhesin deduced from interactions with synthetic oligosaccharides. Eur J Biochem. 258, 677-685 (1998)</ref><ref>Blixt, O., Collins, B. E., van den Nieuwenhof, I. M., Crocker, P. R., Paulson, J. C. Sialoside specificity of the siglec family assessed using novel multivalent probes: identification of potent inhibitors of myelin-associated glycoprotein. J Biol Chem. 278, 31007-31019 (2003)</ref>.

'''Determinant recognized:'''

[[File:Sia3Gal_small.png]]

on glycolipids and/or glycoproteins

'''Specificity for linkage of sialic acid to underlying Gal:'''

about 10-fold better binding to Neu5Acα2,3Gal-R than Neu5Acα2,6Gal-R

'''Underlying glycan structures can enhance binding:'''

Similar binding to the following structures as soluble glycosides<ref name="strenge1998"/>. Enhanced binding to first structure in intact gangliosides<ref name="yang1996">Yang, L. J., Zeller, C.B., Shaper, N.L., Kiso, M., Hasegawa, A., Shapiro, R.E., Schnaar, R.L. Gangliosides are neuronal ligands for myelin-associated glycoprotein. Proc. Natl. Acad. Sci. USA 93, 814-818 (1996).</ref> <ref name="collins1996">Collins, B.E., Ito, H., Sawada, N., Ishida, H., Kiso, M., Schnaar, R.L. Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs. J Biol Chem. 274, 37637-37643 (1999)</ref>.

[[File:Sia3Gal3GalNAc_small.png]]

[[File:Sia3Gal3GlcNAc_small.png]]

[[File:Sia3Gal4GlcNAc_small.png]]

'''Enhanced binding through additional internal sialic acids:'''

[[File:Sia3Gal3GalNAc4(Sia3)Gal_small.png]]

higher binding to

[[File:Sia3Gal3(Sia6)GalNAc_small.png]]

Mouse knockout experiments have implicated MAG-binding brain gangliosides GD1a and/or GT1b as MAG ligands (see "Biosynthesis of Ligands" below). 

=== Cellular expression of GBP and ligands ===
MAG (Siglec-4) is expressed exclusively on myelin, which is produced by oligodendrocytes (the myelinating cells of the central nervous system) and Schwann cells (the myelinating cells of the peripheral nervous system). In both central and peripheral nervous systems, MAG is enriched on the innermost wrap of myelin, directly apposing the axon surface.<ref name="quarles2007">Quarles RH. Myelin-associated glycoprotein (MAG): past, present and beyond. J Neurochem. 100, 1431-1448 (2007).</ref> MAG recognizes as ligands sialoside sequences found on gangliosides that are abundant in axonal membranes<ref name="Schnaar 2009"/>.

=== Biosynthesis of ligands ===
Mice null for the ganglioside-specific N-acetylgalactosaminyltransferase gene B4galnt1 (GM2/GD2 synthase) have similar nervous system phenotypic deficits as MAG-null mice (see "Biological roles of GBP-ligand interaction" below). These data implicate MAG-binding brain gangliosides GD1a and/or GT1b as MAG ligands.<ref name="pan2005">Pan, B., Fromholt, S.E., Hess, E.J., Crawford, T.O., Griffin, J.W., Sheikh, K.A., Schnaar, R.L. Myelin-associated glycoprotein and gangliosides mediate axon-myelin stability: Neuropathology and behavioral deficits in single- and double-null mice. Exp. Neurol. 195, 208-217 (2005)</ref>. 

=== Structure ===

[[File:Siglec-04_cartoon_lg2.jpg]]

 Siglec-4 is a heavily glycosylated protein of about 100kDa with 30% of its mass being made up by carbohydrates distributed over eight glycosylation sites. The extracellular part of Siglec-4 consists of five Ig-like domains (one V-set domain and four C2-set domains). Two splice variants for Siglec-4 are found in mammals, L-MAG (72kDa) and S-MAG (67kDa), which differ in their cytoplasmic domain. L-MAG contains a tyrosine phosphorylation site<ref name="umemori1994">Umemori, H., Sato, S., Yagi, T., Aizawa, S., Yamamoto, T. Initial events of myelination involve Fyn tyrosine kinase signalling. Nature 367, 572-576 (1994)</ref><ref name="jaramillo1994">Jaramillo, M. L., Afar, D. E., Almazan, G., Bell, J. C. Identification of tyrosine 620 as the major phosphorylation site of myelin-associated glycoprotein and its implication in interacting with signaling molecules. J Biol Chem. 269, 27240-27245 (1994)</ref> that is missing in S-MAG.
 

=== Biological roles of GBP-ligand interaction ===
MAG is expressed on the innermost myelin membrane wrap, directly apposed to the axon surface. Although it is not required for myelination, MAG enhances long-term axon survival, helps structure myelin gaps (nodes of Ranvier) essential for rapid nerve conduction, regulates the axon cytoskeleton and protects axons from acute toxic insults. In addition to its role in axon-myelin stabilization, MAG inhibits axon regeneration after injury; MAG on residual myelin membranes at injury sites actively signals axons to halt elongation. Whether MAG's stabilizing effects and its inhibition of axon regeneration are part of the same signaling system is under investigation.
 
MAG has multiple receptors on the axon surface, including gangliosides GD1a/GT1b, the GPI-anchored Nogo receptors (NgR1 and NgR2), and transmembrane proteins PirB and β-integrin. Some of these interactions involve MAG's glycan binding capability, while others may not. The following biological roles of MAG have been experimentally linked to its glycan binding activity using genetic, biochemical, and/or pharmacological criteria:
 
1. Long term axon stabilization: B4galnt1-null mice, which lack the termini of complex gangliosides, display the same progressive axon degeneration phenotype as Mag-null mice. Double null mice (B4galnt1, Mag) are similar. <ref name="pan2005"/>
 
2. Nodes of Ranvier: B4galnt1-null and Mag-null mice have similar deficits in the structures of Nodes of Ranvier<ref name="pernet2008">Pernet V., Joly S., Christ F., Dimou L., Schwab M.E. Nogo-A and myelin-associated
glycoprotein differently regulate oligodendrocyte maturation and myelin formation. J Neurosci. 16, 7435-44 (2008).</ref><ref name="susuki2007">Susuki K., Baba H., Tohyama K., Kanai K., Kuwabara S., Hirata K., Furukawa K., Furukawa K., Rasband M.N., Yuki N. Gangliosides contribute to stability of paranodal junctions and ion channel clusters in myelinated nerve fibers. Glia 55, 746-757. 2007.</ref>
 
3. Cytoskeletal organization: B4galnt1-null, Mag-null and double-null mice have similarly reduced neurofilament spacing and reduced axon diameter.<ref name="pan2005"/>
 
4. Axon protection: MAG-mediated protection of axons from toxic insults is diminished in B4galnt1-null mice or after treatment of axons with sialidase.<ref name="nguyen2009">Nguyen T., Mehta N.R., Conant K., Kim K.J., Jones M., Calabresi P.A., Melli G., Hoke A., Schnaar R.L., Ming G.L., Song H., Keswani S.C., Griffin J.W. Axonal protective effects of the myelin-associated glycoprotein. J Neurosci. 21, 630-637 (2009).</ref><ref name="mehta2010">Mehta N.R., Nguyen T., Bullen J.W., Griffin J.W., Schnaar R.L. Myelin-associated glycoprotein (MAG) protects neurons from acute toxicity using a ganglioside-dependent mechanism. ACS Chem Neurosci. 1, 215-222, 2010.</ref>
 
5. Regulating axon regeneration: MAG-mediated inhibition of axon regeneration is diminished in B4galnt1-null mice, after treatment with sialidase, or by addition of MAG-binding soluble glycans.<ref name="vyas2002">Vyas A.A., Patel H.V., Fromholt S.E., Heffer-Lauc M., Vyas K.A., Dang J., Schachner M., Schnaar R.L. Gangliosides are functional nerve cell ligands for myelin-associated glycoprotein (MAG), an inhibitor of nerve regeneration. Proc Natl Acad Sci U S A 99, 8412-8417, 2002.</ref><ref name="vyas2005"/>
 
MAG signaling is bidirectional,<ref name="quarles2007"/> into the myelinating cells and into myelin-ensheathed axons. Signaling into myelinating cells may involve tyrosine phosphorylation of the MAG intracellular domain downstream of ligand engagement,<ref name="umemori1994"/><ref name="jaramillo1994"/> whereas signals into the axon are likely to involve activation of the small non-receptor GTPase RhoA.<ref name="yiu2006">Yiu G., He Z. Glial inhibition of CNS axon regeneration. Nat. Rev. Neurosci. 7, 617–627, 2006.</ref>
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Siglec-4&maxresults=20 CFG database search results for Siglec-4].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Probes for mouse and human MAG (under the name Siglec-4) have been included on all four versions of the CFG glycogene microarray.
 

=== Knockout mouse lines ===
The CFG has [https://www.functionalglycomics.org/glycomics/publicdata/phenotyping.jsp phenotyped] the MAG-deficient mouse.

=== Glycan array ===
Investigators have used CFG carbohydrate compounds to study MAG ligand specificity.

== Related GBPs ==
Compared to other Siglecs, Siglec-4 is most conserved. Based on sequence similarity orthologous proteins can be identified in all vertebrate genomes available so far (several mammals, chicken, Xenopus, zebrafish and fugu). Sialic acid binding activity selective for 2,3-linked Sia has been shown for the avian ortholog (SMP<ref name="collins1996"/> and fish Siglec-4 from zebrafish (Danio rerio) and fugu (Takifugu rubripes)<ref name=" Lehmann, F.2004"> Lehmann, F., Gäthje, H., Kelm, S., Dietz, F. Evolution of sialic acid-binding proteins: molecular cloning and expression of fish siglec-4. Glycobiology 14, 959-968 (2004)</ref>). Whereas the primary sequences of the Sia-binding N-terminal domains is 97 % identical between rodents and man and share over 50 % sequence identity between fish and mammals, the cytoplasmic tail is much less conserved (20% identical amino acids between fish and mammals<ref name=" Lehmann, F.2004"></ref>).

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Paul Crocker, Sorge Kelm, James Paulson, Ron Schnaar

Siglec-8

2011-03-30T23:32:17Z

Carole Weaver: /* Glycogene microarray */

Siglec-8 is a human siglec expressed predominantly on eosinophils and mast cells, and is a paradigm for the rapidly evolving sub-family of CD33-related siglecs that are expressed on various white blood cells<ref>Crocker, P. R., Paulson, J. C. & Varki, A. Siglecs and their roles in the immune system. Nat Rev Immunol 7, 255-266 (2007).</ref>
<ref name=" Kikly 2009">
Kikly, K.K., Bochner, B.S., et al. [http://www.ncbi.nlm.nih.gov/pubmed/10856141 Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils.] J Allergy Clin Immunol 105, 1093-100 (2000)</ref>
<ref name="Bochner 2009">Bochner, B.S. [http://www.ncbi.nlm.nih.gov/pubmed/19178537 Siglec-8 on human eosinophils and mast cells, and Siglec-F on murine eosinophils, are functionally related inhibitory receptors.] Clin Exp Allergy 39, 317-324 (2009).</ref>
<ref name=" Floyd H 2000"></ref>. A characteristic feature of Siglec-8 and most other CD33-related siglecs is a cytoplasmic domain with a single immunoreceptor tyrosine inhibitory motif (ITIM) and a single ITIM-like motif that participate in siglec-mediated regulation of cell signaling and endocytosis. While there is no clear ortholog in mice, Siglec-F has been documented as a functional paralog that has a similar expression pattern on murine leukocytes and similar ligand specificity<ref name="Bochner 2009"></ref><ref name=" Tateno 1125">Tateno, H., Crocker, P. R. & Paulson, J. C. Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6'-sulfo-sialyl Lewis X as a preferred glycan ligand. Glycobiology 15, 1125-1135 (2005).</ref>
<ref name=" Zhang 2007"></ref>. Siglec-8, and its murine paralog Siglec-F, recognize a ligand containing both sialic acid and sulfate (NeuAcα2-3[6S]Galβ1-4G[Fucα1-3]GlcNAc-), a specificity that is distinct from all other siglecs. Ligation of Siglec-8 (or Siglec-F) with antibodies or polymeric ligands induces apoptosis of eosinophils, suggesting a therapeutic approach for treating eosinophil (or mast cell) mediated disease by targeting Siglec-8<ref>O'Reilly, M. K. & Paulson, J. C. Siglecs as targets for therapy in immune-cell-mediated disease. Trends Pharmacol Sci 30, 240-248 (2009).</ref><ref>Zimmermann, N. et al. Siglec-F antibody administration to mice selectively reduces blood and tissue eosinophils. Allergy 63, 1156-1163 (2008).</ref>
<ref name=" Bochner 4307 ">Bochner, B. S. et al. Glycan array screening reveals a candidate ligand for Siglec-8. J Biol Chem 280, 4307-4312 (2005).</ref>
<ref name=" Nutku, E 2003">
Nutku, E., Aizawa, H., Hudson, S. A. & Bochner, B. S. Ligation of Siglec-8: a selective mechanism for induction of human eosinophil apoptosis. Blood 101, 5014-5020 (2003).</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
Participating Investigators (PIs) of the CFG have made major contributions to the understanding of the biology of Siglec-8 and its murine paralog, Siglec-F. These include: Bruce Bochner, Nicolai Bovin, Paul Crocker, James Paulson, Ronald Schnaar, Ajit Varki

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about Siglec-8, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information about Siglec-8 can be found in its [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Itlect_00146&sideMenu=no GBP Molecule Page] in the CFG database.
=== Carbohydrate ligands ===
The high affinity ligand for Siglec-8 has been deduced from glycan microarray screening on the CFG microarray (click [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_49_09152004 here] and [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v2_19_02202004 here]) to be NeuAcα2-3(6-SO3)Galβ1-4(Fucα1-3)GlcNAc [6'Su-SLeX] <ref name=" Bochner 4307 ">
</ref><ref name=" Tateno 1125"></ref>

[[File:6pso3slex.jpg]]

For Siglec-F, histologic studies suggest the presence of an α2,3-linked sialylated glycoprotein ligand expressed by airway epithelium. Its constitutive expression requires the enzyme St3Gal3.<ref>Guo JP, Brummet ME, Myers AC, Na HJ, Rowland E, Schnaar RL, Zheng T, Zhu Z, Bochner BS. Characterization of expression of glycan ligands for Siglec-F in normal mouse lungs. Am J Respir Cell and Molec Biol 2010 Apr 15 [Epub ahead of print] 2010 and </ref> Levels of this ligand are increased during allergic pulmonary inflammation.
<ref name=" Zhang 2007">Zhang M, Angata T, Cho JY, Miller M, Broide DH, Varki A. Defining the in vivo function of Siglec-F, a CD33-related Siglec expressed on mouse eosinophils. Blood 2007; 109:4280-7</ref>
 

=== Cellular expression of GBP and ligands ===
Siglec-8 is expressed in human eosinophils and mast cells, and weakly in basophils. <ref name=" Kikly 2009"></ref>
<ref name=" Floyd H 2000">Floyd H, Ni J, Cornish AL, Zeng Z, Liu D, Carter KC, Steel J, Crocker PR. Siglec-8: a novel eosinophil-specific member of the immunoglobulin superfamily. J Biol Chem 2000; 275:861-6.</ref><ref>Yokoi H, Myers A, Matsumoto K, Crocker PR, Saito H, Bochner BS. Alteration and acquisition of Siglecs during in vitro maturation of CD34+ progenitors into human mast cells. Allergy 2006; 61:769-76</ref>
 
=== Biosynthesis of ligands ===
Constitutive expression in airway epithelium of the putative α2,3-linked sialylated glycoprotein ligand for Siglec-F requires the enzyme St3Gal3 which has been characterized in
[http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_hum_624&sideMenu=true&pageType=general Human] and
[http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_mou_644&sideMenu=true&pageType=general Mouse].
 

=== Structure ===
[[File:Siglec8 SiglecF.jpg]]
 

=== Biological roles of GBP-ligand interaction ===
'''In vitro'''
Eosinophil apoptosis. <ref name=" Nutku, E 2003"></ref><ref>Nutku E, Hudson SA, Bochner BS. Mechanism of Siglec-8-induced human eosinophil apoptosis: role of caspases and mitochondrial injury. Biochem Biophys Res Commun 2005; 336:918-24</ref><ref>1Nutku-Bilir E, Hudson SA, Bochner BS. Interleukin-5 priming of human eosinophils alters Siglec-8 mediated apoptosis pathways. Am J Respir Cell Mol Biol 2008; 38:121-4</ref>
Inhibition of mast cell mediator release.<ref>Yokoi H, Choi OH, Hubbard W, Lee H-S, Canning BJ, Lee HH, Ryu S-D, Bickel CA, Hudson SA, MacGlashan DW, Jr., Bochner BS. Inhibition of FcεRI-dependent mediator release and calcium flux from human mast cells by Siglec-8 engagement. J Allergy Clin Immunol 2008; 121:499-505</ref>

'''In vivo (for Siglec-F)'''
Antibody administration to mice causes selective depletion of eosinophils in blood and gastrointestinal tissues via apoptosis.<ref>Zimmermann N, McBride ML, Yamada Y, Hudson SA, Jones C, Cromie KD, Crocker PR, Rothenberg ME, Bochner BS. Siglec-F antibody administration to mice selectively reduces blood and tissue eosinophils. Allergy 2008; 63:1156-63</ref> They are also effective in reversing some sequelae of mouse models of eosinophilic gastroenteritis and asthma.<ref>Song DJ, Cho JY, Miller M, Strangman W, Zhang M, Varki A, Broide DH. Anti-Siglec-F antibody inhibits oral egg allergen induced intestinal eosinophilic inflammation in a mouse model. Clin Immunol 2009; 131:157-69</ref><ref>Song DJ, Cho JY, Lee SY, Miller M, Rosenthal P, Soroosh P, Croft M, Zhang M, Varki A, Broide DH. Anti-Siglec-F antibody reduces allergen-induced eosinophilic inflammation and airway remodeling. J Immunol 2009; 183:5333-41</ref>
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Siglec-8&maxresults=20 CFG database search results for Siglec-8].

=== Glycan profiling ===
Glycan structure analysis has been conducted by the CFG for [http://www.functionalglycomics.org/glycomics/common/jsp/samples/searchSample.jsp?5=Human&15=&10=&9=Eosinophils+&operation=refine&templateKey=2&12=CellType&submit=submit human ] and [http://www.functionalglycomics.org/glycomics/common/jsp/samples/searchSample.jsp?5=Mouse&15=&10=&9=Eosinophils+&operation=refine&templateKey=2&12=CellType&submit=submit mouse] eosinophils.

=== Glycogene microarray ===
Analysis has been conducted on glycosyltransferase expression using the glycogene microarray for [http://www.functionalglycomics.org/static/coreE/flash/search.swf?maExpKey=100393 murine eosinophils] that relates to the enzymes required for expression of cis ligands of Siglec-F on these cells. Probes for human Siglec-8 and mouse Siglec-F have been included on all four versions of the CFG glycogene microarray.

=== Knockout mouse lines ===
Mice deficient in Siglec-F have normal blood and bone marrow eosinophils at baseline, but develop exaggerated bone marrow, blood and lung eosinophilia after allergen sensitization and challenge. <ref name=" Zhang 2007"></ref>
 

=== Glycan array ===
The discovery of the [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_49_09152004 ligand for Siglec-8] [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v2_19_02202004] and its murine paralog, [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_47_10132004 Siglec-F ] [http://www.functionalglycomics.org:80/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v2_58_07262004], was made by investigator-initiated resource requests for glycan array analysis and carbohydrate compounds. To see all glycan array results for Siglec-8, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=siglec-8&cat=coreh here].

== Related GBPs ==
hSiglec-3 (CD33), Siglec-5, Siglec-6 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-6&maxresults=20 (CFG data)], Siglec-7 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-7&maxresults=20 (CFG data)], Siglec-9 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-9&maxresults=20 (CFG data)], Siglec-10 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-10&maxresults=20 (CFG data)], Siglec-11, Siglec-F [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-F&maxresults=20 (CFG data)], Siglec-E [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-E&maxresults=20 (CFG data)], Siglec-G [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=siglec-G&maxresults=20 (CFG data)].

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Bruce Bochner, Paul Crocker, James Paulson, Ron Schnaar

Sialoadhesin

2011-03-30T23:30:48Z

Carole Weaver:

Sialoadhesin (Sn), also known as Siglec-1, is an atypical siglec, due to the presence of an unusually large number of Ig domains (17) and the absence of tyrosine-based intracellular signaling motifs. Sn is expressed uniquely by macrophage subsets in vivo and the 17 Ig domains are thought to be important for its ability to mediate sialic acid-dependent adhesive functions. This contrasts with most other siglecs which are much shorter and masked by cis binding to co-expressed sialic acids.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of Sn include: Paul Crocker, Peter Delputte, Soerge Kelm, Ajit Varki

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about sialoadhesin, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Page for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Itlect_267&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_mou_Itlect_193&sideMenu=no mouse] sialoadhesin (a.k.a. Siglec-1) in the CFG database.
=== Carbohydrate ligands ===

[[File:Sia3Gal_small.png]]

Sn is a fairly promiscuous receptor, with a preference for Siaα2-3Gal over Siaα2-6Gal terminated glycans. Sn prefers NeuNAc in α2,3-linkage over α2,6 and α2,8 linkages and does not recognize NeuGc or NeuAc9Ac. In pull-down experiments using Sn-Fc constructs, mucin-like proteins with multiple O-linked glycans seem to be preferred (eg CD43, Muc-1), but whether these represent preferred counterreceptors during cell-cell interactions between Sn+ macrophages and other cells is unknown
 

=== Cellular expression of GBP and ligands ===
Sn is expressed exclusively by cells of the mononuclear phagocyte lineage, including in some cases myeloid dendritic cells as well as classic macrophages. It is expressed constitutively by many tissue macrophages, particularly those in primary and secondary lymphoid organs and may play a role in antigen capture and tolerance. Sn can also be induced on macrophages by IFN-α or agents that induce expression of IFN-α such as LPS or poly-I:C. Ligands for Sn are regulated via expression of sialyltransferases and are found on many cells of the body. Surveys of haemopoietic targets have identified granulocytes as being rich in Sn ligands but the functional significance of this is unclear at present.
 

=== Biosynthesis of ligands ===

 
=== Structure ===
The crystal structure of the N-terminal carbohydrate-binding domain of sialoadhesin in complex with 3'-sialyllactose highlights the roles of three key conserved amino acids, tryptophan 2, arginine 97 and tryptophan 106, in the ligand-binding domains of the siglecs that are involved in interactions with the various characteristic groups that project from the pyranose ring of sialic acid. The structure provides a rationale for why sialoadhesin binds to N-acetyl- but not N-glycolyl neuraminic acid and the limited interactions with the lactose portion of the glycan are consistent with the ability of sialoadhesin to bind both 2-3 and 2-6 linked sialic acid.<ref name"Crocker1998">May, AP, Robinson, RC, Vinson, M, Crocker, PR and Jones, EY (1998) Crystal structure of the N-terminal domain of sialoadhesin in complex with 3 sialyllactose at 1.85 Å Resolution. Molecular Cell 1, 719-728</ref>
 

=== Biological roles of GBP-ligand interaction ===
Sn contributes to proinflammatory immune responses in a variety of autoimmune diseases<ref>Jiang, H. R. et al. Sialoadhesin promotes the inflammatory response in experimental autoimmune uveoretinitis. J Immunol 177, 2258-2264 (2006).</ref><ref>Ip, C. W., Kroner, A., Crocker, P. R., Nave, K. A. & Martini, R. Sialoadhesin deficiency ameliorates myelin degeneration and axonopathic changes in the CNS of PLP overexpressing mice. Neurobiol Dis 25, 105-111 (2007).</ref>, and this may be due to suppression of Treg expansion as demonstrated in experimental allergic encephalomyelitis, a model for multiple sclerosis<ref>Wu, C. et al. Sialoadhesin-positive macrophages bind regulatory T cells, negatively controlling their expansion and autoimmune disease progression. J Immunol 182, 6508-6516 (2009).</ref>. Sn has also been shown to function as a macrophage receptor for the porcine arterivirus<ref>Delputte, P. L. et al. Porcine arterivirus attachment to the macrophage-specific receptor sialoadhesin is
dependent on the sialic acid-binding activity of the N-terminal immunoglobulin domain of sialoadhesin. J Virol 81, 9546-9550 (2007).</ref> and can also promote macrophage uptake of sialylated bacteria such as ''Neisseria meningitidis''<ref>Jones, C., Virji, M. & Crocker, P. R. Recognition of sialylated meningococcal lipopolysaccharide by siglecs expressed on myeloid cells leads to enhanced bacterial uptake. Mol Microbiol 49, 1213-1225 (2003).</ref>.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Sialoadhesin&maxresults=20 CFG database search results for sialoadhesin].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Probes for mouse and human sialoadhesin (under the name Siglec-1) have been included on all four versions of the CFG glycogene microarray.
 

=== Knockout mouse lines ===
The CFG did not undertake creation of knockout mice for sialoadhesin because generation of such mice was already underway. Studies of these mice indicate a role for sialoadhesin in regulation of the cellular and humoral immune response<ref name=”Oetke2006”>Oetke C, Vinson MC, Jones C, Crocker PR (2006) Sialoadhesin-deficient mice exhibit subtle changes in B- and T-cell populations and reduced immunoglobulin M levels. Mol. Cell. Biol. 26, 1549-57</ref>
 

=== Glycan array ===
The CFG glycan array has been probed with both murine and porcine Sn constructs, but no positive signals were obtained (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1383 here]), likely due to the low affinity of Sn for sialylated oligosaccharides.

== Related GBPs ==
None.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Paul Crocker, James Paulson

Sialoadhesin

2011-03-30T23:30:15Z

Carole Weaver: /* Glycogene microarray */

Sialoadhesin (Sn) is an atypical siglec, due to the presence of an unusually large number of Ig domains (17) and the absence of tyrosine-based intracellular signaling motifs. Sn is expressed uniquely by macrophage subsets in vivo and the 17 Ig domains are thought to be important for its ability to mediate sialic acid-dependent adhesive functions. This contrasts with most other siglecs which are much shorter and masked by cis binding to co-expressed sialic acids.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of Sn include: Paul Crocker, Peter Delputte, Soerge Kelm, Ajit Varki

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about sialoadhesin, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Page for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Itlect_267&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_mou_Itlect_193&sideMenu=no mouse] sialoadhesin (a.k.a. Siglec-1) in the CFG database.
=== Carbohydrate ligands ===

[[File:Sia3Gal_small.png]]

Sn is a fairly promiscuous receptor, with a preference for Siaα2-3Gal over Siaα2-6Gal terminated glycans. Sn prefers NeuNAc in α2,3-linkage over α2,6 and α2,8 linkages and does not recognize NeuGc or NeuAc9Ac. In pull-down experiments using Sn-Fc constructs, mucin-like proteins with multiple O-linked glycans seem to be preferred (eg CD43, Muc-1), but whether these represent preferred counterreceptors during cell-cell interactions between Sn+ macrophages and other cells is unknown
 

=== Cellular expression of GBP and ligands ===
Sn is expressed exclusively by cells of the mononuclear phagocyte lineage, including in some cases myeloid dendritic cells as well as classic macrophages. It is expressed constitutively by many tissue macrophages, particularly those in primary and secondary lymphoid organs and may play a role in antigen capture and tolerance. Sn can also be induced on macrophages by IFN-α or agents that induce expression of IFN-α such as LPS or poly-I:C. Ligands for Sn are regulated via expression of sialyltransferases and are found on many cells of the body. Surveys of haemopoietic targets have identified granulocytes as being rich in Sn ligands but the functional significance of this is unclear at present.
 

=== Biosynthesis of ligands ===

 
=== Structure ===
The crystal structure of the N-terminal carbohydrate-binding domain of sialoadhesin in complex with 3'-sialyllactose highlights the roles of three key conserved amino acids, tryptophan 2, arginine 97 and tryptophan 106, in the ligand-binding domains of the siglecs that are involved in interactions with the various characteristic groups that project from the pyranose ring of sialic acid. The structure provides a rationale for why sialoadhesin binds to N-acetyl- but not N-glycolyl neuraminic acid and the limited interactions with the lactose portion of the glycan are consistent with the ability of sialoadhesin to bind both 2-3 and 2-6 linked sialic acid.<ref name"Crocker1998">May, AP, Robinson, RC, Vinson, M, Crocker, PR and Jones, EY (1998) Crystal structure of the N-terminal domain of sialoadhesin in complex with 3 sialyllactose at 1.85 Å Resolution. Molecular Cell 1, 719-728</ref>
 

=== Biological roles of GBP-ligand interaction ===
Sn contributes to proinflammatory immune responses in a variety of autoimmune diseases<ref>Jiang, H. R. et al. Sialoadhesin promotes the inflammatory response in experimental autoimmune uveoretinitis. J Immunol 177, 2258-2264 (2006).</ref><ref>Ip, C. W., Kroner, A., Crocker, P. R., Nave, K. A. & Martini, R. Sialoadhesin deficiency ameliorates myelin degeneration and axonopathic changes in the CNS of PLP overexpressing mice. Neurobiol Dis 25, 105-111 (2007).</ref>, and this may be due to suppression of Treg expansion as demonstrated in experimental allergic encephalomyelitis, a model for multiple sclerosis<ref>Wu, C. et al. Sialoadhesin-positive macrophages bind regulatory T cells, negatively controlling their expansion and autoimmune disease progression. J Immunol 182, 6508-6516 (2009).</ref>. Sn has also been shown to function as a macrophage receptor for the porcine arterivirus<ref>Delputte, P. L. et al. Porcine arterivirus attachment to the macrophage-specific receptor sialoadhesin is
dependent on the sialic acid-binding activity of the N-terminal immunoglobulin domain of sialoadhesin. J Virol 81, 9546-9550 (2007).</ref> and can also promote macrophage uptake of sialylated bacteria such as ''Neisseria meningitidis''<ref>Jones, C., Virji, M. & Crocker, P. R. Recognition of sialylated meningococcal lipopolysaccharide by siglecs expressed on myeloid cells leads to enhanced bacterial uptake. Mol Microbiol 49, 1213-1225 (2003).</ref>.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Sialoadhesin&maxresults=20 CFG database search results for sialoadhesin].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Probes for mouse and human sialoadhesin (under the name Siglec-1) have been included on all four versions of the CFG glycogene microarray.
 

=== Knockout mouse lines ===
The CFG did not undertake creation of knockout mice for sialoadhesin because generation of such mice was already underway. Studies of these mice indicate a role for sialoadhesin in regulation of the cellular and humoral immune response<ref name=”Oetke2006”>Oetke C, Vinson MC, Jones C, Crocker PR (2006) Sialoadhesin-deficient mice exhibit subtle changes in B- and T-cell populations and reduced immunoglobulin M levels. Mol. Cell. Biol. 26, 1549-57</ref>
 

=== Glycan array ===
The CFG glycan array has been probed with both murine and porcine Sn constructs, but no positive signals were obtained (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1383 here]), likely due to the low affinity of Sn for sialylated oligosaccharides.

== Related GBPs ==
None.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Paul Crocker, James Paulson

CD22

2011-03-30T23:29:01Z

Carole Weaver: /* Glycogene microarray */

CD22 is predominantly expressed on B cells and is well documented as a regulator of B cell receptor (BCR) signaling<ref name="Crocker 2007">Crocker PR, Paulson JC, Varki A. [http://www.ncbi.nlm.nih.gov/pubmed/17380156 Siglecs and their roles in the immune system]. ''Nat Rev Immunol'' 2007 Apr;7(4):255-66. Review.</ref>. It is one of four siglecs that are highly conserved among mammals. This paradigm is unique among the siglecs in that the cytoplasmic domain has six conserved tyrosine motifs, including three immunoreceptor tyrosine inhibitory motifs (ITIM), one ITIM-like motif, and a growth factor receptor bound protein2 (GRB2) motif. These tyrosine motifs are involved in regulation of BCR signaling and also mediate its constitutive clathrin mediated endocytosis, an activity believed to be tied to its regulation of cell signaling. The preferred glycan ligand of CD22 differs significantly in humans and mice<ref name="Crocker 2007"/><ref name="Kimura 2007">Kimura N, Ohmori K, Miyazaki K, Izawa M, Matsuzaki Y, Yasuda Y, Takematsu H, Kozutsumi Y, Moriyama A, Kannagi R. [http://www.ncbi.nlm.nih.gov/pubmed/17728258 Human B-lymphocytes express alpha2-6-sialylated 6-sulfo-N-acetyllactosamine serving as a preferred ligand for CD22/Siglec-2]. J'' Biol Chem''. 2007 Nov 2;282(44):32200-7.</ref><ref name="Blixt 2004">Blixt O, Head S, Mondala T, Scanlan C, Huflejt ME, Alvarez R, Bryan MC, Fazio F, Calarese D, Stevens J, Razi N, Stevens DJ, Skehel JJ, van Die I, Burton DR, Wilson IA, Cummings R, Bovin N, Wong CH, Paulson JC. [http://www.ncbi.nlm.nih.gov/pubmed/15563589 Printed covalent glycan array for ligand profiling of diverse glycan binding proteins]. ''Proc Natl Acad Sci U S A''. 2004 Dec 7;101(49):17033-8.</ref>. While both recognize the sequence Siaa-2-6Galb-1-4GlcNAc expressed abundantly on B cells, murine CD22 prefers Neu5Gc (not found in humans) over Neu5Ac, while human CD22 exhibits highest affinity for sulfated sialoside, Neu5Aca-2-6Galb-1-4[6S]GlcNAc, demonstrating significant evolution of ligand specificity with conservation of function. Although CD22 recognizes ligands on the same cell in ''cis'', it also binds to ligands in ''trans'' if expressed on adjacent contacting cells. A major area of investigation is to understand the relative roles of ''cis'' and ''trans'' ligands in CD22 function.

[[Image:SiglecCD22.jpg|right|alt text]]
== CFG Participating Investigators contributing to the understanding of this paradigm ==

CFG Participating Investigators (PIs) have made major contributions to the understanding of the biology of human and murine CD22. These include: Nicolai Bovin, Paul Crocker, Jamey Marth, David Nemazee, Lars Nitschke, Jim Paulson, Ajit Varki

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about CD22, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Page for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Itlect_269&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_mou_Itlect_194&sideMenu=no mouse] CD22 (aka Siglec-2) in the CFG database.
=== Carbohydrate ligands ===
Although CD22 is highly conserved throughout mammalian species, murine and human CD22 are known to exhibit significant differences in their specificities that appear to have evolved to compensate for changes in the glycan ligands expressed on B cells. While both bind Siaα2-6Gal terminated glycans, murine CD22 prefers NeuGc (NeuGcα2-6Galβ1-4GlcNAc), which is not found in humans. In contrast, human human CD22 recognizes NeuAc and NeuGc with equal affinity. In addition, however, human CD22 exhibits highest affinity for a ligand with sulfate at the 6 position of GlcNAc (NeuAcα2-6Galβ1-4[6S]GlcNAc).<ref name="Crocker 2007"/><ref name="Kimura 2007"/> 9-O-acetylation of sialic acid abrogates binding of CD22, which is thought to regulate the binding of ''cis'' ligands on B cells.
 
=== Cellular expression of GBP and ligands ===
CD22 is primarily expressed on mature B cells and to a lesser extent on memory B cells. However, it is not expressed on pre-B cells and differentiated plasma cells. Like many siglecs, CD22 interacts with endogenous ligands on B cells in ''cis'', and on other cells, such as T cells and bone marrow vessel endothelial cells in ''trans''. Although ''cis'' ligands of tend to mask the CD22 binding site, CD22 is able to interact with ''trans'' ligands on contacting cells (B cells and T cells), and to bind to synthetic multivalent ligands that have sufficient avidity.
 
=== Biosynthesis of ligands ===
The ligands of CD22 are predominately the product of a single sialyltransferase, ST6Gal I. Mice deficient in ST6Gal I express no ligands on B cells resulting in an immuno-deficient phenotype.
 
=== Structure ===
Although the crystal structure of CD22 has not yet been elucidated, structures of other siglecs, including sialoadhesin, siglec-5 and siglec-7 have shed insights into the nature of the ligand binding site of CD22.<ref name="Crocker 2007"/>
 
=== Biological roles of GBP-ligand interaction ===

 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CD22&maxresults=20 CFG database search results for CD22].

=== Glycan profiling ===
Both murine and human CD22 recognize the sequence Siaa-2-6Galb-1-4GlcNAc expressed abundantly on [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=b%20AND%20cells&cat=corec B cells], which have been subjected to glycan profiling by the CFG.
 

=== Glycogene microarray ===

The CFG glycogene microarray has been used to show that ST6Gal I is downregulated [https://www.functionalglycomics.org/glycomics/publicdata/microarray.jsp?resReqId=cfg_rRequest_2 'on T cells] upon activation suggesting that B cell ''trans'' ligands are reduced on activated T cells. Probes for mouse and human CD22 have been included on all four versions of the CFG glycogene array.

=== Knockout mouse lines ===
Mice deficient in [https://www.functionalglycomics.org/static/consortium/resources/resourcecoref16.shtml CD22] and the sialyltransferase, ST6Gal I, responsible for synthesis of its ligands ([https://www.functionalglycomics.org/glycomics/publicdata/phenotyping.jsp ST6Gal I]) distributed by the CFG have been instrumental in understanding the biology of CD22.

=== Glycan array ===
The CFG's glycan array was instrumental in identification of the [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1792 high affinity ligands of CD22] as sialylated-sulfated glycans.<ref name="Kimura 2007"/><ref name="Blixt 2004"/>

== Related GBPs ==

This paradigm is unique among the siglecs in that the cytoplasmic domain has six conserved tyrosine motifs, including three immunoreceptor tyrosine inhibitory motifs (ITIM), one ITIM-like motif, and a growth factor receptor bound protein2 (GRB2) motif. However, other members of the homologous siglec family have contributed to an understanding of the glycan binding site of CD22, and general principles governing the interaction of CD22 with ''cis'' and ''trans'' ligands.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Paul Crocker, James Paulson

Cyanovirin-N (CVN)

2011-03-30T23:26:12Z

Carole Weaver: /* Glycogene microarray */

'''Microbial Antiviral Proteins with GBP Activity''' 

Antiviral compounds made by eukaryotes that recognize unique glycan determinants represent a new paradigm in terms of understanding the innate immune system of primitive organisms and how that might relate to mammalian innate immune systems. Many mammalian GBPs act as innate immune defenders, including the C-type lectins [[Ficolins/Mannose-binding protein]] and other collectins. Interestingly, many of these bind glycan determinants often relatively rich in mannose and/or fucose. One promising anti-HIV-1 drug in development is cyanovirin-N, initially isolated from an extract of the cyanobacterium Nostoc ellipsosoprum<ref name="Boyd1997">Boyd, M.R., Gustafson, K.R., McMahon, J.B., Shoemaker, R.H., O'Keefe, B.R., Mori, T., Gulakowski, R.J., Wu, L., Rivera, M.I., Laurencot, C.M., Currens, M.J., Cardellina, J.H., 2nd, Buckheit, R.W., Jr., Nara, P.L., Pannell, L.K., Sowder, R.C., 2nd and Henderson, L.E. 1997. Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob Agents Chemother, 41, 1521-1530</ref>. While CVN was originally thought to be an orphan lectin with little homology to any other known protein family<ref name="Bewley1998">Bewley, C.A., Gustafson, K.R., Boyd, M.R., Covell, D.G., Bax, A., Clore, G.M., and Gronenborn, A.M. 1998. Solution structure of cyanovirin-N, a potent HIV-inactivating protein. Nat Struct Biol 5, 571-578</ref>, a family of CVN homologs, termed CVNHs, has been described<ref>Percudani, R., Montanini, B. and Ottonello, S. 2005. The anti-HIV cyanovirin-N domain is evolutionarily conserved and occurs as a protein module in eukaryotes. Proteins, 60, 670-678</ref>. Members of this family are found in multicellular ascomycetous fungi and in ferns and share a 3-D fold<ref>Koharudin, L.M., Viscomi, A.R., Jee, J.G., Ottonello, S. and Gronenborn, A.M. 2008. The evolutionarily conserved family of cyanovirin-N homologs: structures and carbohydrate specificity. Structure, 16, 570-584</ref>. A CVNH of the toxin-producing cyanobacterium Microcystis aeruginosa also binds high mannose-type glycans and is involved in cell–cell attachment of Microcystis<ref>Kehr, J.C., Zilliges, Y., Springer, A., Disney, M.D., Ratner, D.D., Bouchier, C., Seeberger, P.H., de Marsac, N.T. and Dittmann, E. 2006. A mannan binding lectin is involved in cell-cell attachment in a toxic strain of Microcystis aeruginosa. Mol Microbiol, 59, 893-906</ref>.

Defining the glycan binding specificity and mode of action for virucidal lectins may help to develop new therapeutic approaches directed at combating viral infections.

'''Cyanovirin-N''' (''Nostoc ellipsosporum'' - a cyanobacterium) 

Cyanovirin-N (CVN) was chosen as a paradigm because of its relevance to human disease and as an example of a new virucidal found in nature. CVN was originally isolated from the cyanobacterium Nostoc ellipsosporum, in a screening program for anti-HIV activities<ref name="Boyd1997"/>. CVN is a small protein of 101 amino acids with two internal tandem repeats of ~50 amino acid. Its structure established a novel fold with no significant similarity to any other known protein<ref name="Bewley1998"/>. It exhibits pseudo-symmetry and comprises two domains, each possessing an independent glycan binding site<ref>Bewley, C.A., Kiyonaka, S., and Hamachi, I. (2002). Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9). J Mol Biol 322, 881-889</ref><ref>Barrientos, L.G., Matei, E., Lasala, F., Delgado, R., and Gronenborn, A.M. (2006). Dissecting carbohydrate-Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition. Protein Eng Des Sel 19, 525-535</ref>. The protein can also exist as a domain-swapped dimer<ref>Yang, F., Bewley, C.A., Louis, J.M., Gustafson, K.R., Boyd, M.R., Gronenborn, A.M., Clore, G.M., and Wlodawer, A. (1999). Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping. J. Mol. Biol. 288, 403-412</ref><ref>Barrientos, L.G., Louis, J.M., Botos, I., Mori, T., Han, Z., O'Keefe, B.R., Boyd, M.R., Wlodawer, A., and Gronenborn, A.M. (2002). The domain-swapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of X-ray and NMR structures. Structure 10, 673-686</ref>. CVN inhibits HIV entry into cells by interacting with the high mannose-type N-glycans on the envelope glycoprotein gp120 of HIV-1. CVN also binds to the glycoproteins of other enveloped viruses, such as SIV, Ebola, influenza and hepatitis C. Thus, CVN represents a new paradigm of microbial GBPs, wherein a unique glycan binding domain comprised of approximately 50 amino acids, exhibits specificity toward α1-2-linked mannose residues.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) who have contributed to studies of this paradigmatic protein include: Simone Ottonello (University of Parma, Italy) and Angela M. Gronenborn (University of Pittsburgh, USA).

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
The physiological ligand for Cyanovirin-N is not precisely known. However, based on extensive data on carbohydrate binding studies on this protein by NMR, X-ray, and glycan microarray screening on the CFG microarray, it is expected that CV-N would recognize any glycans that contain highly enriched alpha (1->2) linkage - mannoses.

=== Cellular expression of GBP and ligands ===
The Cyanovirin-N protein is expressed by Cyanobacterium (blue-green alga) Nostoc ellipsosporum. It binds ligands on glycoproteins of enveloped viruses including HIV, SIV, Ebola, influenza and hepatitis C.

=== Biosynthesis of ligands ===
 
=== Structure ===
The structures of Cyanovirin-N can be found at http://www.pdb.org/ [http://www.pdb.org/pdb/results/results.do?outformat=&qrid=620C4AC9&tabtoshow=Current].

Below are a few representations for the available structures of CV-N determined so far:

The solution structure of wild type CV-N as a monomer (PDB:2EZM).

[[File:CVN1.png]]

The crystal structure of P51G mutant of CV-N as a swapped dimer (PDB:1L5B).

[[File:CVN2.png]]

The crystal structure of swapped-dimeric CV-N in complex with hexamannose (PDB:3GXY).

[[File:CVN3.png]]

 

=== Biological roles of GBP-ligand interaction ===
In vitro, low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus<ref name = "Boyd1997"/>.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Cyanovirin-N&maxresults=20 CFG database search results for "cyanovirin-N"].

=== Glycan profiling ===

 

=== Glycogene microarray ===
Analysis has not been conducted. CVN is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.

=== Knockout mouse lines ===
Not applicable.

=== Glycan array ===
The CFG has contributed glycans for various glycan specificity studies.
Glycan specificity analysis has been conducted for [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_3001 Cyanovirin-N] using the CFG glycan microarray as shown below. To see all glycan array results for Cyanovirin-N, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=cyanovirin-N&cat=coreh here].

[[File:CVNglycan.png]]

== Related GBPs ==
A large family of CVNHs has been found in both eukaryotic fungi and cyanobacteria (see refs 3, 4 and 5 below). Click here for [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=CVNH&cat=coreh CFG data] on CVNHs.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Angela Gronenborn

Parainfluenza virus type 3 hemagglutinin-neuraminidase

2011-03-30T23:25:21Z

Carole Weaver: /* Glycogene microarray */

Human parainfluenza viruses (HPIVs) are a group of respiratory viruses associated with human respiratory diseases including bronchitis, bronchiolitis, and pneumonia<ref>Moscona, A. 2005. Entry of parainfluenza virus into cells as a target for interrupting childhood respiratory disease. J Clin Invest 115(7):1688-98.</ref><ref>Sato, M. and P.F. 2008. Current status of vaccines for parainfluenza virus infections. Pediatr Infect Dis J 27(10 Suppl):S123-5.</ref><ref>Johnstone, J., S.R. Majumdar, J.D. Fox, and T.J. Marrie. 2008. Viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation. Chest 134(6):1141-8.</ref>. Paramyxoviruses, including HPIVs, possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor-cleaving as well as receptor-binding activity where the two activities reside on the same glycoprotein unlike influenza which carries hemagglutinin and neuraminidase activities as individual glycoproteins. HN is also essential for activating the fusion protein (F) to mediate merger of the viral envelope with the host cell membrane <ref>Lamb, R. 1993. Paramyxovirus fusion: A hypothesis for changes. Virology 197:1-11.</ref><ref>Iorio, R.M., V.R. Melanson, and P.J. Mahon. 2009. Glycoprotein interactions in paramyxovirus fusion. Future Virol 4(4):335-351.</ref>. For the parainfluenza viruses as well as other HN-containing paramyxoviruses, this single molecule carries out three different but critical activities at specific points in the process of viral entry. The first step in infection by HPIV is binding to the lung cells’ surface via interaction of the viral receptor-binding molecule with sialic acid-containing receptor molecules on the cell surface <ref>Suzuki, T., A. Portner, R.A. Scroggs, M. Uchikawa, N. Koyama, et al. 2001. Receptor specificities of human respiroviruses. J Virol 75(10):4604-13.</ref><ref>Moscona, A., M. Porotto, S. Palmer, C. Tai, L. Aschenbrenner, et al. 2010. A Recombinant Sialidase Fusion Protein Effectively Inhibits Human Parainfluenza Viral Infection In Vitro and In Vivo. J Infect Dis.</ref>.

Structures of three paramyxovirus HNs have been determined; they are Newcastle Disease virus (NDV), HPIV type 3, and HPIV type 5 (formerly called SV5). Determination of the HN structure of hPIV3 (globular domain) show an enzyme active site very similar to that of influenza neuraminidase and PIV5 HN and this appears to also be a binding site. A second site at the dimer interface has been crystallographically determined only for Newcastle Disease virus (NDV) HN, but a rising number of reports postulate the presence of such a second site for other paramyxoviruses <ref>Zaitsev, V., M. von Itzstein, D. Groves, M. Kiefel, T. Takimoto, et al. 2004. Second sialic acid binding site in newcastle disease virus hemagglutinin-neuraminidase: implications for fusion. J Virol 78(7):3733-41.</ref><ref>Lawrence, M.C., N.A. Borg, V.A. Streltsov, P.A. Pilling, V.C. Epa, et al. 2004. Structure of the Haemagglutinin-neuraminidase from Human Parainfluenza Virus Type III. J Mol Biol 335(5):1343-57.</ref><ref>Yuan, P., T.B. Thompson, B.A. Wurzburg, R.G. Paterson, R.A. Lamb, et al. 2005. Structural studies of the parainfluenza virus 5 hemagglutinin-neuraminidase tetramer in complex with its receptor, sialyllactose. Structure 13(5):803-15.</ref><ref>Lamb, R.A., R.G. Paterson, and T.S. Jardetzky. 2005. Paramyxovirus membrane fusion: lessons from the F and HN atomic structures. Virology 344(1):30-7.</ref><ref>Bousse, T. and T. Takimoto. 2006. Mutation at residue 523 creates a second receptor binding site on human parainfluenza virus type 1 hemagglutinin-neuraminidase protein. J Virol 80(18):9009-16.</ref><ref>Porotto, M., M. Fornabaio, G. Kellogg, and A. Moscona. 2007. A second receptor binding site on the human parainfluenza 3 hemagglutinin-neuraminidase contributes to activation of the fusion mechanism. J Virol 81(7):3216-3228.</ref>. Interestingly for NDV HN, functional analysis of the two sites indicated that engagement of the first site activates the second <ref>Porotto, M., M. Fornabaio, O. Greengard, M.T. Murrell, G.E. Kellogg, et al. 2006. Paramyxovirus receptor-binding molecules: engagement of one site on the hemagglutinin-neuraminidase protein modulates activity at the second site. J Virol 80(3):1204-13.</ref><ref>Ryan, C., V. Zaitsev, D.J. Tindal, J.C. Dyason, R.J. Thomson, et al. 2006. Structural analysis of a designed inhibitor complexed with the hemagglutinin-neuraminidase of Newcastle disease virus. Glycoconj J 23(1-2):135-41.</ref>. Further studies on the relationship between the sialic acid binding and cleavage activity of wildtype and mutant HPIV HNs are ongoing among CFG PIs. A region next to the transmembrane domain of HN (stalk region) is still elusive to crystal determination, however several studies showed the importance of this domain in fusion promotion <ref>Melanson, V.R. and R.M. Iorio. 2004. Amino acid substitutions in the F-specific domain in the stalk of the newcastle disease virus HN protein modulate fusion and interfere with its interaction with the F protein. J Virol 78(23):13053-61.</ref><ref>Melanson, V.R. and R.M. Iorio. 2006. Addition of N-glycans in the stalk of the Newcastle disease virus HN protein blocks its interaction with the F protein and prevents fusion. J Virol 80(2):623-33.</ref><ref>Porotto, M., M. Murrell, O. Greengard, and A. Moscona. 2003. Triggering of human parainfluenza virus 3 fusion protein(F) by the hemagglutinin-neuraminidase (HN): an HN mutation diminishing the rate of F activation and fusion. J Virol 77(6):3647-3654.</ref><ref>Bishop, K.A., A.C. Hickey, D. Khetawat, J.R. Patch, K.N. Bossart, et al. 2008. Residues in the stalk domain of the hendra virus g glycoprotein modulate conformational changes associated with receptor binding. J Virol 82(22):11398-409.</ref>.

To further emphasize the importance of understanding GBP paradigms, CFG PIs have shown that HPIV3 infection in cultured monolayer cells greatly differs from infection in human airway epithelial (HAE) cell cultures or in animal models <ref>Zhang, L., M.E. Peeples, R.C. Boucher, P.L. Collins, and R.J. Pickles. 2002. Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J Virol 76(11):5654-66.</ref><ref>Mellow, T.E., P.C. Murphy, J.L. Carson, T.L. Noah, L. Zhang, et al. 2004. The effect of respiratory synctial virus on chemokine release by differentiated airway epithelium. Exp Lung Res 30(1):43-57.</ref><ref>Zhang, L., A. Bukreyev, C.I. Thompson, B. Watson, M.E. Peeples, et al. 2005. Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium. J Virol 79(2):1113-24.</ref><ref>Thompson, C.I., W.S. Barclay, M.C. Zambon, and R.J. Pickles. 2006. Infection of human airway epithelium by human and avian strains of influenza A virus. J Virol 80(16):8060-8.</ref>. HPIV3 with a single amino acid mutation in the HN glycoprotein with better than wildtype growth in cell culture had a disadvantage in an ex vivo or in vivo system, revealing a gap in our understanding of the biology of these viruses in their natural host <ref>Palermo, L., M. Porotto, C. Yokoyama, S. Palmer, B. Mungall, et al. 2009. Human parainfluenza virus infection of the airway epithelium: the viral hemagglutinin-neuraminidase regulates fusion protein activation and modulates infectivity. J Virol 83(13):6900-6908.</ref>. This suggests that even slight variations in receptor types may influence HPIV infectivity. Recently a series of studies using glycoarray analysis started to navigate the complexity of the interaction between these viruses and glycomolecules <ref>Amonsen, M., D.F. Smith, R.D. Cummings, and G.M. Air. 2007. Human parainfluenza viruses hPIV1 and hPIV3 bind oligosaccharides with alpha2-3-linked sialic acids that are distinct from those bound by H5 avian influenza virus hemagglutinin. J Virol 81(15):8341-5.</ref>. The three functions of HN depend upon interaction with glycomolecules, therefore understanding whether glycomolecules are preferentially bound, cleave, or activate the fusion process will unravel the biology of these viruses and will help in developing targeted antivirals.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of parainfluenza virus type 3 HN include: Gillian Air, Theodore Jardetsky, Matteo Porotto, Charles Russell

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

Parainfluenza virus type 3 hemagglutinin-neuraminidase binds sialylated glycans. The sialic acid is linked α2-3 to galactose. The minimal binding motif is a pentasaccharide if there are no modifications, but smaller units bind if there is sulfation or fucosylation, as shown in the figure below <ref>Amonsen, M., D.F. Smith, R.D. Cummings, and G.M. Air. 2007. Human parainfluenza viruses hPIV1 and hPIV3 bind oligosaccharides with alpha2-3-linked sialic acids that are distinct from those bound by H5 avian influenza virus hemagglutinin. J Virol 81(15):8341-5.</ref>
 [[File: PIV3glycans.png]]
[[Media:Example.ogg]]

=== Cellular expression of GBP and ligands ===

Parainfluenza virus type 3 hemagglutinin-neuraminidase is expressed by HPIV paramyxoviruses that bind to sialic acid-containing receptor molecules on the surface of host lung cells.

=== Biosynthesis of ligands ===

 
=== Structure ===
The crystal structure of a hPIV3 HN has been determined in dimer form <ref>Lawrence, M.C., N.A. Borg, V.A. Streltsov, P.A. Pilling, V.C. Epa, et al. 2004. Structure of the Haemagglutinin-neuraminidase from Human Parainfluenza Virus Type III. J Mol Biol 335(5):1343-57.</ref> and serves as the model for glycan binding and neuraminidase studies.
 
The subunits are colored green and blue. A molecule of inhibitor 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid is bound to the active site of each subunit (stick model: C, O and N atoms are gray, red and blue respectively). The figure was made using PyMol (Delano Scientific) from PDB file 1V3D.
 
[[file: 1V3D.png]]
 

=== Biological roles of GBP-ligand interaction ===
hPIV HN plays important roles in several distinct steps associated with viral entry, which causes human respiratory infections.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=parainfluenza&maxresults=20 CFG database search results for "parainfluenza"].

=== Glycan profiling ===

 
=== Glycogene microarray ===
hPIV HN is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===

 
=== Glycan array ===
There have been many resource requests for glycan array screening of paramyxovirus hemagglutinin-neuraminidase (for example, click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2490 here]). To see all glycan array results for parainfluenza hemagglutinin-neuraminidase, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=parainfluenza&cat=coreh here].

== Related GBPs ==
* Other paramyxovirus HNs: some appear to have one site that carries out both activities; others appear to have separate sites. For glycan array results of other paramyxovirus HNs, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=paramyxovirus&cat=coreh here].
* Human parainfluenza types 1, 2, 4 and 5
* Newcastle Disease virus
* Mumps virus

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Gillian Air, James Paulson, Matteo Porotto

[[Category:Introduction]]

Parvovirus Minute Virus of Mice (MVM)

2011-03-30T23:24:36Z

Carole Weaver: /* Glycogene microarray */

The '''Parvoviridae''' is a family of small non-enveloped ssDNA viruses with a broad range of natural vertebrate and invertebrate hosts, including humans, monkeys, dogs, cats, mice, and insects. Pathogenic members cause severe disease in the young and immunocompromised adults. As examples, the newly discovered human bocavirus causes respiratory tract infections and gastroenteritis in young children and human parvovirus B19, which causes a mild rash in children infects, can cause acute severe or chronic anemia. Severe anemia due to B19 infection of an unborn baby can result in miscarriage in ~5% of pregnant women who are not immune to the virus. Non-pathogenic members, such as the Adeno-associated viruses (AAVs), are being developed for therapeutic gene deliver applications.

The ssDNA parvovirus genome, ~5000 bases, is packaged into a T=1 capsid that is ~260 Å in diameter<ref>Chapman, M.S. and M. Agbandje-McKenna, Atomic structure if viral particles., in Parvoviruses, J.R. Kerr, et al., Editors. 2006, Edward Aenold Ltd. New York: New York. p. 107-123.</ref>. The capsid is assembled from 60 copies (in total) of the common C-terminal region (~520 aa) of two to four overlapping capsid viral proteins (VPs), depending on the family member, with the larger proteins having N-terminal extensions which are not required for capsid assembly, but perform other functions in the life cycle, including a PLA2 domain required for endosomal escape during cellular trafficking. Thus these capsids are assembled from essentially one protein which is responsible for performing the multitude of functions required for successful host infection, including host cell receptor attachment, an essential first step in the life cycle<ref>Agbandje-McKenna, M. and M.S. Chapman, Correlating structure with function in the viral capsid, in Parvoviruses, J.R. Kerr, et al., Editors. 2006, Edward Arnold, New York: New York.</ref>. While the cell surface receptors are not known for the majority of the Parvoviridae, recognition of cell surface glycans, in the context of proteins or lipids, have been reported to be an important first step in infection. This glycan recognition also plays a role in (I) tissue tropism and pathogenicity differences between highly homologous strains for several pathogenic members and (II) tissue tropism and transduction efficiency in viral gene delivery vectors. Thus characterizing the interaction(s) of these viruses with their receptors is important for understanding the capsid determinants of tissue tropism and pathogenicity, and for manipulating the capsids for improved efficacy in corrective gene delivery applications. Minute Virus of Mice (MVM), is assembled from three VPs, VP1, VP2, and VP3, with the entire sequence of the smallest VP3 contained within VP2 which is in turn contained with VP1; VP1 has a unique N-terminal PLA2 region. VP3, which is only generated from VP2 following genome packaging, forms the majority of the capsid protein content, at ~90%, and contains the receptor recognition site. Significantly, this receptor-recognition site is only created in the assembled capsid with amino acid contributions from icosahedral symmetry related VPs<ref name="Kontou 2005">Kontou, M., L. Govindasamy, H.-J. Nam, N. Bryant, A. L. Llamas-Saiz, C. Foces-Foces, E. Hernando, M.-P Rubio, R. McKenna, J. M. Almendral., M. Agbandje-McKenna. 2005. Structural determinants of tissue tropism and in vivo pathogenicity for the parvovirus minute virus of mice. J. Virol., 79:10931-10943.</ref>.

The parvoviruses provide an excellent example of genomic economy in the coding of a “single” viral protein which assembles a multifunctional capsid, including a receptor recognition site. '''MVM''' has proved to be an ideal model for studying the capsid determinant of tissue tropism, pathogenicity, and host range adaption dictated by glycan receptor interaction(s) for this family. Interaction with cell surface sialic acid, in the context of a glycoprotein, is an essential first step in cellular recognition and tropism by MVM<ref name="Cotmore 1987">Cotmore, S. F., and P. Tattersall. 1987. The autonomously replicating parvoviruses of vertebrates. Adv. Virus Res. 33:91–174. </ref><ref name="Lopez-Bueno 2006">López-Bueno, A, M-P. Rubio, N. Bryant, R. McKenna, M. Agbandje-McKenna, J. M. Almendral. 2006. Host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence. J. Virol., 80: 1563-1573. </ref>. Two homologous MVM strains (MVMp, the prototype non-pathogenic strain and MVMi, the immunosuppressive pathogenic strain) that are 97% identical have pronounced differences in tissue tropism and in vivo pathogenesis<ref>Brownstein, D. G., A. L. Smith, R. O. Jacoby, E. A. Johnson, G. Hansen, and P. Tattersall. 1991. Pathogenesis of infection with a virulent allotropic variant of minute virus of mice and regulation by host genotype. Lab. Investig. 65:357–363.</ref><ref>Brownstein, D. G., A. L. Smith, E. A. Johnson, D. J. Pintel, L. K. Naeger, and P. Tattersall. 1992. The pathogenesis of infection with minute virus of mice depends on expression of the small nonstructural protein NS2 and on the genotype of the allotropic determinants VP1 and VP2. J. Virol. 66:3118– 3124. </ref><ref>Ramı´rez, J. C., A. Fiaren, and J. M. Almendral. 1996. Parvovirus minute virus of mice strain I multiplication and pathogenesis in the newborn mouse brain are restricted to proliferative areas and to migratory cerebral young neurons. J. Virol. 70:8109–8116.</ref><ref>Segovia, J. C., J. A. Bueren, and J. M. Almendral. 1995. Myeloid depression follows infection of susceptible newborn mice with the parvovirus minute virus of mice (strain i). J. Virol. 69:3229–3232.</ref><ref>Segovia, J. C., J. M. Gallego, J. A. Bueren, and J. M. Almendral. 1999. Severe leukopenia and dysregulated erythropoiesis in SCID mice persistently infected with the parvovirus minute virus of mice. J. Virol. 73:1774–1784. </ref>. Their phenotypes are associated with one or two VP amino acid differences resulting in local structural variations that alter MVM – infectious sialic acid receptor interactions and utilization<ref name="Nam 2006">Nam, H.-J., B. Gurda-Whitaker, W. Y. Gan, S. Ilaria, R. McKenna, P. Mehta, R. A. Alvarez, M. Agbandje-McKenna. 2006. Identification of the sialic acid structures recognized by minuterole of binding affinity in virulence adaptation. J. Bio. Chem., 281:25670-25677.</ref><ref name="Lopez-Bueno 2006"/>. Studies of the MVMp, in which virulent mutations were observed, also provided information on capsid adaptations, associated with altered receptor affinity, which confer a pathogenic phenotype<ref name="Nam 2006"/>. In addition, MVM has served as a model for studying capsid adaptations, involving the sialic acid receptor recognition site, which enables the infection of a new host<ref>Etingov I, R. Itah, M. Mincberg, A. Keren-Naus, H.-J. Nam, M. Agbandje-McKenna, C. Davis C. 2008. An extension of the Minute Virus of Mice tissue tropism. Virology, 379:245-255.</ref>. While it is known that a limited number of amino acids differences can also dictate tropism and pathogenicity disparities between homologous strains of other Parvoviridae members, unlike MVM, these have not been extensively studied with respect to the contribution of glycans interactions in dictating these differences.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) contributing to the understanding of MVM include: Mavis McKenna
* CFG PIs contributing to the understanding of AAV capsid include: Aravind Asokan, Regine Heilbronn

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
MVM interacts with cell surface sialic acid, in the context of a glycoprotein<ref name="Cotmore 1987"/><ref name="Lopez-Bueno 2006"/>. This glycan is also recognized by several members of the Parvoviridae, including H1 (rat parvovirus) and members of the AAV[13] (and unpublished data).
 

=== Cellular expression of GBP and ligands ===
MVM is a member of the Parvoviridae, a group of small non-enveloped ssDNA family of viruses with a T=1 icosahedral capsid. Members of this family infect a broad range of vertebrate and invertebrate hosts, including humans, dogs, cats, mice, and insects and have tropism for different tissues and organs In vitro MVM strains grow in mouse fibroblasts, T lymphocytes, and hematopoietic precursors. In vivo MVM replicates in many organs, including the kidneys, liver, and the brain (for MVM strain i)
 

=== Biosynthesis of ligands ===
Sialylated glycoproteins recognized by MVM are synthesized by host cells.
 

=== Structure ===

MVM is assembled from three VPs, VP1, VP2, and VP3, with the entire sequence of the smallest VP3 contained within VP2 which is in turn contained with VP1; VP1 has a unique N-terminal PLA2 region. VP3, which is only generated from VP2 following genome packaging, forms the majority of the capsid protein content, at ~90%, and contains the receptor recognition site. Significantly, this receptor-recognition site is only created in the assembled capsid with amino acid contributions from icosahedral symmetry related VPs<ref name="Kontou 2005"/>.
 [[File:MVMMonoCapsid.jpg]]
 
The glycan structures below have been observed to interact with MVM capsids using the CFG glycan array
 
[[File:structuresGD3SiaLN.jpg]]
 

=== Biological roles of GBP-ligand interaction ===
Glycan recognition by members of the Parvoviridae family dictates tissue tropism and is involved in tissue tropism and pathogenicity differences between highly homologous strains for several pathogenic members.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=parvovirus&maxresults=20 CFG database search results for "parvovirus"].

Three CFG resources have been used in the characterization of MVM glycan interactions and ''in vitro'' tissue tropism.
=== Glycan profiling ===
TThe CFG performed glycan profiling of three different cell lines, A9 fibroblasts, EL4T lymphocytes, and NB324K cells used for in vitro studies of MVM, in order to correlate the glycan screening results with the glycans present on the cells that are permissive to the MVM strains (unpublished data).

=== Glycogene microarray ===
MVM is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
N/A
 

=== Glycan array ===
The glycan recognition properties of the two MVM strains (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_35_08102004 here]), as well as virulent mutants arising from ''in vivo'' studies of MVMp, were further defined using the CFG glycan array<ref name="Nam 2006"/> (and unpublished data). These capsids were the first intact virus capsids to be screened by the CFG. This was done using plate arrays versions 2 and 3<ref name="Nam 2006"/>. The glycans identified in the array screening were then provided by the CFG for X-ray crystallographic studies to map the receptor binding site on the virus capsids (unpublished data). Mutations of capsid surface amino acids residues at the mapped binding site results in reduced sialic acid affinity<ref name="Lopez-Bueno 2006"/><ref name="Nam 2006"/>. CFG PIs have also used the CFG glycan array to screen AAV capsids. To see all glycan array results for MVM, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=MVM&cat=coreh here].

== Related GBPs ==
Other parvovirus GBPs have been studied by the CFG, including H1 (rat parvovirus) and several AAV serotypes: AAV1 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV1&maxresults=20 (CFG data)], AAV2, AAV4-AAV9<ref name="Wu 2006">Wu Z, E. Miller E, M. Agbandje-McKenna, R. J. Samulski. 2006. {alpha}2,3 and {alpha}2,6 N Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6. J. Virol., 80:9093-9103. </ref> (and unpublished data; CFG data: [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV5&maxresults=20 AAV5,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV6&maxresults=20 AAV6,] [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=AAV7&cat=coreh AAV7,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV8&maxresults=20 AAV8,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV9&maxresults=20 AAV9.]) Glycan array analyses have been used to identify glycans recognized by the capsids of these viruses, which includes terminal sialic acid containing oligosaccharides as well as those which terminate in galatose <ref name="Wu 2006"/>(and unpublished data).

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Mavis Agbandje-McKenna, Thilo Stehle

Reovirus hemagglutinin (sigma 1)

2011-03-30T23:24:06Z

Carole Weaver: /* Glycogene microarray */

'''Mammalian orthoreoviruses (reoviruses)''' are useful models for studies of viral receptor recognition and the pathogenesis of viral disease. Reovirus also efficiently lyses tumor cells in experimental animals<ref>Duncan, M.R., Stanish, S.M., and Cox, D.C. Differential sensitivity of normal and transformed human cells to reovirus infection. J. Virol. 28:444-449, 1978.</ref><ref> Coffey, M.C., Strong, J.E., Forsyth, P.A., and Lee, P.W. Reovirus therapy of tumors with activated Ras pathway. Science 282:1332-1334, 1998.</ref> and has shown efficacy in clinical trials for aggressive and refractory human tumors<ref>Stoeckel, J., and Hay, J.G. Drug evaluation: Reolysin--wild-type reovirus as a cancer therapeutic. Curr. Opin. Mol. Ther. 8:249-260, 2006.</ref><ref>Twigger, K., Vidal, L., White, C.L., De Bono, J.S., Bhide, S., Coffey, M., Thompson, B., Vile, R.G., Heinemann, L., Pandha, H.S., et al. Enhanced in vitro and in vivo cytotoxicity of combined reovirus and radiotherapy. Clin. Cancer Res. 14:912-923, 2008. </ref>. Reovirus forms double-shelled particles<ref>Dryden, K.A., Wang, G., Yeager, M., Nibert, M.L., Coombs, K.M., Furlong, D.B., Fields, B.N., and Baker, T.S. Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction. J. Cell Biol. 122:1023-1041, 1993.</ref> that contain a segmented dsRNA genome. The reovirus sigma 1 protein is a long, fiber-like molecule that extends from the virion surface<ref>Furlong, D.B., Nibert, M.L., and Fields, B.N. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles. J. Virol. 62:246-256, 1988.</ref> and mediates viral attachment<ref>Weiner, H.L., Ault, K.A., and Fields, B.N. Interaction of reovirus with cell surface receptors. I. Murine and human lymphocytes have a receptor for the hemagglutinin of reovirus type 3. J. Immunol. 124:2143-2148, 1980.</ref><ref>Lee, P.W.K., Hayes, E.C., and Joklik, W.K. Protein σ1 is the reovirus cell attachment protein. Virology 108:156-163, 1981.</ref>. The three human serotypes (T1, T2, and T3) differ in cell tropism, which correlates directly with receptor-binding properties of sigma 1. Sialic acid serves as an essential receptor for T3 reovirus on murine erythroleukemia (MEL) cells<ref name="Rubin1992">Rubin, D.H., Wetzel, J.D., Williams, W.V., Cohen, J.A., Dworkin, C., and Dermody, T.S. Binding of type 3 reovirus by a domain of the σ1 protein important for hemagglutination leads to infection of murine erythroleukemia cells. J. Clin. Invest. 90:2536-2542, 1992.</ref>, and it functions as a coreceptor on murine L929 (L) cells<ref name="Gentsch1985">Gentsch, J.R., and Pacitti, A.F. Effect of neuraminidase treatment of cells and effect of soluble glycoproteins on type 3 reovirus attachment to murine L cells. J. Virol. 56:356-364, 1985.</ref><ref name="Pacitti1987">Pacitti, A., and Gentsch, J.R. Inhibition of reovirus type 3 binding to host cells by sialylated glycoproteins is mediated through the viral attachment protein. J. Virol. 61:1407-1415, 1987. </ref><ref name="name4">.</ref><ref name="Rubin1992"></ref><ref name="Nibert1995">Nibert, M.L., Chappell, J.D., and Dermody, T.S. Infectious subvirion particles of reovirus type 3 Dearing exhibit a loss in infectivity and contain a cleaved σ1 protein. J. Virol. 69:5057-5067, 1995.</ref>. Residues involved in sialic acid-binding map to the center of the long fiber, close to the midpoint of the molecule<ref name="Chappell2002">Chappell, J.D., Prota, A., Dermody, T.S., and Stehle, T. Crystal structure of reovirus attachment protein σ1 reveals evolutionary relationship to adenovirus fiber. EMBO J. 21:1-11, 2002.</ref>, in a repetitive structural region known as the triple β-spiral. The T1 sigma 1 protein binds to cell-surface glycans of unknown structure.

The triple β-spiral of '''sigma 1''' functions as a trimerization domain and defines a novel carbohydrate-recognition motif. Other carbohydrate-recognition domains, such as those of the C-type lectin superfamily<ref>Weis, W.I., Taylor, M.E., and Drickamer, K. The C-type lectin superfamily in the immune system. Immunol. Rev. 163:19-34, 1998.</ref> or the sialic acid-binding domains in the Siglec family of adhesion proteins<ref>Crocker, P.R., and Varki, A. Siglecs in the immune system. Immunology 103:137-145, 2001.</ref> (see [http://glycobank.mit.edu/glycoWiki/Main_Page Siglec paradigms]), have been described, but none are formed by a repetitive, fiber-like structure such as the one present in sigma 1. In fact, the domain in sigma 1 that binds sialic acid constitutes a carbohydrate-binding “cassette” that could be endowed with altered ligand-binding properties or grafted onto other trimeric structures and used to create avidity for carbohydrates. For example, the adenovirus fiber shaft could be licensed with sialic acid-binding capacity using this approach. These properties render the sigma 1 protein unique among the structurally known glycan-binding moieties.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of sigma 1 include: Terence Dermody, Thilo Stehle

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
Reovirus strains of all three serotypes are capable of binding to carbohydrates, which is a property mediated by viral attachment protein sigma 1<ref name="Chappell2000">Chappell, J.D., Duong, J.L., Wright, B.W., and Dermody, T.S. Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses. J. Virol. 74:8472-8479, 2000.</ref>. Substantial evidence indicates that serotype 3 reoviruses bind to sialic acid, whereas the identity of carbohydrates bound by serotype 1 and 2 reoviruses is less clear. Serotype 3 reoviruses bind to alpha-linked sialic acid in either alpha2,3 or alpha2,6 linkages on a variety of cell types<ref name="Chappell2000">.</ref><ref name="Gentsch1985">.</ref><ref name="name4">Paul, R.W., Choi, A.H., and Lee, P.W.K. The a-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus. Virology 172:382-385, 1989.</ref><ref>Dermody, T.S., Nibert, M.L., Bassel-Duby, R., and Fields, B.N. Sequence diversity in S1 genes and S1 translation products of 11 serotype 3 reovirus strains. J. Virol. 64:4842-4850, 1990.</ref><ref name="Chappell1997">Chappell, J.D., Gunn, V.L., Wetzel, J.D., Baer, G.S., and Dermody, T.S. Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein s1. J. Virol. 71:1834-1841, 1997.</ref>. Hemagglutination by serotype 3 reoviruses is mediated by interactions with alpha-linked sialic acid on several glycosylated erythrocyte proteins such as glycophorin A<ref>Gentsch, J.R., and Pacitti, A.F. Differential interaction of reovirus type 3 with sialylated receptor components on animal cells. Virology 161:245-248, 1987.</ref><ref>Paul, R.W., and Lee, P.W.K. Glycophorin is the reovirus receptor on human erythrocytes. Virology 159:94-101, 1987.</ref>. Reovirus strain T3D binds to sialoglycophorin, but not to asialoglycophorin, with an avidity of ~ 5 x 10e-9 M<ref name="Barton2001a">Barton, E.S., Connolly, J.L., Forrest, J.C., Chappell, J.D., and Dermody, T.S. Utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening. J. Biol. Chem. 276:2200-2211, 2001.</ref>.

Serotype 1 reoviruses also appear to bind to sialic acid in some contexts. Reovirus strain T1L, but not strain T3D, binds to the apical surface of microfold (M) cells, but not to enterocytes, in tissue sections of rabbit Peyer’s patches<ref name="name10">Helander, A., Silvey, K.J., Mantis, N.J., Hutchings, A.B., Chandran, K., Lucas, W.T., Nibert, M.L., and Neutra, M.R. The viral s1 protein and glycoconjugates containing a2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces. J. Virol. 77:7964-7977, 2003.</ref>. Binding is inhibited by pre-incubation of the tissue sections with neuraminidase or with lectins that specifically recognize alpha2-3-linked sialic acid.

=== Cellular expression of GBP and ligands ===
Sialic acid serves as an essential receptor for type 3 reoviruses on murine erythroleukemia (MEL) cells<ref name="Chappell1997">.</ref><ref name="Rubin1992"></ref>. Sialic acid also functions as a coreceptor on murine L929 cells<ref name="Gentsch1985">.</ref><ref name="name4">.</ref><ref name="Rubin1992">.</ref><ref name="Pacitti1987">.</ref><ref name="Nibert1995">.</ref> and human HeLa cells<ref name="Barton2001a">.</ref>. Although not all serotype 3 strains are capable of binding to sialic acid, the majority bind to this carbohydrate.

Serotype 1 reoviruses are incapable of infecting MEL cells, which support infection only by sialic-acid-binding strains<ref name="Rubin1992">.</ref>. Serotype 1 reoviruses also are insensitive to the growth-inhibitory effects of neuraminidase treatment of L929 cells<ref name="Nibert1995">.</ref>. However, binding of serotype 1 reoviruses to intestinal M cells is diminished by neuraminidase treatment<ref name="name10">.</ref>. The explanation for this discrepancy is not known.

Reovirus employs a multi-step mechanism of viral attachment in which a low-affinity interaction with sialic acid serves to tether the virion to target cells and precedes a high-affinity interaction with JAM-A<ref name="Barton2001a">.</ref>, an immunoglobulin superfamily protein engaged by reovirus<ref name="Barton2001b">Barton, E.S., Forrest, J.C., Connolly, J.L., Chappell, J.D., Liu, Y., Schnell, F., Nusrat, A., Parkos, C.A., and Dermody, T.S. Junction adhesion molecule is a receptor for reovirus. Cell 104:441-451, 2001.</ref><ref>Prota, A.E., Campbell, J.A., Schelling, P., Forrest, J.C., Peters, T.R., Watson, M.J., Aurrand-Lions, M., Imhof, B., Dermody, T.S., and Stehle, T. Crystal structure of human junctional adhesion molecule 1: implications for reovirus binding. Proc. Natl. Acad. Sci. U. S. A. 100:5366-5371, 2003.</ref><ref>Campbell, J.A., Shelling, P., Wetzel, J.D., Johnson, E.M., Wilson, G.A.R., Forrest, J.C., Aurrand-Lions, M., Imhof, B., Stehle, T., and Dermody, T.S. Junctional adhesion molecule-A serves as a receptor for prototype and field-isolate strains of mammalian reovirus. J. Virol. 79:7967-7978, 2005.</ref>. By virtue of its rapid association rate, virus binding to sialic acid adheres the virion to the cell surface, thereby enabling it to diffuse laterally until it encounters JAM-A. Such lateral diffusion has been reported for influenza virus<ref>Sagik, B., Puck, T., and Levine, S. Quantitative aspects of the spontaneous elution of influenza virus from red cells. J. Exp. Med. 99:251-260, 1954.</ref> and phage T4<ref>Wilson, J.H., Luftig, R.B., and Wood, W.B. Interaction of bacteriophage T4 tail fiber components with a lipopolysaccharide fraction from Escherichia coli. J. Mol. Biol. 51:423-434, 1970.</ref>. After attachment, reovirus is internalized by receptor-mediated endocytosis<ref>Borsa, J., Morash, B.D., Sargent, M.D., Copps, T.P., Lievaart, P.A., and Szekely, J.G. Two modes of entry of reovirus particles into L cells. J. Gen. Virol. 45:161-170, 1979.</ref><ref>Borsa, J., Sargent, M.D., Lievaart, P.A., and Copps, T.P. Reovirus: evidence for a second step in the intracellular uncoating and transcriptase activation process. Virology 111:191-200, 1981.</ref><ref>Ehrlich, M., Boll, W., Van Oijen, A., Hariharan, R., Chandran, K., Nibert, M.L., and Kirchhausen, T. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118:591-605, 2004.</ref><ref name="Maginnis2006">Maginnis, M.S., Forrest, J.C., Kopecky-Bromberg, S.A., Dickeson, S.K., Santoro, S.A., Zutter, M.M., Nemerow, G.R., Bergelson, J.M., and Dermody, T.S. b1 integrin mediates internalization of mammalian reovirus. J. Virol. 80:2760-2770, 2006.</ref><ref>Sturzenbecker, L.J., Nibert, M.L., Furlong, D.B., and Fields, B.N. Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle. J. Virol. 61:2351-2361, 1987.</ref><ref name="Maginnis2008">Maginnis, M.S., Mainou, B.A., Derdowski, A.M., Johnson, E.M., Zent, R., and Dermody, T.S. NPXY motifs in the b1 integrin cytoplasmic tail are required for functional reovirus entry. J. Virol. 82:3181-3191, 2008.</ref> using a mechanism dependent on beta 1 integrin<ref name="Maginnis2006">.</ref><ref name="Maginnis2008">.</ref>.
=== Biosynthesis of ligands ===
 
=== Structure ===
Structural analysis of the C-terminal half of reovirus attachment protein sigma 1 from strain T3D (residues 246-455) reveals a trimeric structure, in which each monomer is composed of a slender tail and a compact head<ref name="Chappell2002">.</ref>. The C-terminal residues that form the head domain (310-455) consist of two Greek-key motifs that fold into a beta-barrel. The sigma 1 head domain binds to JAM-A<ref name="Barton2001b">.</ref><ref>Kirchner, E., Guglielmi, K.M., Strauss, H.M., Dermody, T.S., and Stehle, T. Structure of reovirus s1 in complex with its receptor junctional adhesion molecule-A. PLoS Path. 4:e1000235, 2008.</ref>. N-terminal residues in the crystallized fragment form a portion of the tail, residues 246-309, which consists of three beta-spiral repeats. Each repeat is composed of two short beta-strands connected by a four-residue beta-turn that has either a proline or a glycine residue at its third position<ref name="Chappell2002">.</ref>. A surface-exposed, variable loop links successive repeats, and trimerization generates an unusual triple beta-spiral motif that also is observed in the adenovirus fiber<ref>van Raaij, M.J., Mitraki, A., Lavigne, G., and Cusack, S. A triple b-spiral in the adenovirus fibre shaft reveals a new structural motif for a fibrous protein. Nature 401:935-938, 1999.</ref>, bacteriophage PRD1 P5 protein<ref>Merckel, M.C., Huiskonen, J.T., Bamford, D.H., Goldman, A., and Tuma, R. The structure of the bacteriophage PRD1 spike sheds light on the evolution of viral capsid architecture. Mol. Cell 18:161-170, 2005.</ref>, and avian reovirus attachment protein sigma C<ref>Guardado, C.P., Fox, G.C., Hermo Parrado, X.L., Llamas-Saiz, A.L., Costas, C., Martinez-Costas, J., Benavente, J., and van Raaij, M.J. Structure of the carboxy-terminal receptor-binding domain of avian reovirus fibre sigmaC. J. Mol. Biol. 354:137-149, 2005.</ref>.

In addition to the three beta-spiral repeats observed in the crystallized sigma 1 fragment, sequence analysis suggests that residues 167-249 in the T3D sigma 1 tail form an additional five N-terminal beta-spiral repeats<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">Guglielmi, K.M., Johnson, E.M., Stehle, T., and Dermody, T.S. Attachment and cell entry of mammalian orthoreovirus. Curr. Top. Microbiol. Immunol. 309:1-38, 2006.</ref>. Alternatively, these residues may form a combination of beta-spiral repeats and alpha-helical coiled-coil, as suggested by sequence analysis<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">.</ref><ref name="Nibert1990">Nibert, M.L., Dermody, T.S., and Fields, B.N. Structure of the reovirus cell-attachment protein: a model for the domain organization of s1. J. Virol. 64:2976-2989, 1990.</ref> and an observed narrowing in this region in a composite negative-stain electron micrograph<ref>Fraser, R.D.B., Furlong, D.B., Trus, B.L., Nibert, M.L., Fields, B.N., and Steven, A.C. Molecular structure of the cell-attachment protein of reovirus: correlation of computer-processed electron micrographs with sequence-based predictions. J. Virol. 64:2990-3000, 1990.</ref>. The structure of N-terminal residues 1-160 of sigma 1 is unknown. However, a repeating heptad sequence motif is predictive of an amphipathic alpha-helix, which likely assembles into an alpha-helical coiled-coil in the trimer<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">.</ref><ref name="Nibert1990">.</ref>.

Although the structure of sigma 1 in complex with sialic acid is not yet available, studies using expressed proteins indicate that the region of T3D sigma 1 required for sialic binding resides near the midpoint of the tail, whereas a region just N-terminal to the head domain of T1L sigma 1 binds to carbohydrate<ref name="Chappell2000">.</ref>. In both T1L and T3D sigma 1, interactions with carbohydrate are mediated by a region of predicted beta-spiral<ref name="Chappell2002">.</ref>. Adaptation of non-sialic-acid-binding reoviruses to growth in MEL cells results in amino acid substitutions at residues 198, 202, and 204 of sigma 1 that confer sialic-acid-binding capacity on the resultant viruses<ref name="Chappell1997">.</ref>. Molecular modeling of the sigma 1 tail, based on available structure and sequence data, suggests that these residues are surface-exposed and proximal to one another in the predicted beta-spiral region<ref name="Chappell2002">.</ref>. Thus, residues 198, 202, and 204 are likely to contribute to a sialic-acid-binding site in T3D sigma 1.

=== Biological roles of GBP-ligand interaction ===
Sialic acid binding serves an important role in reovirus tropism and pathogenesis in vivo<ref>Barton, E.S., Youree, B.E., Ebert, D.H., Forrest, J.C., Connolly, J.L., Valyi-Nagy, T., Washington, K., Wetzel, J.D., and Dermody, T.S. Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease. J. Clin. Invest. 111:1823-1833, 2003.</ref>. Strain T3SA+, which binds to sialic acid, and strain T3SA-, which does not bind to this carbohydrate, produce equivalent titers in the intestine of newborn mice following peroral inoculation. However, T3SA+ spreads more rapidly from the intestine to sites of secondary replication and produces higher titers in the spleen, liver, and brain. Mice infected with T3SA+, but not T3SA-, develop clinical evidence of bile duct obstruction including jaundice and steatorrhea. Liver tissue from mice infected with T3SA+ show intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in human infants, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ binds 100-fold more efficiently than T3SA- to human cholangiocarcinoma cells. Thus, binding to sialic acid targets reovirus to bile duct epithelium and produces a disease reminiscent of infantile biliary atresia.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=reovirus&maxresults=20 CFG database search results for "reovirus"].

=== Glycan profiling ===
Not performed.

=== Glycogene microarray ===
Sigma 1 is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.

=== Knockout mouse lines ===
Not applicable.

=== Glycan array ===
Experiments in progress. No definitive results have been obtained thus far.

== Related GBPs ==
The attachment protein of adenovirus, fiber, is a structural homolog of sigma 1. At least one adenovirus serotype (Ad37; [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=ad37&maxresults=20 CFG data]) is known to bind glycan receptors via residues in the fiber protein<ref>Burmeister WP, Guilligay D, Cusack S, Wadell G, Arnberg N: Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites. Journal of Virology 78:7727-7736, 2004.</ref>. The actual binding site is not homologous. However, information about reovirus glycan binding could also be used to engineer adenovirus fiber proteins (or other trimeric fiber-like proteins) that possess novel glycan-binding properties.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Terence Dermody, Mavis McKenna, Thilo Stehle.

Polyomavirus capsid protein (VP1)

2011-03-30T23:23:38Z

Carole Weaver: /* Glycogene microarray */

'''Polyomaviruses''' are a group of small, non-enveloped DNA viruses that can infect birds, rodents, and primates. Members of the group include simian virus 40 (SV40) and murine polyomavirus (mPyV) as well as a number of human polyomaviruses such as the BK and JC viruses (BKV and JCV, respectively). Recently, a new human polyomavirus was found to be linked to Merkel cell carcinoma, an aggressive type of skin cancer<ref name=" Feng H1096">
Feng H, Shuda M, Chang Y, Moore PS: Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 2008, 319:1096-1100.</ref>. All polyomavirus capsids are constructed from 360 copies of the major coat protein, VP1, arranged in pentamers on a T=7 icosahedral lattice<ref>Liddington RC, Yan Y, Moulai J, Sahli R, Benjamin TL, Harrison SC: Structure of simian virus 40 at 3.8-A resolution. Nature 1991, 354:278-284.</ref>. The cell-surface receptors for SV40, mPyV, BKV, JCV, and possibly other polyomaviruses are gangliosides, which are complex, sialic acid-containing sphingolipids that reside primarily in lipid rafts. SV40 uses the ganglioside GM1, BKV binds GD1b and GT1b, and mPyV attaches to GD1a and GT1b<ref name=" Tsai B4346 ">Tsai B, Gilbert JM, Stehle T, Lencer W, Benjamin TL, Rapoport TA: Gangliosides are receptors for murine polyoma virus and SV40. Embo J 2003, 22:4346-4355.</ref><ref name=" Low JA1361 ">Low JA, Magnuson B, Tsai B, Imperiale MJ: Identification of gangliosides GD1b and GT1b as receptors for BK virus. J Virol 2006, 80:1361-1366.</ref>. The glycan-binding properties of the human polyomaviruses are currently being investigated.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of VP1 include: Niklas Arnberg, Ten Feizi, Thilo Stehle

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

The polyomaviruses use a range of glycans for attachment and cell entry, depending on the virus type. For the simian virus SV40 and murine polyomavirus, the receptors have been identified as gangliosides GM1 and GT1b/GD1a<ref name=" Tsai B4346 "></ref>, respectively. The human BK polyomavirus binds to sialylated glycans that include gangliosides GD1b and GT1b <ref name=" Low JA1361 "></ref><ref name=" Dugan AS 14442">Dugan AS, Eash S, Atwood WJ (2005) An N-linked glycoprotein with alpha(2,3)-linked sialic acid is a receptor for BK virus. J Virol 79: 14442-14445.</ref>. The human JC polyomavirus uses sialylated glycans<ref>Dugan AS, Gasparovic ML, Atwood WJ (2008) Direct correlation between sialic acid binding and infection of cells by two human polyomaviruses (JC virus and BK virus). J Virol 82: 2560-2564.</ref><ref>Komagome R, Sawa H, Suzuki T, Suzuki Y, Tanaka S, et al. (2002) Oligosaccharides as receptors for JC virus. J Virol 76: 12992-13000.</ref><ref>Liu CK, Wei G, Atwood WJ (1998) Infection of glial cells by the human polyomavirus JC is mediated by an N-linked glycoprotein containing terminal alpha(2-6)-linked sialic acids. J Virol 72: 4643-4649.</ref>as well as the serotonin receptor 5HT2aR<ref>Elphick GF, Querbes W, Jordan JA, Gee GV, Eash S, et al. (2004) The human polyomavirus, JCV, uses serotonin receptors to infect cells. Science 306: 1380-1383.</ref>as attachment receptors. The receptors for other polyomaviruses, such as the recently identified Merkel Cell Polyomavirus<ref name=" Feng H1096"></ref>, have not been characterized, but at least some of these are likely to also use glycans for cell attachment. Participating investigators (PIs) of the CFG have made major contributions to our understanding of the structural and functional basis of attachment of polyomaviruses to their receptors<ref name=" Tsai B4346 "></ref><ref name=" Stehle T 5139">Stehle T, Harrison SC: High-resolution structure of a polyomavirus VP1-oligosaccharide complex: implications for assembly and receptor binding. The EMBO Journal 1997, 16:5139-5148.</ref><ref>Stehle T, Harrison SC (1996) Crystal structures of murine polyomavirus in complex with straight-chain and branched-chain sialyloligosaccharide receptor fragments. Structure, Folding and Design 4: 183-194.</ref><ref name=" Neu U 5219">Neu U, Woellner K, Gauglitz G, Stehle T: Structural basis of GM1 ganglioside recognition by simian virus 40. Proc Natl Acad Sci U S A 2008, 105:5219-5224.</ref><ref> Neu U, Stehle T, Atwood WJ (2009) The Polyomaviridae: Contributions of virus structure to our understanding of virus receptors and infectious entry. Virology 384: 389-399.</ref>

=== Cellular expression of GBP and ligands ===

Varies, depending on virus type:
 
JC Polyomavirus: persists in the kidney <ref> Dorries K (1998) Molecular biology and pathogenesis of human polyomavirus infections. Dev Biol Stand 94: 71-79.</ref>. In immunocompromised individuals, the virus infects glial cells, including astrocytes and the myelin-producing oligodendrocytes, resulting in the fatal disease PML (Progressive Multifocal Leukoecenlopathy) <ref>Silverman L, Rubinstein LJ (1965) Electron microscopic observations on a case of progressive multifocal leukoencephalopathy. Acta Neuropathol 5: 215-224.</ref><ref> Seth P, Diaz F, Major EO (2003) Advances in the biology of JC virus and induction of progressive multifocal leukoencephalopathy. J Neurovirol 9: 236-246.</ref><ref> Khalili K, White MK (2006) Human demyelinating disease and the polyomavirus JCV. Mult Scler 12: 133-142.</ref>.
 
BK Polyomavirus: Infects genitourinary tract <ref> Hirsch HH, Steiger J (2003) Polyomavirus BK. Lancet Infect Dis 3: 611-623.</ref><ref> Shinohara T, Matsuda M, Cheng SH, Marshall J, Fujita M, et al. (1993) BK virus infection of the human urinary tract. J Med Virol 41: 301-305.</ref><ref> Nickeleit V, Hirsch HH, Binet IF, Gudat F, Prince O, et al. (1999) Polyomavirus infection of renal allograft recipients: from latent infection to manifest disease. J Am Soc Nephrol 10: 1080-1089.</ref>, causes PVN (Polyomavirus-Associated Nephropathy).

=== Biosynthesis of ligands ===

Gangliosides, sialylated oligosaccharides are synthesized by the host.

=== Structure ===
Crystal structures are available for complete mPyV particles and for mPyV VP1 pentamers in complex with ganglioside receptor fragments<ref>Stehle T, Yan Y, Benjamin TL, Harrison SC: Structure of murine polyomavirus complexed with an oligosaccharide receptor fragment. Nature 1994, 369:160-163.</ref><ref name=" Stehle T 5139"></ref> as well as for the SV40 VP1 pentamer in complex with GM1 <ref name=" Neu U 5219"></ref>. The available structures show that '''VP1''' forms a scaffold that can modulate the specificity of interaction through small changes in surface loops. We plan to generate a set of perhaps five different structures that highlight conserved as well as non-conserved interactions with different gangliosides, thereby providing a platform for understanding and altering receptor-binding properties. This work is important, as there are few known examples of viral proteins from non-enveloped viruses with a common fold for which subtle modulations of surface properties result in altered glycan-binding specificities. 

A selection of gangliosides is shown below. GM1, GD1a, GD1b and GT1b are used by several polyomaviruses as receptors.
 
[[File:gangliosidesGM1.jpg]]

=== Biological roles of GBP-ligand interaction ===

 
Cell attachment, required for entry and infectivity, determinant of tropism<ref name=" Tsai B4346 "></ref> <ref name=" Dugan AS 14442"></ref><ref name=" Low JA1361 "></ref>. Recent data show that the structure of ganglioside GM1 determines SV40-induced membrane invagination and infection<ref>Ewers H, Romer W, Smith AE, Bacia K, Dmitrieff S, et al. (2010) GM1 structure determines SV40-induced membrane invagination and infection. Nat Cell Biol 12: 11-18; sup pp 11-12.</ref>. Subsequent entry processes depend for many polyomaviruses on proteins in the endoplasmic reticulum<ref> Schelhaas M, Malmstrom J, Pelkmans L, Haugstetter J, Ellgaard L, et al. (2007) Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells. Cell 131: 516-529.</ref>.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=polyoma&maxresults=20 CFG database search results for "polyoma"].

=== Glycan profiling ===

 
N/A

=== Glycogene microarray ===
VP1 is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===

 
None

=== Glycan array ===
The CFG glycan array was used to determine the [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2_405_07312006 ligand specificity for SV40]. This information was then used to crystallize SV40 VP1 in complex with the oligosaccharide portion of the GM1 gangloside. To see all glycan array results for VP1, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=VP1&cat=coreh here].

== Related GBPs ==
Functionally (but not structurally) related are the pentameric B proteins of the AB5-type toxins, such as [[Subtilase cytotoxin (SubAB)]]. These also interact with gangliosides.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Mavis McKenna, Thilo Stehle

C. difficile toxin A (TcdA)

2011-03-30T23:23:07Z

Carole Weaver: /* Glycogene microarray */

'''The large clostridial cytotoxins (LCT)''' are a family of structurally and functionally related exotoxins from ''Clostridium difficile'' (toxins A and B [also termed TcdA and TcdB, respectively), ''C. sordellii'' (lethal and hemorrhagic toxin) and ''C. novyi'' (alpha-toxin). The LCTs are major virulence factors, which in addition to their in vivo effects, are cytotoxic to cultured cell lines, causing reorganization of the cytoskeleton accompanied by morphological changes<ref name="Just 1">Just. I., Gerhard, R. (2004). Large clostridial cytotoxins. Rev. Physiol. Biochem. Pharmacol. 152:23-47.</ref>. The LCTs are single-chain protein toxins, comprising three domains: receptor-binding, translocation and catalysis, which mediate cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and enzymatic cytotoxic activity, respectively. Enzymatic activity involves transfer of a glucosyl moiety from UDP-glucose (or the N-acetyl-glucosaminyl moiety from UDP-GlcNAc in the case of alpha toxin) to a conserved threonine within the effector regions of the intracellular Rho and Ras GTPases<ref name="Just 1"/>. The C-terminal receptor-binding domain comprises up to one third of the LCT molecule, and contains repetitive peptide elements called combined repetitive oligopetides (CROPs). The repeating units binding regions of certain streptococcal glycosyltransferases<ref name="Just 1"/>.

'''TcdA''' is the better characterized of the two LCTs produced by ''C. difficile.'' Collectively, TcdA and TcdB are directly responsible for the increasingly common and serious human gastrointestinal disease caused when antibiotic treatment enables ''C. difficile'' to out-compete commensal gut microflora. Recently, the crystal structure of a C-terminal fragment of TcdA has been solved, revealing a solenoid-like structure, which consists of 32 short repeats with 15–21 residues and seven long repeats with 30 residues<ref name="Jank 2007">Thomas Jank, Torsten Giesemann and Klaus Aktories. (2007). Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function. Glycobiology. 17: 15R–22R.
</ref>. Solenoid structures are often found in bacterial surface proteins, and their extended surface allows protein–protein or protein–carbohydrate interactions. TcdA is known to bind to Galα1-3Galβ1-4GlcNAc- glycans, although this structure is not present in humans. However, it is also known to recognize a variety of other structures with a Galβ[1→4]GlcNAcβ- backbone, including the fucosylated blood group antigens Lewisx and Lewisy, both of which are present in human intestinal epithelium<ref name="Voth 2005">Voth, D. E., and Ballard, J. D. (2005). Clostridium difficile toxins: mechanism of action and role in disease. Clin. Microbiol. Rev. 18, 247-263.</ref>. Clearly, a better understanding of the identity/structure of the human receptor(s) for these toxins will facilitate design of novel therapeutics capable of blocking toxin binding. Glycan array analysis conducted by the CFG showed that TcdA bound most strongly to glycans terminating in Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4-GlcNAc-, which mimics the blood group antigen Lewisa; other bound structures included Galα1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ-, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-, and Fucαa1-2[GalNAcαa1-3]Galβ1-4GlcNAcβ1-3Galβb1-4GlcNAcβ-.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) contributing to the understanding of TcdA include: Borden Lacy, Yashwant Mahida, Kenneth Ng
* Non-CFG PIs include: Brian Dieckgraefe

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
*Galα1-3Galβ1-4GlcNAc- glycans (not present in humans). 
*Other structures with a Galβ[1→4]GlcNAcβ- backbone, including the fucosylated blood group antigens Lewisx and Lewisy, both of which are present in human intestinal epithelium<ref name="Voth 2005"/>. 
*Strongest binding: glycans terminating in Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4-GlcNAc-, which mimics the blood group antigen Lewisa. 
*Also binds: Galα1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ-, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-, and Fucαa1-2[GalNAcαa1-3]Galβ1-4GlcNAcβ1-3Galβb1-4GlcNAcβ-. 
 
=== Cellular expression of GBP and ligands ===
TcdA is produced by ''Clostridium difficile''. Structurally and functionally related exotoxins in the LCT family are produced by ''C. sordellii'' (lethal and hemorrhagic toxin) and ''C. novyi'' (alpha-toxin).
 
=== Biosynthesis of ligands ===

 
=== Structure ===
Members of the LCT family are single-chain protein toxins with receptor-binding, translocation and catalysis domains, which mediate cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and enzymatic cytotoxic activity, respectively. Up to one third of the LCT molecule is comprised of the C-terminal receptor-binding domain, which contains repetitive peptide elements called combined repetitive oligopeptides (CROPs).
 
X-ray crystallography has revealed the three-dimensional structures of several recombinant fragments of TcdA and related toxins<ref name="Jank 2007"/>. The N-terminal domain consists of a GT-A family glucosyltransferase that catalyzes the transfer of glucose from UDP-glucose to specific threonine residues of small GTPases regulating the structure and dynamics of the cytoskeleton. Although the three-dimensional structure of the GT domain of TcdA has not been solved by crystallography, the structures of closely related GT domains from TcdB and the letahal toxin from ''Clostridium sordellii'' have been determined.
 
The crystallographic structure of a cysteine protease domain bound to inositol hexakisphosphate has also been determined. This domain follows the N-terminal GT domain and is responsible for the autoproteolysis of TcdA that is important for releasing the GT domain into the cytoplasm.
 
Crystallographic structures of several recombinant fragments from the highly repetitive C-terminal domain have revealed an extended beta-solenoid structure that presents seven binding sites for complex oligosaccharide receptors. The bulk of this carbohydrate-binding domain is comprised of multiple copies of short repeats that interspersed with single copies of long repeats. Carbohydrate-binding sites appear to be formed at the junctions of long repeats and the preceding and following short repeats.
 
Crystallographic structures of recombinant fragments of TcdA have been determined with oligosaccharides, including the Gal-α(1,3)-LacNAc trisaccharide shown below<ref name = "Greco 2006">Greco, A et al. (2006). Carbohydrate recognition by ''Clostridium difficile'' toxin A. Nat. Struct. Mol. Biol. 13:460-461.</ref>. This trisaccharide was determined to be a likely native receptor for TcdA in the brush border membranes of intestinal epithelial cells from hamster<ref name = "Krivan 1986">Krivan, HC et al (1986). Cell-surface binding site for ''Clostridium difficile'' enterotoxin: evidence for a glycoconjugate containing the sequence Galα1,3-Galβ1,4-GlcNAc. Infect. Imm. 53:573-581.</ref>, an organism which is susceptible to ''C. difficile'' infection (CDI) and is often used as a model for studying the disease.
 
[[File:CD_fig1.png]]

=== Biological roles of GBP-ligand interaction ===
TcdA and TcdB, the other LCT produced by ''C. difficile'', cause the serious human gastrointestinal disease that results when antibiotic treatment enables ''C. difficile'' to out-compete commensal gut microflora. The LCTs are major virulence factors, which in addition to their in vivo effects, are cytotoxic to cultured cell lines, causing reorganization of the cytoskeleton accompanied by morphological changes<ref name="Just 1"/>.
 
== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=difficile&maxresults=20 CFG database search results for "difficile"].

=== Glycan profiling ===

 
=== Glycogene microarray ===
TcdA is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG glycan array has been used to examine the [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2693 glycan-binding specificity of TcdA]. In addition, the CFG's glycan synthesis capability is being used to prepare synthetic oligosaccharides indentified in the glycan array screens (with or without biotin tags) to further probe TcdA-receptor interactions.

== Related GBPs ==
''C. difficile'' toxin B (TcdB; [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=TcdB&maxresults=20 CFG data]), ''C. sordellii'' lethal toxin and hemorrhagic toxin, ''C. novyi'' alpha-toxin.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, Kenneth Ng, James Paton

Botulinum toxin serotype A (BoNT/A)

2011-03-30T23:22:41Z

Carole Weaver: /* Glycogene microarray */

'''The clostridial neurotoxins''' are the most lethal protein toxins for humans and recently have been utilized as therapeutic agents to treat numerous human neurological inflictions. The Botulinum neurotoxins produced by ''Clostridium botulinum'' and Tetanus toxin produced by ''C. tetani'' are members of the family of clostridial neurotoxins. The clostridial neurotoxins are di-chain toxins with the N-terminal catalytic domains (Light Chain, LC) possessing metalloprotease activity that is disulfide linked to the C-terminal
domain (Heavy Chain, HC). The neurological toxicity and therapeutic utility of the clostridial neurotoxins is due to the HC’s tropism for neuronal receptors and the LC’s cleavage of neuron-specific target proteins, termed SNARE proteins. SNARE proteins are responsible for the fusion of neurotransmitter vesicles with the plasma membrane. There are seven serotypes of the BoNTs that share primary and ternary structure-function properties. Each BoNT serotype utilizes dual receptors for entry into neurons and each cleaves a specific SNARE protein or a unique site on a specific SNARE protein.

'''The Botulinum neurotoxins''' elicit flaccid paralysis, while tetanus toxin elicits spastic paralysis. The differential toxicity is due to the unique trafficking of these toxins in motor neurons. Botulinum neurotoxin HC binds to dual host receptors on the surface of motor neurons to deliver the BoNT to acidified vesicles where the LC is translocated into the cytoplasm of the neuron. Upon entry into the cytoplasm, LC cleaves a SNARE protein which inhibits fusion of neurotransmitter vesicles to the plasma membrane, inhibiting the release of neurotransmitter molecules. In contrast, TeNT HC binds to dual host receptors on the surface of motor neurons to deliver the TeNT to neutral vesicles for retrograde trafficking to the central nervous system. Following this transcytosis, Tetanus toxin binds dual receptors on inhibitory neurons to deliver TeNT to acidified endosomes where the LC is translocated into the cytoplasm of the neuron. TeNT LC cleaves the SNARE proteins, VAMP-2, which inhibits the release of neurotransmitters from inhibitory motor neurons. The inability to release neurotransmitter from inhibitory motor neurons yields spastic paralysis.

'''Botulinum toxin serotype A (BoNT/A)''' was chosen as the paradigm for the clostridial neurotoxins, since the dual host receptors for BoNT/A have been determined and the basis for recognizing cleavage of the SNARES substrate SNAP25 has been characterized. In addition, BoNT/A is the most common serotype used in clinical therapies. It is anticipated that understanding BoNT/A action will provide new information relevant to the entire family of clostridial neurotoxins. These studies will enhance the understanding of the unique
properties of each BoNT serotype and Tetanus toxin to extend their utility in human inflictions. The CFG has been used to facilitate the characterization of the ganglioside binding pocket of these neurotoxins. Prior studies utilized low throughput analyses that provided limited insight into interactions between these neurotoxins and gangliosides, and but high throughput analysis of the CFG core provided a better understanding of the biochemical and structural interactions of the neurotoxins with glycans. For example, array analysis showed that the dual receptors for Tetanus toxin recognized unique components of gangliosides.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of BoNT/A include: Joseph Barbieri, Edwin Chapman, Minoru Fukuda, Raymond Stevens, Willie Vann

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

 
=== Cellular expression of GBP and ligands ===
Botulinum toxins produced by ''Clostridium botulinum'' bind to dual host receptors on the surface of motorneurons.
 
=== Biosynthesis of ligands ===

 
=== Structure ===

 
=== Biological roles of GBP-ligand interaction ===
Botulinum toxins cause neurotoxicity by cleaving SNARE proteins, which normally allow neurotransmitter-containing vesicles to fuse with the neuronal plasma membrane.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=botulinum&maxresults=20 CFG database search results for botulinum].

=== Glycan profiling ===

 
=== Glycogene microarray ===
BoNT/A is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG synthesized ganglioside derivates that have been used in co-crystallization studies with the clostridial neurotoxins. The CFG glycan array was used to identify the ganglioside binding specificity to the clostridial neurotoxins ([http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2011 BoNT/C], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1972 BoNT/D], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1681 BoNT/F]).

== Related GBPs ==

Botulinum neurotoxins serotypes B-G (CFG data: [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FB&maxresults=20 BoNT/B,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FC&maxresults=20 BoNT/C,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FD&maxresults=20 BoNT/D,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FE&maxresults=20 BoNT/E,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Botulinum+AND+neurotoxin+AND+F&maxresults=20 BoNT/F,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FG&maxresults=20 BoNT/G)], Tetanus toxin [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=tetanus+AND+toxin&maxresults=20 (CFG data)]

== References ==
<references/>
* C. Chen, Z. Fu, J-J. P. Kim, J.T. Barbieri, and M. R. Baldwin. 2009. Gangliosides as High Affinity Receptors for Tetanus Neurotoxin. J Biol Chem. 284: 26569-77. PMC2785345
* M. Dong, W. H. Tepp, H. Liu, E. A. Johnson, and E. R. Chapman. 2007. Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons. J. Cell Biol. 179: 1511-1522. PMC2373501

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton

Subtilase cytotoxin (SubAB)

2011-03-30T23:22:15Z

Carole Weaver: /* Glycogene microarray */

'''AB5 toxins''' are an important family of bacterial toxins, so termed because they comprise a catalytic A subunit, non-covalently linked to a pentameric B subunit that binds to specific host cell surface glycans. There are three well-characterised AB5 toxin sub-families: (i) cholera toxin (Ctx) and the closely related E. coli heat labile enterotoxins (LT-I and LT-II); (ii) pertussis toxin (Ptx); and (iii) Shiga toxin (Stx). In each case, these AB5 toxins are key virulence factors of the bacteria that produce them: Vibrio cholerae and enterotoxigenic E. coli [ETEC] (Ctx and LT-I&II, respectively); Bordetella pertussis (Ptx); Shiga toxigenic E. coli [STEC] and Shigella dysenteriae (Stx). A fourth sub-family, subtilise cytotoxin (SubAB), also produced by STEC, has been described relatively recently. The AB5 toxins from each sub-family possess unique properties that arise from differing catalytic activities of the A subunit and/or differing receptor specificities of the B subunit. The A subunits of the Ctx/LT and Ptx families ADP-ribosylate the Gsα and Giα proteins, respectively, disrupting signal transduction pathways. This results in an increase in intracellular cAMP levels and disregulation of ion transport mechanisms. Stx family A subunits have RNA N-glycosidase activity, and inhibit eukaryotic protein synthesis by cleaving a specific adenine base from 28S rRNA, while SubA is a highly specific subtilase-like serine protease that cleaves the essential endoplasmic reticulum chaperone BiP/GRP78. Binding of AB5 toxin B subunits to cognate host glycan receptors triggers internalization by receptor- mediated endocytosis, followed by trafficking to the appropriate intracellular compartment. The glycan receptors for AB5 toxin B subunit pentamers are displayed either on glycolipids (for Ctx/LT and Stx) or on glycoproteins (for Ptx and SubAB). Glycan receptor specificity is critical for the pathogenic process, as it determines host susceptibility, tissue tropism, and the nature and spectrum of the resultant pathology. Knowledge of the molecular/structural basis for B subunit pentamer/glycan interactions is providing a rational framework for design of specific toxin inhibitors with considerable potential as anti-infective therapeutic agents<ref name="Fan 1">Fan, E. et al. (2000). AB5 toxins: structures and inhibitor design. Curr. Opin. Struct. Biol. 10: 680-686.</ref>.

'''SubAB''' is a recently-discovered prototype of a new AB5 toxin sub-family, with a highly novel mode of inducing cytotoxicity<ref>Paton, A.W., Srimanote, P., Talbot, U.M., Wang, H., and Paton, J.C. (2004). A new family of potent AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. J. Exp. Med. 200: 35-46.</ref><ref>Paton, A.W., Beddoe, T., Thorpe, C.M., Whisstock, J.C., Wilce, M.C.J., Rossjohn, J., Talbot, U.M. and Paton J.C. (2006). AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443: 548-552.</ref>. It has been selected as a paradigm because the other AB5 toxin sub-families referred to above have been known for many years, and a substantial body of information on toxin-receptor interactions had been built up using conventional biochemical techniques<ref name="Fan 1"/><ref>Merrit, E. A. and Hol, W. G. J. (1995). AB5 toxins. Curr. Opin. Struct. Biol. 5: 165-171.</ref>. SubB has recently been shown to bind to N-linked glycans displayed on several glycoproteins on the surface of Vero and HeLa cells, including α2β1integrin<ref name="Yahiro 2006">Yahiro, K., Morinaga, N., Satoh, M., Matsuura, G., Tomonaga, T., Nomura, F., Moss, J., Noda, M. (2006). Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin. Mol. Microbiol. 62: 480–490.</ref>. However, nothing was known about the identity of the cognate glycan structures prior to accessing the CFG Core H glycan array facilities. Thus, the CFG has enabled seminal studies on SubB-host receptor interactions. Glycan array analysis showed that SubB has a high degree of specificity for glycans terminating with α2-3-linked N-glycolylneuraminic acid (Neu5Gc), with little discrimination for the penultimate moiety<ref name="Byres 6">Byres, E., Paton, A.W., Paton, J.C., Löfling, J.C., Smith, D.F., Wilce, M.C.J., Talbot, U.M., Chong, D.C., Yu, H., Huang, S., Chen, X., Varki, N.M., Varki, A., Rossjohn, J., and Beddoe, T. (2008). Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. Nature. 456: 648-652.</ref>. Roughly 20-fold weaker binding was seen with otherwise identical glycans that terminated in α2-3-linked N-acetylneuraminic acid (Neu5Ac), which differs by one hydroxyl group from Neu5Gc. Binding was reduced over 30-fold if the Neu5Gc linkage was changed from α2-3 to α2-6, and 100-fold if the terminal sialic acid was removed. This high specificity for Neu5Gc-terminating glycans is believed to be unique amongst bacterial toxins<ref name="Byres 6"/>. Identification of the SubB receptor glycan informed structural analysis of SubB in complex with synthetic oligosaccharides provided by another CFG PI. This showed that Neu5Gc bound to a shallow pocket halfway down the sides of the SubB pentamer, whereas identical experiments using Neu5Ac failed to show any binding<ref name="Byres 6"/>. In contrast, CtxB whose receptor is a ganglioside rather than a glycoprotein, has a deep receptor binding pocket located on the base of the pentamer, juxtaposed to the membrane<ref name="Merritt 1997">Merritt, E.A.; Sarfarty, S.; Jobling, M.G.; Chang, T.; Holmes, R.K.; Hirst, T.R.; Hol, W.G.
Structural studies of receptor binding by cholera toxin mutants. Protein Sci. 1997, 6, 1516–1528.</ref>. In the SubB-Neu5Gc complex, Neu5Gc makes key interactions with the side chains of Asp8, Ser12, Glu36 and Tyr78<ref name="Byres 6"/>. Neu5Gc differs from Neu5Ac by the addition of a hydroxyl on the methyl group of the N-Acetyl moiety, which makes additional crucial interactions with SubB; namely the extra hydroxyl points towards and interacts with Tyr78OH and also hydrogen bonds with the main chain of Met10. These key interactions could not occur with Neu5Ac, thus explaining the marked preference for Neu5Gc6. The biological relevance of the structural analysis has been confirmed by further interacting residues<ref name="Byres 6"/>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) have made seminal contributions to understanding the biology of this highly potent toxin, in particular the molecular interactions between the binding subunit SubB and cognate host cell glycans described above. These include Xi Chen, Adrienne Paton, James Paton, David Smith, and Ajit Varki. Importantly, on-going collaborations have been established between these PIs as a consequence of involvement in the CFG, and these have already generated one collaborative paper in ''Nature''<ref name="Byres 6"/>.

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about SubAB, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===
SubB binds N-linked glycans displayed on glycoproteins on the surface of Vero and HeLa cells, including α2β1integrin<ref name="Yahiro 2006"/>. Screening with the CFG glycan array showed that SubB has a high degree of specificity for glycans terminating with α2-3-linked N-glycolylneuraminic acid (Neu5Gc), with little discrimination for the penultimate moiety<ref name="Byres 6"/>. Roughly 20-fold weaker binding was seen with otherwise identical glycans that terminated in α2-3-linked N-acetylneuraminic acid (Neu5Ac), which differs by one hydroxyl group from Neu5Gc. Binding was reduced over 30-fold if the Neu5Gc linkage was changed from α2-3 to α2-6, and 100-fold if the terminal sialic acid was removed. This high specificity for Neu5Gc-terminating glycans is believed to be unique amongst bacterial toxins<ref name="Byres 6"/>.
 
=== Cellular expression of GBP and ligands ===
Subtilase cytotoxin, SubAB, is produced by Shiga toxigenic E. coli. Other members of the AB5 toxin family are expressed as follows: Cholera toxin (Ctx) is produced by Vibrio cholerae, the heat-labile enterotoxins LT-I and LT-II are produced by enterotoxigenic E. coli (ETEC), pertussis toxin (Ptx) is produced by Bordetella pertussis, and shiga toxin (Stx) is produced by Shiga toxigenic E. coli (STEC) and Shigella dysenteriae.
 
AB5 toxin family members bind to glycan receptors in the host. The glycan receptors for AB5 toxin B subunit pentamers are displayed either on glycolipids (for Ctx/LT and Stx) or on glycoproteins (for Ptx and SubAB). 
=== Biosynthesis of ligands ===

 
=== Structure ===
Structural analysis of SubB in complex with synthetic oligosaccharides showed that Neu5Gc binds to a shallow pocket halfway down the sides of the SubB pentamer, whereas identical experiments using Neu5Ac failed to show any binding<ref name="Byres 6"/>. In contrast, CtxB, whose receptor is a ganglioside rather than a glycoprotein, has a deep receptor binding pocket located on the base of the pentamer, juxtaposed to the membrane<ref name="Merritt 1997"/>. In the SubB-Neu5Gc complex, Neu5Gc makes key interactions with the side chains of Asp8, Ser12, Glu36 and Tyr78<ref name="Byres 6"/>. Neu5Gc differs from Neu5Ac by the addition of a hydroxyl on the methyl group of the N-Acetyl moiety, which makes additional crucial interactions with SubB; namely, the extra hydroxyl points towards and interacts with Tyr78OH and also hydrogen bonds with the main chain of Met10. These key interactions could not occur with Neu5Ac, thus explaining the marked preference for Neu5Gc6.
 
=== Biological roles of GBP-ligand interaction ===
Glycan receptor specificity is critical for the pathogenic process, as it determines host susceptibility, tissue tropism, and the nature and spectrum of the resultant pathology.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=SubAB&maxresults=20 SubAB] and [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=subtilase&maxresults=20 subtilase].

=== Glycan profiling ===

 
=== Glycogene microarray ===
SubAB is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG glycan array was fundamental in the identification and characterization of [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_759 SubB receptor specificity]. This information then permitted structural analysis of protein-glycan complexes. Glycan array analysis was also critical for investigating the influence of mutation of SubB residues predicted to be critical for Neu5Gc-specific binding on the repertoire of glycan structures engaged by the toxin. To see all glycan array results for subtilase cytotoxin, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=subtilase%20AND%20cytotoxin&cat=coreh here].

== Related GBPs ==

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton

F17G/GafD

2011-03-30T23:21:24Z

Carole Weaver: /* Glycogene microarray */

The F17-G (GafD) adhesin at the tip of flexible F17 fimbriae of enterotoxigenic ''Escherichia coli'' mediates binding to N-acetyl-β-D-glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants, leading to diarrhea or septicaemia. F17-G belong to two-domain adhesins (TDA)s consisting of a pilin domain and a lectin domain, both having an Ig-fold joined via a short interdomain linker
<ref name=" Buts, L2003">
Buts, L., Bouckaert, J., De Gents, E., Loris, R., Oscarson, S., Lahmann, M., Messens, J., Brosens, E., Wyns, L. & De Greve, H. (2003). The fimbrial adhesin F17-G of enterotoxigenic Escherichia coli has an immunoglobulin-like lectin domain that binds N-acetylglucosamine. Mol. Microb. 49, 705-715.</ref><ref>Merckel, M. C., Tanskanen, J., Edelman, S., Westerlund-Wilkström, B., Korhonen, T. K. & Goldman, A. (2003). The structural basis of receptor-binding by Escherichia coli associaed with diarrhea and septicemia. J. Mol. Biol. 331, 897-905.</ref>. Related adhesins have been characterized in enteropathogenic ''E. coli'' ( FedF on F18 fimbriae<ref>Coddens, A., Diswall, M., Angstrom, J., Breimer, M. E., Goddeeris, B., Cox, E. & Teneberg, S. (2009). Recognition of blood group ABH type 1 determinants by the FedF adhesin of F18-fimbriated Escherichia coli. J Biol Chem 284, 9713-26.</ref> and CfaE on CFA/I pili<ref>Poole, S. T., McVeigh, A. L., Anantha, R. P., Lee, L. H., Akay, Y. M., Pontzer, E. A., Scott, D. A., Bullitt, E. & Savarino, S. J. (2007). Donor strand complementation governs intersubunit interaction of fimbriae of the alternate chaperone pathway. Mol Microbiol 63, 1372-84.</ref>) ) and uropathogenic ones (FimH on type 1 fimbriae<ref>Bouckaert, J., Berglund, J., Schembri, M., De Gents, E., Cools, L., Wuhrer, M., Hung, C.-S., Pinkner, J., Slättegard, R., Savialov, A., Choudhury, D., Langermann, S., Hultgren, S. J., Wyns, L., Klemm, P., Oscarson, S., Knight, S. D. & De Greve, H. (2005). Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin. Mol. Microb. 55, 441-455.</ref> and PapG on P-pili<ref>Dodson, K. W., Pinkner, J. S., Rose, T., Magnusson, G., Hultgren, S. J. & Waksman, G. (2001). Structural basis of the interaction of the pyelonephritic E. coli adhesin to ist human kideny receptor. Cell 105, 733-743.</ref>). Fimbrial adhesins from other organisms, such as CupB6 from ''Pseudomonas aeruginosa'' are also investigated. All share the immunoglobulin-like fold of the two structural components, despite lack of any sequence identity and diversity in carbohydrate specificity and binding site, and the corresponding pili are assembled by the chaperone-usher pathway<ref>De Greve, H., Wyns, L. & Bouckaert, J. (2007). Combining sites of bacterial fimbriae. Curr Opin Struct Biol 17, 506-12.</ref><ref>Sauer, F. G., Barnhart, M., Choudhury, D., Knight, S. D., Waksman, G. & Hultgren, S. J. (2000). Chaperone-assisted pilus assembly and bacterial attachment. Curr Opin Struct Biol 10, 548-56.</ref>. The paradigm is unique among TAD for his specificity toward GlcNAc. The binding site is located laterally and not at the tip of the pili, therefore the long and flexible F17 fimbriae could intrude between the microvilli of the epithelium, with the binding site of the lectin domain interacting laterally with GlcNAc-containing receptors. Five naturally occurring variants, differing in 1-18 amino acids of the adhesion domain have been identified<ref>De Kerpel, M., Van Molle, I., Brys, L., Wyns, L., De Greve, H. & Bouckaert, J. (2006). N-terminal truncation enables crystallization of the receptor-binding domain of the FedF bacterial adhesin. Acta Crystallogr Sect F Struct Biol Cryst Commun 62, 1278-82.</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
This is an emerging field of investigation and contributions arose from a small number of CFG Participating Investigators (PIs). These include: Esther Bullit, Eric Cox, Anne Imberty, Remy Loris, James Nataro

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about F17G/GafD, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===

The F17G adhesin is most specific for the disaccharide GlcNAcb1,3Gal that can be recognised as a terminal or internal sequence in bovine glycophorin <ref>Mouricout, M., Milhavet, M., Durié, C., Grange, P. Characterization of glycoprotein glycan receptors for Escherichia coli F17 fimbrial lectin, Microb. Pathog. (1995) 18, 297-306</ref>:
 
[[File:carbSynthe_0691_D000.jpg]]
 
The branched form gives a closely similar fluorescent signal:
 
[[File:carbSynthe_0897_D000.jpg]]
 
The presence of the b1,3 linkage of N-acetyl glucosamine to galactose enhances the affinity for F17G at least 2-fold, compared to the monosaccharide N-acetyl glucosamine, as validated using surface plasmon resonance measurements. Second best binders are the b1,4 and b1,6 galactose linked disaccharides, whereas chitobiose, that is also a characterized inhibitor of F17G-mediated bacterial adhesion, is clearly lagging behind. F17G can thus be ranked under glycan binding proteins that display high selectivity.

=== Cellular expression of GBP and ligands ===

F17G adhesins are expressed on enterotoxigenic E. coli infecting neonatal lambs, calves, and goat kids.
The F17G ligand GlcNAcb1,3Gal occurs universally but mostly internally in the sequence of poly-lactosaminyl glycans and blood group antigens.
These glycan structures are widely expressed on mamalian cell surfaces .

=== Biosynthesis of ligands ===
 
F17-fimbriated E. coli predominantly colonize neonatal animals, but also are a major causal agent (55%) of mastitis in bovines <ref>Lipman, L.J.A., de Nijs A., Gaastra W.. Isolation and identification of fimbriae and toxin production by Escherichia coli strains from cows with clinical mastitis, Vet. Microbiology 47 (1995) p. 1-7 </ref>. Congruent with the glycans recognized by F17G on the printed array versions 2.1 and 4.1, the N-acetyl glucosamine residue of GlcNAcb1,3Gal may be unsubstituted at the early life stage of calves, that are at the same time protected from bacterial infections by glycans secreted in the cow's milk.

=== Structure ===

The F17G adhesin is a two-domain adhesin (TDA) located at the F17 fimbrial tip. The determination of the crystal structure of the F17G lectin domain led to the discovery of the variable immunoglobulin-like structure
as a paradigm for bacterial fimbrial TDAs <ref name=" Buts, L2003">
</ref>. F17G has a shallow groove for carbohydrate recognition on its flank.

 
[[File:F17G_Igfold.jpg]]
 

=== Biological roles of GBP-ligand interaction ===

The F17G fimbrial lectin enhances intestinal colonization in the early life of ruminants. The long and flexible F17 fimbriae can penetrate deep between intestinal microvilli, where the fimbrial tip adhesin finds its glycan receptors. The subsequent secretion of heat stable and heat labile toxins can lead to severe diarrhea.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=fimbriae&maxresults=20 fimbriae] and [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=pili&maxresults=20 pili].

=== Glycan profiling ===

 
=== Glycogene microarray ===
F17G/GafD is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
F17G adhesins have been screened for their glycan specificity (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2_177_11182005 here]). To see all glycan array results for F17G adhesin, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=F17G&cat=coreh here].

== Related GBPs ==
FedF [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=FedF&maxresults=20 (CFG data)], CfaE [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CfaE&maxresults=20 (CFG data)], FimH [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=FimH&maxresults=20 (CFG data)], PapG, CupB6 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CupB6&maxresults=20 (CFG data)].

The specificity of some of the other fimbrial tip adhesins was determined by CFG glycan array analysis ([http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2358 ''P. gingivalis'' fimbriae], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2_178_11182005 ''E. coli'' FedF adhesin], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1106 ''E. coli'' CfaE adhesin from CFA/I pili])

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Alisdair Boraston, Julie Bouckaert, Anne Imberty

CBM47

2011-03-30T23:20:55Z

Carole Weaver: /* Glycogene microarray */

Enzymes that degrade host glycans are increasingly being found as virulence factors in pathogenic bacteria<ref>Shelburne, S. A., Davenport, M. T., Keith, D. B. & Musser, J. M. (2008). The role of complex carbohydrate catabolism in the pathogenesis of invasive streptococci. Trends Microbiol 16, 318-25.</ref><ref name="Hava 16">Hava, D. L. & Camilli, A. (2002). Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol Microbiol 45, 1389-406.</ref>. A common property of extracellular glycan degrading enzymes found in such bacteria is multi-modularity; these enzymes often comprise a large number of modules with a variety of functions. The most common class of ancillary module are carbohydrate-binding modules (CBMs)<ref>Boraston, A. B., Bolam, D. N., Gilbert, H. J. & Davies, G. J. (2004). Carbohydrate-binding modules: fine tuning polysaccharide recognition. Biochem J 382, 769-782.</ref>, which are alternatively referred to as lectin-domains. These modules are responsible for targeting carbohydrate-degrading enzymes to a glycan substrate or, when the enzymes are attached to the bacterial cell-surface, likely also function to adhere the bacterium to a glycan<ref>Ficko-Blean, E., Gregg, K. J., Adams, J. J., Hehemann, J. H., Czjzek, M., Smith, S. P. & Boraston, A. B. (2009). Portrait of an enzyme, a complete structural analysis of a multimodular {beta}-N-acetylglucosaminidase from Clostridium perfringens. J Biol Chem 284, 9876-84.</ref>. The presence of these lectin-domains in multi-modular proteins and their contribution of glycan binding function to catalytically active proteins distinguishes these modules from other bacterial glycan-binding proteins (GBPs). The CBM47 modules from the ''Streptococcus pneumoniae'' enzyme SpGH98 (or "fucolectin-related protein") are specific to the Lewisy antigen<ref name ="Boraston 2006">Boraston, A. B., Wang, D. & Burke, R. D. (2006). Blood group antigen recognition by a Streptococcus pneumoniae virulence factor. J Biol Chem 281, 35263-35271.</ref>, which is quite rare among all GBPs, and function to target this enzyme to this antigen when present on epithelial cells<ref>Higgins, M. A., Whitworth, G. E., El Warry, N., Randriantsoa, M., Samain, E., Burke, R. D., Vocadlo, D. J. & Boraston, A. B. (2009). Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases. J Biol Chem 284, 26161-73.</ref>. Recognition and destruction of this antigen appears to be a critical process in pneumococcal virulence<ref name="Hava 16"/><ref>Embry, A., Hinojosa, E. & Orihuela, C. J. (2007). Regions of Diversity 8, 9 and 13 contribute to Streptococcus pneumoniae virulence. BMC Microbiol 7, 80.</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
This is a very new area of investigation. CFG Participating Investigators (PIs) that have screened other CBMs or proteins containing CBMs include: Alisdair Boraston, Garry Taylor, Warren Wakarchuck

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about CBM47, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===
CBM47 is a fucose specific binding module that is related to ''Anguilla anguilla'' fucolectin. The high affinity ligand for CBM47 has been determined from glycan microarray screening on the CFG microarray[http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_34_08192004#]to be Fucα1-2Galβ1-4(Fucα1-3)GlcNAc [Lewis y]<ref name="Boraston 2006"/>.

[[File:Lewisy.jpg]]
 

=== Cellular expression of GBP and ligands ===

 
=== Biosynthesis of ligands ===

 
=== Structure ===

CBM47 originates from a multimodular ''S. pneumoniae'' that has an N-terminal, Lewisy degrading catalytic module. Indeed, three CBM47 modules are found in tandem.

[[File:modular.jpg]]

The high resolution X-ray structures of the N-terminal and C-terminal CBM47 modules have been determined and the N-terminal module in complex with the Lewisy tetrasaccharide<ref name="Boraston 2006"/>.

[[File:CBM47.jpg]]

 

=== Biological roles of GBP-ligand interaction ===

The primary role of CBM47, and indeed other CBMs found in carbohydrate-active enzymes, is to direct the entire enzyme to its glycan substrate. However, CBMs found in carbohydrate-active enzymes attached to microbial cell surfaces may also play a role in the adhesion of the bacterium to host glycan-bearing tissues.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CBM&maxresults=20 CFG database search results for CBM].

=== Glycan profiling ===

 
=== Glycogene microarray ===
CBM47 is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The specificity of several CBMs have been investigated by CFG glycan array analysis (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1831 here] for example). Isolated glycans for structural and quantitative binding studies have also been obtained from the CFG.

== Related GBPs ==

There are presently over 55 families of CBMs that are defined on the basis of amino acid sequence similarity; however, the majority of these CBMs families contain members specific for plant cell wall polysaccharides. CFG resources have been instrumental in studying the subset of CBMs that recognize complex glycans. In addition to CBM47 there are a number of CBMs belonging to family 32 and 51 that share the property of binding complex glycans. Though unrelated at the amino acid sequence level CBMs in families 32, 47, and 51 are structurally related and functionally related. It is important to note, however, that the diversity of complex glycan binding among these family members is considerable and only coming to light through glycan array screening. The availability of purfied glycans is facilitating structural and quantitative studies of glycan binding by CBMs in families 32, 47, and 51.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Alisdair Boraston, Anne Imberty

PA-IIL

2011-03-30T23:20:18Z

Carole Weaver: /* Glycogene microarray */

PA-IIL (Pseudomonas lectin II, LecB) is a cytoplasmic lectin produced in ''Pseudomonas aruginosa'' under the control of quorum sensing. It is a virulence factor involved in binding of human tissues such as epithelial lung of cystic fibrosis patients<ref name="Imberty 2004">Imberty, A., Wimmerova, M., Mitchell, E. P. & Gilboa-Garber, N. (2004). Structures of the lectins from Pseudomonas aeruginosa: Insights into molecular basis for host glycan recognition. Microb. Infect. 6, 222-229.</ref>. The role of PA-IIL in pathogenesis has been highlighted, since interfering with PA-IIL binding site reduces the mortality of ''P. aeruginosa'' induced-pneumonia in a murine model<ref name="Chemani 2009">Chemani, C., Imberty, A., de Bentzman, S., Pierre, P., Wimmerová, M., Guery, B. P. & Faure, K. (2009). Role of LecA and LecB lectins in Pseudomonas aeruginosa induced lung injury and effect of carbohydrates ligands. Infect. Immun. 77, 2065-2075.</ref>. The paradigm is unique among glycan-binding proteins (GBPs) in that it contains two cationic ions in the carbohydrate binding site that result in unusual high affinity for the carbohydrate target<ref>Loris, R., Tielker, D., Jaeger, K.-E. & Wyns, L. (2003). Structural basis of carbohydrate recognition by the lectin LecB from Pseudomonas aeruginosa. J. Mol. Biol. 331, 861-870.</ref><ref name="test 1">Mitchell, E., Houles, C., Sudakevitz, D., Wimmerova, M., Gautier, C., Pérez, S., Wu, A. M., Gilboa-Garber, N. & Imberty, A. (2002). Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. Nature Struct. Biol. 9, 918-921.</ref><ref>Mitchell, E. P., Sabin, C., Šnajdrová, L., Pokorná, M., Perret, S., Gautier, C., Hofr, C., Gilboa-Garber, N., Koča, J., Wimmerová, M. & Imberty, A. (2005). High affinity fucose binding of Pseudomonas aeruginosa lectin PA-IIL: 1.0 Å resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches. Proteins: Struct. Funct. Bioinfo. 58, 735-748.</ref>. The preferred glycan ligand of PA-IIL is the trisaccharide Lewis a<ref name="test 2">Perret, S., Sabin, C., Dumon, C., Pokorná, M., Gautier, C., Galanina, O., Ilia, S., Bovin, N., Nicaise, M., Desmadril, M., Gilboa-Garber, N., Wimmerova, M., Mitchell, E. P. & Imberty, A. (2005). Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa. Biochem. J. 389, 325-332.</ref>. PA-IIL-like lectins have been characterized in other opportunistic bacteria such as ''Ralstonia solanacearum''<ref>Sudakevitz, D., Kostlanova, N., Blatman-Jan, G., Mitchell, E. P., Lerrer, B., Wimmerova, M., Katcof, f. D. J., Imberty, A. & Gilboa-Garber, N. (2004). A new Ralstonia solanacearum high affinity mannose-binding lectin RS-IIL structurally resembling the Pseudomonas aeruginosa fucose-specific lectin PA-IIL. Mol. Microbiol. 52, 691-700.</ref>, ''Chromobacterium violaceum''<ref name="Pokorna 11">Pokorná, M., Cioci, G., Perret, S., Rebuffet, E., Kostlánová, N., Adam, J., Gilboa-Garber, N., Mitchell, E. P., Imberty, A. & Wimmerová, M. (2006). Unusual entropy driven affinity of Chromobacterium violaceum lectin CV-IIL towards fucose and mannose. Biochemistry 45, 7501-7510.</ref>, and ''Burkholderia cenocepacia''<ref>Lameignere, E., Malinovská, L., Sláviková, M., Duchaud, E., Mitchell, E. P., Varrot, A., Šedo, O., Imberty, A. & Wimmerová, M. (2008). Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia. Biochem. J. 411, 307-318.</ref><ref>Lameignere, E., Shiao, T. C., Roy, R., Wimmerová, M., Dubreuil, F., Varrot, A. & Imberty, A. (2010). Structural basis of the affinity for oligomannosides and analogs displayed by BC2L-A, a Burkholderia cenocepacia soluble lectin. Glycobiology 20, 87-98.</ref>, albeit with some variations in the fine specificity. Starting from the results obtained with the help of CFG tools, major efforts are devoided in collaboration with carbohydrate chemists in order to design anti-bacterial new glyco-derived compounds that bind to PA-IIL with high affinity<ref>Imberty, A., Chabre, Y. M. & Roy, R. (2008). Glycomimetics and glycodendrimers as high affinity microbial antiadhesins. Chemistry 14, 7490-7499.</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) have made major contribution to the understanding of the structure/specificity relationship of PA-IIL and PA-IIL-like proteins. These include: Anne Imberty, Remy Loris, Michaela Wimmerova

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about PA-IIL, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===

PA-IIL is a fucose-specific lectin that also recognises mannose residues. 
The high affinity ligand for PA-IIL has been deduced from glycan microarray screening on the CFG microarray[http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_221_02142006#] to be Galβ1-4(Fucα1-4)GlcNAc [Lewis a] <ref name="test 1"></ref><ref name="test 2"></ref><ref>Marotte K, Sabin C, Preville C, Moume-Pymbock M, Wimmerova M, Mitchell EP, Imberty A, Roy R. (2008) X-ray Structures and thermodynamics of the interaction of PA-IIL from Pseudomonas aeruginosa with disaccharide derivatives. ChemMedChem 10,1328-1338.</ref>

[[File:lewisa.jpg]]

=== Cellular expression of GBP and ligands ===
PA-IIL is produced in ''Pseudomonas aruginosa,'' and binds ligands in human tissues, including epithelial lung.
 
=== Biosynthesis of ligands ===

 
=== Structure ===
PA-IIL is a tetrameric lectin. Each monomer consists of a β-sandwich and contains two calcium ions that interact directly with the carbohydrate ligand. Crystal structures have been determined for PA-IIL alone, and its complex with fucose, with related monosaccharides, and with complex fucosylated oligosaccharides.

[[File:pa2ls.jpg]]
 
Information on crystal structures of PA-IIL and links to PDB are available at PA-IIL page of [http://www.cermav.cnrs.fr/cgi-bin/lectines/menu.cgi?Bacterial%20lectins+2-CA%20b-sandwich+Pseudomonas%20PA-IIL 3D-lectin database]

=== Biological roles of GBP-ligand interaction ===
PA-IIL, a virulence factor that binds human tissues such as epithelial lung of cystic fibrosis patients<ref name="Imberty 2004"/>, has roles in pathogenesis <ref name="Chemani 2009"/>.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=PA-IIL&maxresults=20 CFG database search results for PA-IIL].

=== Glycan profiling ===

 
=== Glycogene microarray ===
PA-IIL is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The specificity of PA-IIl and related proteins was determined through glycan array analysis (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2_230_02102006 here] and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_221_02142006# here]).

== Related GBPs ==
CV-IIL [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CV-IIL&maxresults=20 (CFG data)], RS-IIL [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=RS-IIL&cat=coreh (CFG data)], BC2L-A, BC2L-B [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BC2L-B&maxresults=20 (CFG data)], BC2L-C [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BC2L-C&maxresults=20 (CFG data)].

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Anne Imberty and Micha Wimmerova

Reovirus hemagglutinin (sigma 1)

2011-03-28T19:12:19Z

Carole Weaver: /* Glycogene microarray */

'''Mammalian orthoreoviruses (reoviruses)''' are useful models for studies of viral receptor recognition and the pathogenesis of viral disease. Reovirus also efficiently lyses tumor cells in experimental animals<ref>Duncan, M.R., Stanish, S.M., and Cox, D.C. Differential sensitivity of normal and transformed human cells to reovirus infection. J. Virol. 28:444-449, 1978.</ref><ref> Coffey, M.C., Strong, J.E., Forsyth, P.A., and Lee, P.W. Reovirus therapy of tumors with activated Ras pathway. Science 282:1332-1334, 1998.</ref> and has shown efficacy in clinical trials for aggressive and refractory human tumors<ref>Stoeckel, J., and Hay, J.G. Drug evaluation: Reolysin--wild-type reovirus as a cancer therapeutic. Curr. Opin. Mol. Ther. 8:249-260, 2006.</ref><ref>Twigger, K., Vidal, L., White, C.L., De Bono, J.S., Bhide, S., Coffey, M., Thompson, B., Vile, R.G., Heinemann, L., Pandha, H.S., et al. Enhanced in vitro and in vivo cytotoxicity of combined reovirus and radiotherapy. Clin. Cancer Res. 14:912-923, 2008. </ref>. Reovirus forms double-shelled particles<ref>Dryden, K.A., Wang, G., Yeager, M., Nibert, M.L., Coombs, K.M., Furlong, D.B., Fields, B.N., and Baker, T.S. Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction. J. Cell Biol. 122:1023-1041, 1993.</ref> that contain a segmented dsRNA genome. The reovirus sigma 1 protein is a long, fiber-like molecule that extends from the virion surface<ref>Furlong, D.B., Nibert, M.L., and Fields, B.N. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles. J. Virol. 62:246-256, 1988.</ref> and mediates viral attachment<ref>Weiner, H.L., Ault, K.A., and Fields, B.N. Interaction of reovirus with cell surface receptors. I. Murine and human lymphocytes have a receptor for the hemagglutinin of reovirus type 3. J. Immunol. 124:2143-2148, 1980.</ref><ref>Lee, P.W.K., Hayes, E.C., and Joklik, W.K. Protein σ1 is the reovirus cell attachment protein. Virology 108:156-163, 1981.</ref>. The three human serotypes (T1, T2, and T3) differ in cell tropism, which correlates directly with receptor-binding properties of sigma 1. Sialic acid serves as an essential receptor for T3 reovirus on murine erythroleukemia (MEL) cells<ref name="Rubin1992">Rubin, D.H., Wetzel, J.D., Williams, W.V., Cohen, J.A., Dworkin, C., and Dermody, T.S. Binding of type 3 reovirus by a domain of the σ1 protein important for hemagglutination leads to infection of murine erythroleukemia cells. J. Clin. Invest. 90:2536-2542, 1992.</ref>, and it functions as a coreceptor on murine L929 (L) cells<ref name="Gentsch1985">Gentsch, J.R., and Pacitti, A.F. Effect of neuraminidase treatment of cells and effect of soluble glycoproteins on type 3 reovirus attachment to murine L cells. J. Virol. 56:356-364, 1985.</ref><ref name="Pacitti1987">Pacitti, A., and Gentsch, J.R. Inhibition of reovirus type 3 binding to host cells by sialylated glycoproteins is mediated through the viral attachment protein. J. Virol. 61:1407-1415, 1987. </ref><ref name="name4">.</ref><ref name="Rubin1992"></ref><ref name="Nibert1995">Nibert, M.L., Chappell, J.D., and Dermody, T.S. Infectious subvirion particles of reovirus type 3 Dearing exhibit a loss in infectivity and contain a cleaved σ1 protein. J. Virol. 69:5057-5067, 1995.</ref>. Residues involved in sialic acid-binding map to the center of the long fiber, close to the midpoint of the molecule<ref name="Chappell2002">Chappell, J.D., Prota, A., Dermody, T.S., and Stehle, T. Crystal structure of reovirus attachment protein σ1 reveals evolutionary relationship to adenovirus fiber. EMBO J. 21:1-11, 2002.</ref>, in a repetitive structural region known as the triple β-spiral. The T1 sigma 1 protein binds to cell-surface glycans of unknown structure.

The triple β-spiral of '''sigma 1''' functions as a trimerization domain and defines a novel carbohydrate-recognition motif. Other carbohydrate-recognition domains, such as those of the C-type lectin superfamily<ref>Weis, W.I., Taylor, M.E., and Drickamer, K. The C-type lectin superfamily in the immune system. Immunol. Rev. 163:19-34, 1998.</ref> or the sialic acid-binding domains in the Siglec family of adhesion proteins<ref>Crocker, P.R., and Varki, A. Siglecs in the immune system. Immunology 103:137-145, 2001.</ref> (see [http://glycobank.mit.edu/glycoWiki/Main_Page Siglec paradigms]), have been described, but none are formed by a repetitive, fiber-like structure such as the one present in sigma 1. In fact, the domain in sigma 1 that binds sialic acid constitutes a carbohydrate-binding “cassette” that could be endowed with altered ligand-binding properties or grafted onto other trimeric structures and used to create avidity for carbohydrates. For example, the adenovirus fiber shaft could be licensed with sialic acid-binding capacity using this approach. These properties render the sigma 1 protein unique among the structurally known glycan-binding moieties.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of sigma 1 include: Terence Dermody, Thilo Stehle

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
Reovirus strains of all three serotypes are capable of binding to carbohydrates, which is a property mediated by viral attachment protein sigma 1<ref name="Chappell2000">Chappell, J.D., Duong, J.L., Wright, B.W., and Dermody, T.S. Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses. J. Virol. 74:8472-8479, 2000.</ref>. Substantial evidence indicates that serotype 3 reoviruses bind to sialic acid, whereas the identity of carbohydrates bound by serotype 1 and 2 reoviruses is less clear. Serotype 3 reoviruses bind to alpha-linked sialic acid in either alpha2,3 or alpha2,6 linkages on a variety of cell types<ref name="Chappell2000">.</ref><ref name="Gentsch1985">.</ref><ref name="name4">Paul, R.W., Choi, A.H., and Lee, P.W.K. The a-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus. Virology 172:382-385, 1989.</ref><ref>Dermody, T.S., Nibert, M.L., Bassel-Duby, R., and Fields, B.N. Sequence diversity in S1 genes and S1 translation products of 11 serotype 3 reovirus strains. J. Virol. 64:4842-4850, 1990.</ref><ref name="Chappell1997">Chappell, J.D., Gunn, V.L., Wetzel, J.D., Baer, G.S., and Dermody, T.S. Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein s1. J. Virol. 71:1834-1841, 1997.</ref>. Hemagglutination by serotype 3 reoviruses is mediated by interactions with alpha-linked sialic acid on several glycosylated erythrocyte proteins such as glycophorin A<ref>Gentsch, J.R., and Pacitti, A.F. Differential interaction of reovirus type 3 with sialylated receptor components on animal cells. Virology 161:245-248, 1987.</ref><ref>Paul, R.W., and Lee, P.W.K. Glycophorin is the reovirus receptor on human erythrocytes. Virology 159:94-101, 1987.</ref>. Reovirus strain T3D binds to sialoglycophorin, but not to asialoglycophorin, with an avidity of ~ 5 x 10e-9 M<ref name="Barton2001a">Barton, E.S., Connolly, J.L., Forrest, J.C., Chappell, J.D., and Dermody, T.S. Utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening. J. Biol. Chem. 276:2200-2211, 2001.</ref>.

Serotype 1 reoviruses also appear to bind to sialic acid in some contexts. Reovirus strain T1L, but not strain T3D, binds to the apical surface of microfold (M) cells, but not to enterocytes, in tissue sections of rabbit Peyer’s patches<ref name="name10">Helander, A., Silvey, K.J., Mantis, N.J., Hutchings, A.B., Chandran, K., Lucas, W.T., Nibert, M.L., and Neutra, M.R. The viral s1 protein and glycoconjugates containing a2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces. J. Virol. 77:7964-7977, 2003.</ref>. Binding is inhibited by pre-incubation of the tissue sections with neuraminidase or with lectins that specifically recognize alpha2-3-linked sialic acid.

=== Cellular expression of GBP and ligands ===
Sialic acid serves as an essential receptor for type 3 reoviruses on murine erythroleukemia (MEL) cells<ref name="Chappell1997">.</ref><ref name="Rubin1992"></ref>. Sialic acid also functions as a coreceptor on murine L929 cells<ref name="Gentsch1985">.</ref><ref name="name4">.</ref><ref name="Rubin1992">.</ref><ref name="Pacitti1987">.</ref><ref name="Nibert1995">.</ref> and human HeLa cells<ref name="Barton2001a">.</ref>. Although not all serotype 3 strains are capable of binding to sialic acid, the majority bind to this carbohydrate.

Serotype 1 reoviruses are incapable of infecting MEL cells, which support infection only by sialic-acid-binding strains<ref name="Rubin1992">.</ref>. Serotype 1 reoviruses also are insensitive to the growth-inhibitory effects of neuraminidase treatment of L929 cells<ref name="Nibert1995">.</ref>. However, binding of serotype 1 reoviruses to intestinal M cells is diminished by neuraminidase treatment<ref name="name10">.</ref>. The explanation for this discrepancy is not known.

Reovirus employs a multi-step mechanism of viral attachment in which a low-affinity interaction with sialic acid serves to tether the virion to target cells and precedes a high-affinity interaction with JAM-A<ref name="Barton2001a">.</ref>, an immunoglobulin superfamily protein engaged by reovirus<ref name="Barton2001b">Barton, E.S., Forrest, J.C., Connolly, J.L., Chappell, J.D., Liu, Y., Schnell, F., Nusrat, A., Parkos, C.A., and Dermody, T.S. Junction adhesion molecule is a receptor for reovirus. Cell 104:441-451, 2001.</ref><ref>Prota, A.E., Campbell, J.A., Schelling, P., Forrest, J.C., Peters, T.R., Watson, M.J., Aurrand-Lions, M., Imhof, B., Dermody, T.S., and Stehle, T. Crystal structure of human junctional adhesion molecule 1: implications for reovirus binding. Proc. Natl. Acad. Sci. U. S. A. 100:5366-5371, 2003.</ref><ref>Campbell, J.A., Shelling, P., Wetzel, J.D., Johnson, E.M., Wilson, G.A.R., Forrest, J.C., Aurrand-Lions, M., Imhof, B., Stehle, T., and Dermody, T.S. Junctional adhesion molecule-A serves as a receptor for prototype and field-isolate strains of mammalian reovirus. J. Virol. 79:7967-7978, 2005.</ref>. By virtue of its rapid association rate, virus binding to sialic acid adheres the virion to the cell surface, thereby enabling it to diffuse laterally until it encounters JAM-A. Such lateral diffusion has been reported for influenza virus<ref>Sagik, B., Puck, T., and Levine, S. Quantitative aspects of the spontaneous elution of influenza virus from red cells. J. Exp. Med. 99:251-260, 1954.</ref> and phage T4<ref>Wilson, J.H., Luftig, R.B., and Wood, W.B. Interaction of bacteriophage T4 tail fiber components with a lipopolysaccharide fraction from Escherichia coli. J. Mol. Biol. 51:423-434, 1970.</ref>. After attachment, reovirus is internalized by receptor-mediated endocytosis<ref>Borsa, J., Morash, B.D., Sargent, M.D., Copps, T.P., Lievaart, P.A., and Szekely, J.G. Two modes of entry of reovirus particles into L cells. J. Gen. Virol. 45:161-170, 1979.</ref><ref>Borsa, J., Sargent, M.D., Lievaart, P.A., and Copps, T.P. Reovirus: evidence for a second step in the intracellular uncoating and transcriptase activation process. Virology 111:191-200, 1981.</ref><ref>Ehrlich, M., Boll, W., Van Oijen, A., Hariharan, R., Chandran, K., Nibert, M.L., and Kirchhausen, T. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118:591-605, 2004.</ref><ref name="Maginnis2006">Maginnis, M.S., Forrest, J.C., Kopecky-Bromberg, S.A., Dickeson, S.K., Santoro, S.A., Zutter, M.M., Nemerow, G.R., Bergelson, J.M., and Dermody, T.S. b1 integrin mediates internalization of mammalian reovirus. J. Virol. 80:2760-2770, 2006.</ref><ref>Sturzenbecker, L.J., Nibert, M.L., Furlong, D.B., and Fields, B.N. Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle. J. Virol. 61:2351-2361, 1987.</ref><ref name="Maginnis2008">Maginnis, M.S., Mainou, B.A., Derdowski, A.M., Johnson, E.M., Zent, R., and Dermody, T.S. NPXY motifs in the b1 integrin cytoplasmic tail are required for functional reovirus entry. J. Virol. 82:3181-3191, 2008.</ref> using a mechanism dependent on beta 1 integrin<ref name="Maginnis2006">.</ref><ref name="Maginnis2008">.</ref>.
=== Biosynthesis of ligands ===
 
=== Structure ===
Structural analysis of the C-terminal half of reovirus attachment protein sigma 1 from strain T3D (residues 246-455) reveals a trimeric structure, in which each monomer is composed of a slender tail and a compact head<ref name="Chappell2002">.</ref>. The C-terminal residues that form the head domain (310-455) consist of two Greek-key motifs that fold into a beta-barrel. The sigma 1 head domain binds to JAM-A<ref name="Barton2001b">.</ref><ref>Kirchner, E., Guglielmi, K.M., Strauss, H.M., Dermody, T.S., and Stehle, T. Structure of reovirus s1 in complex with its receptor junctional adhesion molecule-A. PLoS Path. 4:e1000235, 2008.</ref>. N-terminal residues in the crystallized fragment form a portion of the tail, residues 246-309, which consists of three beta-spiral repeats. Each repeat is composed of two short beta-strands connected by a four-residue beta-turn that has either a proline or a glycine residue at its third position<ref name="Chappell2002">.</ref>. A surface-exposed, variable loop links successive repeats, and trimerization generates an unusual triple beta-spiral motif that also is observed in the adenovirus fiber<ref>van Raaij, M.J., Mitraki, A., Lavigne, G., and Cusack, S. A triple b-spiral in the adenovirus fibre shaft reveals a new structural motif for a fibrous protein. Nature 401:935-938, 1999.</ref>, bacteriophage PRD1 P5 protein<ref>Merckel, M.C., Huiskonen, J.T., Bamford, D.H., Goldman, A., and Tuma, R. The structure of the bacteriophage PRD1 spike sheds light on the evolution of viral capsid architecture. Mol. Cell 18:161-170, 2005.</ref>, and avian reovirus attachment protein sigma C<ref>Guardado, C.P., Fox, G.C., Hermo Parrado, X.L., Llamas-Saiz, A.L., Costas, C., Martinez-Costas, J., Benavente, J., and van Raaij, M.J. Structure of the carboxy-terminal receptor-binding domain of avian reovirus fibre sigmaC. J. Mol. Biol. 354:137-149, 2005.</ref>.

In addition to the three beta-spiral repeats observed in the crystallized sigma 1 fragment, sequence analysis suggests that residues 167-249 in the T3D sigma 1 tail form an additional five N-terminal beta-spiral repeats<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">Guglielmi, K.M., Johnson, E.M., Stehle, T., and Dermody, T.S. Attachment and cell entry of mammalian orthoreovirus. Curr. Top. Microbiol. Immunol. 309:1-38, 2006.</ref>. Alternatively, these residues may form a combination of beta-spiral repeats and alpha-helical coiled-coil, as suggested by sequence analysis<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">.</ref><ref name="Nibert1990">Nibert, M.L., Dermody, T.S., and Fields, B.N. Structure of the reovirus cell-attachment protein: a model for the domain organization of s1. J. Virol. 64:2976-2989, 1990.</ref> and an observed narrowing in this region in a composite negative-stain electron micrograph<ref>Fraser, R.D.B., Furlong, D.B., Trus, B.L., Nibert, M.L., Fields, B.N., and Steven, A.C. Molecular structure of the cell-attachment protein of reovirus: correlation of computer-processed electron micrographs with sequence-based predictions. J. Virol. 64:2990-3000, 1990.</ref>. The structure of N-terminal residues 1-160 of sigma 1 is unknown. However, a repeating heptad sequence motif is predictive of an amphipathic alpha-helix, which likely assembles into an alpha-helical coiled-coil in the trimer<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">.</ref><ref name="Nibert1990">.</ref>.

Although the structure of sigma 1 in complex with sialic acid is not yet available, studies using expressed proteins indicate that the region of T3D sigma 1 required for sialic binding resides near the midpoint of the tail, whereas a region just N-terminal to the head domain of T1L sigma 1 binds to carbohydrate<ref name="Chappell2000">.</ref>. In both T1L and T3D sigma 1, interactions with carbohydrate are mediated by a region of predicted beta-spiral<ref name="Chappell2002">.</ref>. Adaptation of non-sialic-acid-binding reoviruses to growth in MEL cells results in amino acid substitutions at residues 198, 202, and 204 of sigma 1 that confer sialic-acid-binding capacity on the resultant viruses<ref name="Chappell1997">.</ref>. Molecular modeling of the sigma 1 tail, based on available structure and sequence data, suggests that these residues are surface-exposed and proximal to one another in the predicted beta-spiral region<ref name="Chappell2002">.</ref>. Thus, residues 198, 202, and 204 are likely to contribute to a sialic-acid-binding site in T3D sigma 1.

=== Biological roles of GBP-ligand interaction ===
Sialic acid binding serves an important role in reovirus tropism and pathogenesis in vivo<ref>Barton, E.S., Youree, B.E., Ebert, D.H., Forrest, J.C., Connolly, J.L., Valyi-Nagy, T., Washington, K., Wetzel, J.D., and Dermody, T.S. Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease. J. Clin. Invest. 111:1823-1833, 2003.</ref>. Strain T3SA+, which binds to sialic acid, and strain T3SA-, which does not bind to this carbohydrate, produce equivalent titers in the intestine of newborn mice following peroral inoculation. However, T3SA+ spreads more rapidly from the intestine to sites of secondary replication and produces higher titers in the spleen, liver, and brain. Mice infected with T3SA+, but not T3SA-, develop clinical evidence of bile duct obstruction including jaundice and steatorrhea. Liver tissue from mice infected with T3SA+ show intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in human infants, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ binds 100-fold more efficiently than T3SA- to human cholangiocarcinoma cells. Thus, binding to sialic acid targets reovirus to bile duct epithelium and produces a disease reminiscent of infantile biliary atresia.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=reovirus&maxresults=20 CFG database search results for "reovirus"].

=== Glycan profiling ===
Not performed.

=== Glycogene microarray ===
Not applicable, as the CFG microarrays only contain probes for mouse and human glycogenes.

=== Knockout mouse lines ===
Not applicable.

=== Glycan array ===
Experiments in progress. No definitive results have been obtained thus far.

== Related GBPs ==
The attachment protein of adenovirus, fiber, is a structural homolog of sigma 1. At least one adenovirus serotype (Ad37; [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=ad37&maxresults=20 CFG data]) is known to bind glycan receptors via residues in the fiber protein<ref>Burmeister WP, Guilligay D, Cusack S, Wadell G, Arnberg N: Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites. Journal of Virology 78:7727-7736, 2004.</ref>. The actual binding site is not homologous. However, information about reovirus glycan binding could also be used to engineer adenovirus fiber proteins (or other trimeric fiber-like proteins) that possess novel glycan-binding properties.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Terence Dermody, Mavis McKenna, Thilo Stehle.

C. difficile toxin A (TcdA)

2011-03-28T19:11:26Z

Carole Weaver: /* Glycogene microarray */

'''The large clostridial cytotoxins (LCT)''' are a family of structurally and functionally related exotoxins from ''Clostridium difficile'' (toxins A and B [also termed TcdA and TcdB, respectively), ''C. sordellii'' (lethal and hemorrhagic toxin) and ''C. novyi'' (alpha-toxin). The LCTs are major virulence factors, which in addition to their in vivo effects, are cytotoxic to cultured cell lines, causing reorganization of the cytoskeleton accompanied by morphological changes<ref name="Just 1">Just. I., Gerhard, R. (2004). Large clostridial cytotoxins. Rev. Physiol. Biochem. Pharmacol. 152:23-47.</ref>. The LCTs are single-chain protein toxins, comprising three domains: receptor-binding, translocation and catalysis, which mediate cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and enzymatic cytotoxic activity, respectively. Enzymatic activity involves transfer of a glucosyl moiety from UDP-glucose (or the N-acetyl-glucosaminyl moiety from UDP-GlcNAc in the case of alpha toxin) to a conserved threonine within the effector regions of the intracellular Rho and Ras GTPases<ref name="Just 1"/>. The C-terminal receptor-binding domain comprises up to one third of the LCT molecule, and contains repetitive peptide elements called combined repetitive oligopetides (CROPs). The repeating units binding regions of certain streptococcal glycosyltransferases<ref name="Just 1"/>.

'''TcdA''' is the better characterized of the two LCTs produced by ''C. difficile.'' Collectively, TcdA and TcdB are directly responsible for the increasingly common and serious human gastrointestinal disease caused when antibiotic treatment enables ''C. difficile'' to out-compete commensal gut microflora. Recently, the crystal structure of a C-terminal fragment of TcdA has been solved, revealing a solenoid-like structure, which consists of 32 short repeats with 15–21 residues and seven long repeats with 30 residues<ref name="Jank 2007">Thomas Jank, Torsten Giesemann and Klaus Aktories. (2007). Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function. Glycobiology. 17: 15R–22R.
</ref>. Solenoid structures are often found in bacterial surface proteins, and their extended surface allows protein–protein or protein–carbohydrate interactions. TcdA is known to bind to Galα1-3Galβ1-4GlcNAc- glycans, although this structure is not present in humans. However, it is also known to recognize a variety of other structures with a Galβ[1→4]GlcNAcβ- backbone, including the fucosylated blood group antigens Lewisx and Lewisy, both of which are present in human intestinal epithelium<ref name="Voth 2005">Voth, D. E., and Ballard, J. D. (2005). Clostridium difficile toxins: mechanism of action and role in disease. Clin. Microbiol. Rev. 18, 247-263.</ref>. Clearly, a better understanding of the identity/structure of the human receptor(s) for these toxins will facilitate design of novel therapeutics capable of blocking toxin binding. Glycan array analysis conducted by the CFG showed that TcdA bound most strongly to glycans terminating in Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4-GlcNAc-, which mimics the blood group antigen Lewisa; other bound structures included Galα1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ-, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-, and Fucαa1-2[GalNAcαa1-3]Galβ1-4GlcNAcβ1-3Galβb1-4GlcNAcβ-.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) contributing to the understanding of TcdA include: Borden Lacy, Yashwant Mahida, Kenneth Ng
* Non-CFG PIs include: Brian Dieckgraefe

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
*Galα1-3Galβ1-4GlcNAc- glycans (not present in humans). 
*Other structures with a Galβ[1→4]GlcNAcβ- backbone, including the fucosylated blood group antigens Lewisx and Lewisy, both of which are present in human intestinal epithelium<ref name="Voth 2005"/>. 
*Strongest binding: glycans terminating in Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4-GlcNAc-, which mimics the blood group antigen Lewisa. 
*Also binds: Galα1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ-, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-, and Fucαa1-2[GalNAcαa1-3]Galβ1-4GlcNAcβ1-3Galβb1-4GlcNAcβ-. 
 
=== Cellular expression of GBP and ligands ===
TcdA is produced by ''Clostridium difficile''. Structurally and functionally related exotoxins in the LCT family are produced by ''C. sordellii'' (lethal and hemorrhagic toxin) and ''C. novyi'' (alpha-toxin).
 
=== Biosynthesis of ligands ===

 
=== Structure ===
Members of the LCT family are single-chain protein toxins with receptor-binding, translocation and catalysis domains, which mediate cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and enzymatic cytotoxic activity, respectively. Up to one third of the LCT molecule is comprised of the C-terminal receptor-binding domain, which contains repetitive peptide elements called combined repetitive oligopeptides (CROPs).
 
X-ray crystallography has revealed the three-dimensional structures of several recombinant fragments of TcdA and related toxins<ref name="Jank 2007"/>. The N-terminal domain consists of a GT-A family glucosyltransferase that catalyzes the transfer of glucose from UDP-glucose to specific threonine residues of small GTPases regulating the structure and dynamics of the cytoskeleton. Although the three-dimensional structure of the GT domain of TcdA has not been solved by crystallography, the structures of closely related GT domains from TcdB and the letahal toxin from ''Clostridium sordellii'' have been determined.
 
The crystallographic structure of a cysteine protease domain bound to inositol hexakisphosphate has also been determined. This domain follows the N-terminal GT domain and is responsible for the autoproteolysis of TcdA that is important for releasing the GT domain into the cytoplasm.
 
Crystallographic structures of several recombinant fragments from the highly repetitive C-terminal domain have revealed an extended beta-solenoid structure that presents seven binding sites for complex oligosaccharide receptors. The bulk of this carbohydrate-binding domain is comprised of multiple copies of short repeats that interspersed with single copies of long repeats. Carbohydrate-binding sites appear to be formed at the junctions of long repeats and the preceding and following short repeats.
 
Crystallographic structures of recombinant fragments of TcdA have been determined with oligosaccharides, including the Gal-α(1,3)-LacNAc trisaccharide shown below<ref name = "Greco 2006">Greco, A et al. (2006). Carbohydrate recognition by ''Clostridium difficile'' toxin A. Nat. Struct. Mol. Biol. 13:460-461.</ref>. This trisaccharide was determined to be a likely native receptor for TcdA in the brush border membranes of intestinal epithelial cells from hamster<ref name = "Krivan 1986">Krivan, HC et al (1986). Cell-surface binding site for ''Clostridium difficile'' enterotoxin: evidence for a glycoconjugate containing the sequence Galα1,3-Galβ1,4-GlcNAc. Infect. Imm. 53:573-581.</ref>, an organism which is susceptible to ''C. difficile'' infection (CDI) and is often used as a model for studying the disease.
 
[[File:CD_fig1.png]]

=== Biological roles of GBP-ligand interaction ===
TcdA and TcdB, the other LCT produced by ''C. difficile'', cause the serious human gastrointestinal disease that results when antibiotic treatment enables ''C. difficile'' to out-compete commensal gut microflora. The LCTs are major virulence factors, which in addition to their in vivo effects, are cytotoxic to cultured cell lines, causing reorganization of the cytoskeleton accompanied by morphological changes<ref name="Just 1"/>.
 
== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=difficile&maxresults=20 CFG database search results for "difficile"].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Not applicable, as the CFG microarrays only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG glycan array has been used to examine the [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2693 glycan-binding specificity of TcdA]. In addition, the CFG's glycan synthesis capability is being used to prepare synthetic oligosaccharides indentified in the glycan array screens (with or without biotin tags) to further probe TcdA-receptor interactions.

== Related GBPs ==
''C. difficile'' toxin B (TcdB; [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=TcdB&maxresults=20 CFG data]), ''C. sordellii'' lethal toxin and hemorrhagic toxin, ''C. novyi'' alpha-toxin.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, Kenneth Ng, James Paton

Botulinum toxin serotype A (BoNT/A)

2011-03-28T19:11:02Z

Carole Weaver: /* Glycogene microarray */

'''The clostridial neurotoxins''' are the most lethal protein toxins for humans and recently have been utilized as therapeutic agents to treat numerous human neurological inflictions. The Botulinum neurotoxins produced by ''Clostridium botulinum'' and Tetanus toxin produced by ''C. tetani'' are members of the family of clostridial neurotoxins. The clostridial neurotoxins are di-chain toxins with the N-terminal catalytic domains (Light Chain, LC) possessing metalloprotease activity that is disulfide linked to the C-terminal
domain (Heavy Chain, HC). The neurological toxicity and therapeutic utility of the clostridial neurotoxins is due to the HC’s tropism for neuronal receptors and the LC’s cleavage of neuron-specific target proteins, termed SNARE proteins. SNARE proteins are responsible for the fusion of neurotransmitter vesicles with the plasma membrane. There are seven serotypes of the BoNTs that share primary and ternary structure-function properties. Each BoNT serotype utilizes dual receptors for entry into neurons and each cleaves a specific SNARE protein or a unique site on a specific SNARE protein.

'''The Botulinum neurotoxins''' elicit flaccid paralysis, while tetanus toxin elicits spastic paralysis. The differential toxicity is due to the unique trafficking of these toxins in motor neurons. Botulinum neurotoxin HC binds to dual host receptors on the surface of motor neurons to deliver the BoNT to acidified vesicles where the LC is translocated into the cytoplasm of the neuron. Upon entry into the cytoplasm, LC cleaves a SNARE protein which inhibits fusion of neurotransmitter vesicles to the plasma membrane, inhibiting the release of neurotransmitter molecules. In contrast, TeNT HC binds to dual host receptors on the surface of motor neurons to deliver the TeNT to neutral vesicles for retrograde trafficking to the central nervous system. Following this transcytosis, Tetanus toxin binds dual receptors on inhibitory neurons to deliver TeNT to acidified endosomes where the LC is translocated into the cytoplasm of the neuron. TeNT LC cleaves the SNARE proteins, VAMP-2, which inhibits the release of neurotransmitters from inhibitory motor neurons. The inability to release neurotransmitter from inhibitory motor neurons yields spastic paralysis.

'''Botulinum toxin serotype A (BoNT/A)''' was chosen as the paradigm for the clostridial neurotoxins, since the dual host receptors for BoNT/A have been determined and the basis for recognizing cleavage of the SNARES substrate SNAP25 has been characterized. In addition, BoNT/A is the most common serotype used in clinical therapies. It is anticipated that understanding BoNT/A action will provide new information relevant to the entire family of clostridial neurotoxins. These studies will enhance the understanding of the unique
properties of each BoNT serotype and Tetanus toxin to extend their utility in human inflictions. The CFG has been used to facilitate the characterization of the ganglioside binding pocket of these neurotoxins. Prior studies utilized low throughput analyses that provided limited insight into interactions between these neurotoxins and gangliosides, and but high throughput analysis of the CFG core provided a better understanding of the biochemical and structural interactions of the neurotoxins with glycans. For example, array analysis showed that the dual receptors for Tetanus toxin recognized unique components of gangliosides.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of BoNT/A include: Joseph Barbieri, Edwin Chapman, Minoru Fukuda, Raymond Stevens, Willie Vann

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

 
=== Cellular expression of GBP and ligands ===
Botulinum toxins produced by ''Clostridium botulinum'' bind to dual host receptors on the surface of motorneurons.
 
=== Biosynthesis of ligands ===

 
=== Structure ===

 
=== Biological roles of GBP-ligand interaction ===
Botulinum toxins cause neurotoxicity by cleaving SNARE proteins, which normally allow neurotransmitter-containing vesicles to fuse with the neuronal plasma membrane.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=botulinum&maxresults=20 CFG database search results for botulinum].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Not applicable, as the CFG microarrays only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG synthesized ganglioside derivates that have been used in co-crystallization studies with the clostridial neurotoxins. The CFG glycan array was used to identify the ganglioside binding specificity to the clostridial neurotoxins ([http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2011 BoNT/C], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1972 BoNT/D], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1681 BoNT/F]).

== Related GBPs ==

Botulinum neurotoxins serotypes B-G (CFG data: [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FB&maxresults=20 BoNT/B,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FC&maxresults=20 BoNT/C,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FD&maxresults=20 BoNT/D,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FE&maxresults=20 BoNT/E,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Botulinum+AND+neurotoxin+AND+F&maxresults=20 BoNT/F,][http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BoNT%2FG&maxresults=20 BoNT/G)], Tetanus toxin [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=tetanus+AND+toxin&maxresults=20 (CFG data)]

== References ==
<references/>
* C. Chen, Z. Fu, J-J. P. Kim, J.T. Barbieri, and M. R. Baldwin. 2009. Gangliosides as High Affinity Receptors for Tetanus Neurotoxin. J Biol Chem. 284: 26569-77. PMC2785345
* M. Dong, W. H. Tepp, H. Liu, E. A. Johnson, and E. R. Chapman. 2007. Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons. J. Cell Biol. 179: 1511-1522. PMC2373501

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton

Subtilase cytotoxin (SubAB)

2011-03-28T19:10:33Z

Carole Weaver: /* Glycogene microarray */

'''AB5 toxins''' are an important family of bacterial toxins, so termed because they comprise a catalytic A subunit, non-covalently linked to a pentameric B subunit that binds to specific host cell surface glycans. There are three well-characterised AB5 toxin sub-families: (i) cholera toxin (Ctx) and the closely related E. coli heat labile enterotoxins (LT-I and LT-II); (ii) pertussis toxin (Ptx); and (iii) Shiga toxin (Stx). In each case, these AB5 toxins are key virulence factors of the bacteria that produce them: Vibrio cholerae and enterotoxigenic E. coli [ETEC] (Ctx and LT-I&II, respectively); Bordetella pertussis (Ptx); Shiga toxigenic E. coli [STEC] and Shigella dysenteriae (Stx). A fourth sub-family, subtilise cytotoxin (SubAB), also produced by STEC, has been described relatively recently. The AB5 toxins from each sub-family possess unique properties that arise from differing catalytic activities of the A subunit and/or differing receptor specificities of the B subunit. The A subunits of the Ctx/LT and Ptx families ADP-ribosylate the Gsα and Giα proteins, respectively, disrupting signal transduction pathways. This results in an increase in intracellular cAMP levels and disregulation of ion transport mechanisms. Stx family A subunits have RNA N-glycosidase activity, and inhibit eukaryotic protein synthesis by cleaving a specific adenine base from 28S rRNA, while SubA is a highly specific subtilase-like serine protease that cleaves the essential endoplasmic reticulum chaperone BiP/GRP78. Binding of AB5 toxin B subunits to cognate host glycan receptors triggers internalization by receptor- mediated endocytosis, followed by trafficking to the appropriate intracellular compartment. The glycan receptors for AB5 toxin B subunit pentamers are displayed either on glycolipids (for Ctx/LT and Stx) or on glycoproteins (for Ptx and SubAB). Glycan receptor specificity is critical for the pathogenic process, as it determines host susceptibility, tissue tropism, and the nature and spectrum of the resultant pathology. Knowledge of the molecular/structural basis for B subunit pentamer/glycan interactions is providing a rational framework for design of specific toxin inhibitors with considerable potential as anti-infective therapeutic agents<ref name="Fan 1">Fan, E. et al. (2000). AB5 toxins: structures and inhibitor design. Curr. Opin. Struct. Biol. 10: 680-686.</ref>.

'''SubAB''' is a recently-discovered prototype of a new AB5 toxin sub-family, with a highly novel mode of inducing cytotoxicity<ref>Paton, A.W., Srimanote, P., Talbot, U.M., Wang, H., and Paton, J.C. (2004). A new family of potent AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. J. Exp. Med. 200: 35-46.</ref><ref>Paton, A.W., Beddoe, T., Thorpe, C.M., Whisstock, J.C., Wilce, M.C.J., Rossjohn, J., Talbot, U.M. and Paton J.C. (2006). AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443: 548-552.</ref>. It has been selected as a paradigm because the other AB5 toxin sub-families referred to above have been known for many years, and a substantial body of information on toxin-receptor interactions had been built up using conventional biochemical techniques<ref name="Fan 1"/><ref>Merrit, E. A. and Hol, W. G. J. (1995). AB5 toxins. Curr. Opin. Struct. Biol. 5: 165-171.</ref>. SubB has recently been shown to bind to N-linked glycans displayed on several glycoproteins on the surface of Vero and HeLa cells, including α2β1integrin<ref name="Yahiro 2006">Yahiro, K., Morinaga, N., Satoh, M., Matsuura, G., Tomonaga, T., Nomura, F., Moss, J., Noda, M. (2006). Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin. Mol. Microbiol. 62: 480–490.</ref>. However, nothing was known about the identity of the cognate glycan structures prior to accessing the CFG Core H glycan array facilities. Thus, the CFG has enabled seminal studies on SubB-host receptor interactions. Glycan array analysis showed that SubB has a high degree of specificity for glycans terminating with α2-3-linked N-glycolylneuraminic acid (Neu5Gc), with little discrimination for the penultimate moiety<ref name="Byres 6">Byres, E., Paton, A.W., Paton, J.C., Löfling, J.C., Smith, D.F., Wilce, M.C.J., Talbot, U.M., Chong, D.C., Yu, H., Huang, S., Chen, X., Varki, N.M., Varki, A., Rossjohn, J., and Beddoe, T. (2008). Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. Nature. 456: 648-652.</ref>. Roughly 20-fold weaker binding was seen with otherwise identical glycans that terminated in α2-3-linked N-acetylneuraminic acid (Neu5Ac), which differs by one hydroxyl group from Neu5Gc. Binding was reduced over 30-fold if the Neu5Gc linkage was changed from α2-3 to α2-6, and 100-fold if the terminal sialic acid was removed. This high specificity for Neu5Gc-terminating glycans is believed to be unique amongst bacterial toxins<ref name="Byres 6"/>. Identification of the SubB receptor glycan informed structural analysis of SubB in complex with synthetic oligosaccharides provided by another CFG PI. This showed that Neu5Gc bound to a shallow pocket halfway down the sides of the SubB pentamer, whereas identical experiments using Neu5Ac failed to show any binding<ref name="Byres 6"/>. In contrast, CtxB whose receptor is a ganglioside rather than a glycoprotein, has a deep receptor binding pocket located on the base of the pentamer, juxtaposed to the membrane<ref name="Merritt 1997">Merritt, E.A.; Sarfarty, S.; Jobling, M.G.; Chang, T.; Holmes, R.K.; Hirst, T.R.; Hol, W.G.
Structural studies of receptor binding by cholera toxin mutants. Protein Sci. 1997, 6, 1516–1528.</ref>. In the SubB-Neu5Gc complex, Neu5Gc makes key interactions with the side chains of Asp8, Ser12, Glu36 and Tyr78<ref name="Byres 6"/>. Neu5Gc differs from Neu5Ac by the addition of a hydroxyl on the methyl group of the N-Acetyl moiety, which makes additional crucial interactions with SubB; namely the extra hydroxyl points towards and interacts with Tyr78OH and also hydrogen bonds with the main chain of Met10. These key interactions could not occur with Neu5Ac, thus explaining the marked preference for Neu5Gc6. The biological relevance of the structural analysis has been confirmed by further interacting residues<ref name="Byres 6"/>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) have made seminal contributions to understanding the biology of this highly potent toxin, in particular the molecular interactions between the binding subunit SubB and cognate host cell glycans described above. These include Xi Chen, Adrienne Paton, James Paton, David Smith, and Ajit Varki. Importantly, on-going collaborations have been established between these PIs as a consequence of involvement in the CFG, and these have already generated one collaborative paper in ''Nature''<ref name="Byres 6"/>.

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about SubAB, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===
SubB binds N-linked glycans displayed on glycoproteins on the surface of Vero and HeLa cells, including α2β1integrin<ref name="Yahiro 2006"/>. Screening with the CFG glycan array showed that SubB has a high degree of specificity for glycans terminating with α2-3-linked N-glycolylneuraminic acid (Neu5Gc), with little discrimination for the penultimate moiety<ref name="Byres 6"/>. Roughly 20-fold weaker binding was seen with otherwise identical glycans that terminated in α2-3-linked N-acetylneuraminic acid (Neu5Ac), which differs by one hydroxyl group from Neu5Gc. Binding was reduced over 30-fold if the Neu5Gc linkage was changed from α2-3 to α2-6, and 100-fold if the terminal sialic acid was removed. This high specificity for Neu5Gc-terminating glycans is believed to be unique amongst bacterial toxins<ref name="Byres 6"/>.
 
=== Cellular expression of GBP and ligands ===
Subtilase cytotoxin, SubAB, is produced by Shiga toxigenic E. coli. Other members of the AB5 toxin family are expressed as follows: Cholera toxin (Ctx) is produced by Vibrio cholerae, the heat-labile enterotoxins LT-I and LT-II are produced by enterotoxigenic E. coli (ETEC), pertussis toxin (Ptx) is produced by Bordetella pertussis, and shiga toxin (Stx) is produced by Shiga toxigenic E. coli (STEC) and Shigella dysenteriae.
 
AB5 toxin family members bind to glycan receptors in the host. The glycan receptors for AB5 toxin B subunit pentamers are displayed either on glycolipids (for Ctx/LT and Stx) or on glycoproteins (for Ptx and SubAB). 
=== Biosynthesis of ligands ===

 
=== Structure ===
Structural analysis of SubB in complex with synthetic oligosaccharides showed that Neu5Gc binds to a shallow pocket halfway down the sides of the SubB pentamer, whereas identical experiments using Neu5Ac failed to show any binding<ref name="Byres 6"/>. In contrast, CtxB, whose receptor is a ganglioside rather than a glycoprotein, has a deep receptor binding pocket located on the base of the pentamer, juxtaposed to the membrane<ref name="Merritt 1997"/>. In the SubB-Neu5Gc complex, Neu5Gc makes key interactions with the side chains of Asp8, Ser12, Glu36 and Tyr78<ref name="Byres 6"/>. Neu5Gc differs from Neu5Ac by the addition of a hydroxyl on the methyl group of the N-Acetyl moiety, which makes additional crucial interactions with SubB; namely, the extra hydroxyl points towards and interacts with Tyr78OH and also hydrogen bonds with the main chain of Met10. These key interactions could not occur with Neu5Ac, thus explaining the marked preference for Neu5Gc6.
 
=== Biological roles of GBP-ligand interaction ===
Glycan receptor specificity is critical for the pathogenic process, as it determines host susceptibility, tissue tropism, and the nature and spectrum of the resultant pathology.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=SubAB&maxresults=20 SubAB] and [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=subtilase&maxresults=20 subtilase].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Not applicable, as the CFG microarrays only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG glycan array was fundamental in the identification and characterization of [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_759 SubB receptor specificity]. This information then permitted structural analysis of protein-glycan complexes. Glycan array analysis was also critical for investigating the influence of mutation of SubB residues predicted to be critical for Neu5Gc-specific binding on the repertoire of glycan structures engaged by the toxin. To see all glycan array results for subtilase cytotoxin, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=subtilase%20AND%20cytotoxin&cat=coreh here].

== Related GBPs ==

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton

Subtilase cytotoxin (SubAB)

2011-03-28T19:10:15Z

Carole Weaver: /* Glycogene microarray */

'''AB5 toxins''' are an important family of bacterial toxins, so termed because they comprise a catalytic A subunit, non-covalently linked to a pentameric B subunit that binds to specific host cell surface glycans. There are three well-characterised AB5 toxin sub-families: (i) cholera toxin (Ctx) and the closely related E. coli heat labile enterotoxins (LT-I and LT-II); (ii) pertussis toxin (Ptx); and (iii) Shiga toxin (Stx). In each case, these AB5 toxins are key virulence factors of the bacteria that produce them: Vibrio cholerae and enterotoxigenic E. coli [ETEC] (Ctx and LT-I&II, respectively); Bordetella pertussis (Ptx); Shiga toxigenic E. coli [STEC] and Shigella dysenteriae (Stx). A fourth sub-family, subtilise cytotoxin (SubAB), also produced by STEC, has been described relatively recently. The AB5 toxins from each sub-family possess unique properties that arise from differing catalytic activities of the A subunit and/or differing receptor specificities of the B subunit. The A subunits of the Ctx/LT and Ptx families ADP-ribosylate the Gsα and Giα proteins, respectively, disrupting signal transduction pathways. This results in an increase in intracellular cAMP levels and disregulation of ion transport mechanisms. Stx family A subunits have RNA N-glycosidase activity, and inhibit eukaryotic protein synthesis by cleaving a specific adenine base from 28S rRNA, while SubA is a highly specific subtilase-like serine protease that cleaves the essential endoplasmic reticulum chaperone BiP/GRP78. Binding of AB5 toxin B subunits to cognate host glycan receptors triggers internalization by receptor- mediated endocytosis, followed by trafficking to the appropriate intracellular compartment. The glycan receptors for AB5 toxin B subunit pentamers are displayed either on glycolipids (for Ctx/LT and Stx) or on glycoproteins (for Ptx and SubAB). Glycan receptor specificity is critical for the pathogenic process, as it determines host susceptibility, tissue tropism, and the nature and spectrum of the resultant pathology. Knowledge of the molecular/structural basis for B subunit pentamer/glycan interactions is providing a rational framework for design of specific toxin inhibitors with considerable potential as anti-infective therapeutic agents<ref name="Fan 1">Fan, E. et al. (2000). AB5 toxins: structures and inhibitor design. Curr. Opin. Struct. Biol. 10: 680-686.</ref>.

'''SubAB''' is a recently-discovered prototype of a new AB5 toxin sub-family, with a highly novel mode of inducing cytotoxicity<ref>Paton, A.W., Srimanote, P., Talbot, U.M., Wang, H., and Paton, J.C. (2004). A new family of potent AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. J. Exp. Med. 200: 35-46.</ref><ref>Paton, A.W., Beddoe, T., Thorpe, C.M., Whisstock, J.C., Wilce, M.C.J., Rossjohn, J., Talbot, U.M. and Paton J.C. (2006). AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443: 548-552.</ref>. It has been selected as a paradigm because the other AB5 toxin sub-families referred to above have been known for many years, and a substantial body of information on toxin-receptor interactions had been built up using conventional biochemical techniques<ref name="Fan 1"/><ref>Merrit, E. A. and Hol, W. G. J. (1995). AB5 toxins. Curr. Opin. Struct. Biol. 5: 165-171.</ref>. SubB has recently been shown to bind to N-linked glycans displayed on several glycoproteins on the surface of Vero and HeLa cells, including α2β1integrin<ref name="Yahiro 2006">Yahiro, K., Morinaga, N., Satoh, M., Matsuura, G., Tomonaga, T., Nomura, F., Moss, J., Noda, M. (2006). Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin. Mol. Microbiol. 62: 480–490.</ref>. However, nothing was known about the identity of the cognate glycan structures prior to accessing the CFG Core H glycan array facilities. Thus, the CFG has enabled seminal studies on SubB-host receptor interactions. Glycan array analysis showed that SubB has a high degree of specificity for glycans terminating with α2-3-linked N-glycolylneuraminic acid (Neu5Gc), with little discrimination for the penultimate moiety<ref name="Byres 6">Byres, E., Paton, A.W., Paton, J.C., Löfling, J.C., Smith, D.F., Wilce, M.C.J., Talbot, U.M., Chong, D.C., Yu, H., Huang, S., Chen, X., Varki, N.M., Varki, A., Rossjohn, J., and Beddoe, T. (2008). Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. Nature. 456: 648-652.</ref>. Roughly 20-fold weaker binding was seen with otherwise identical glycans that terminated in α2-3-linked N-acetylneuraminic acid (Neu5Ac), which differs by one hydroxyl group from Neu5Gc. Binding was reduced over 30-fold if the Neu5Gc linkage was changed from α2-3 to α2-6, and 100-fold if the terminal sialic acid was removed. This high specificity for Neu5Gc-terminating glycans is believed to be unique amongst bacterial toxins<ref name="Byres 6"/>. Identification of the SubB receptor glycan informed structural analysis of SubB in complex with synthetic oligosaccharides provided by another CFG PI. This showed that Neu5Gc bound to a shallow pocket halfway down the sides of the SubB pentamer, whereas identical experiments using Neu5Ac failed to show any binding<ref name="Byres 6"/>. In contrast, CtxB whose receptor is a ganglioside rather than a glycoprotein, has a deep receptor binding pocket located on the base of the pentamer, juxtaposed to the membrane<ref name="Merritt 1997">Merritt, E.A.; Sarfarty, S.; Jobling, M.G.; Chang, T.; Holmes, R.K.; Hirst, T.R.; Hol, W.G.
Structural studies of receptor binding by cholera toxin mutants. Protein Sci. 1997, 6, 1516–1528.</ref>. In the SubB-Neu5Gc complex, Neu5Gc makes key interactions with the side chains of Asp8, Ser12, Glu36 and Tyr78<ref name="Byres 6"/>. Neu5Gc differs from Neu5Ac by the addition of a hydroxyl on the methyl group of the N-Acetyl moiety, which makes additional crucial interactions with SubB; namely the extra hydroxyl points towards and interacts with Tyr78OH and also hydrogen bonds with the main chain of Met10. These key interactions could not occur with Neu5Ac, thus explaining the marked preference for Neu5Gc6. The biological relevance of the structural analysis has been confirmed by further interacting residues<ref name="Byres 6"/>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) have made seminal contributions to understanding the biology of this highly potent toxin, in particular the molecular interactions between the binding subunit SubB and cognate host cell glycans described above. These include Xi Chen, Adrienne Paton, James Paton, David Smith, and Ajit Varki. Importantly, on-going collaborations have been established between these PIs as a consequence of involvement in the CFG, and these have already generated one collaborative paper in ''Nature''<ref name="Byres 6"/>.

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about SubAB, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===
SubB binds N-linked glycans displayed on glycoproteins on the surface of Vero and HeLa cells, including α2β1integrin<ref name="Yahiro 2006"/>. Screening with the CFG glycan array showed that SubB has a high degree of specificity for glycans terminating with α2-3-linked N-glycolylneuraminic acid (Neu5Gc), with little discrimination for the penultimate moiety<ref name="Byres 6"/>. Roughly 20-fold weaker binding was seen with otherwise identical glycans that terminated in α2-3-linked N-acetylneuraminic acid (Neu5Ac), which differs by one hydroxyl group from Neu5Gc. Binding was reduced over 30-fold if the Neu5Gc linkage was changed from α2-3 to α2-6, and 100-fold if the terminal sialic acid was removed. This high specificity for Neu5Gc-terminating glycans is believed to be unique amongst bacterial toxins<ref name="Byres 6"/>.
 
=== Cellular expression of GBP and ligands ===
Subtilase cytotoxin, SubAB, is produced by Shiga toxigenic E. coli. Other members of the AB5 toxin family are expressed as follows: Cholera toxin (Ctx) is produced by Vibrio cholerae, the heat-labile enterotoxins LT-I and LT-II are produced by enterotoxigenic E. coli (ETEC), pertussis toxin (Ptx) is produced by Bordetella pertussis, and shiga toxin (Stx) is produced by Shiga toxigenic E. coli (STEC) and Shigella dysenteriae.
 
AB5 toxin family members bind to glycan receptors in the host. The glycan receptors for AB5 toxin B subunit pentamers are displayed either on glycolipids (for Ctx/LT and Stx) or on glycoproteins (for Ptx and SubAB). 
=== Biosynthesis of ligands ===

 
=== Structure ===
Structural analysis of SubB in complex with synthetic oligosaccharides showed that Neu5Gc binds to a shallow pocket halfway down the sides of the SubB pentamer, whereas identical experiments using Neu5Ac failed to show any binding<ref name="Byres 6"/>. In contrast, CtxB, whose receptor is a ganglioside rather than a glycoprotein, has a deep receptor binding pocket located on the base of the pentamer, juxtaposed to the membrane<ref name="Merritt 1997"/>. In the SubB-Neu5Gc complex, Neu5Gc makes key interactions with the side chains of Asp8, Ser12, Glu36 and Tyr78<ref name="Byres 6"/>. Neu5Gc differs from Neu5Ac by the addition of a hydroxyl on the methyl group of the N-Acetyl moiety, which makes additional crucial interactions with SubB; namely, the extra hydroxyl points towards and interacts with Tyr78OH and also hydrogen bonds with the main chain of Met10. These key interactions could not occur with Neu5Ac, thus explaining the marked preference for Neu5Gc6.
 
=== Biological roles of GBP-ligand interaction ===
Glycan receptor specificity is critical for the pathogenic process, as it determines host susceptibility, tissue tropism, and the nature and spectrum of the resultant pathology.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=SubAB&maxresults=20 SubAB] and [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=subtilase&maxresults=20 subtilase].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Not applicable, as the microarrays only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The CFG glycan array was fundamental in the identification and characterization of [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_759 SubB receptor specificity]. This information then permitted structural analysis of protein-glycan complexes. Glycan array analysis was also critical for investigating the influence of mutation of SubB residues predicted to be critical for Neu5Gc-specific binding on the repertoire of glycan structures engaged by the toxin. To see all glycan array results for subtilase cytotoxin, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=subtilase%20AND%20cytotoxin&cat=coreh here].

== Related GBPs ==

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton

F17G/GafD

2011-03-28T19:09:38Z

Carole Weaver: /* Glycogene microarray */

The F17-G (GafD) adhesin at the tip of flexible F17 fimbriae of enterotoxigenic ''Escherichia coli'' mediates binding to N-acetyl-β-D-glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants, leading to diarrhea or septicaemia. F17-G belong to two-domain adhesins (TDA)s consisting of a pilin domain and a lectin domain, both having an Ig-fold joined via a short interdomain linker
<ref name=" Buts, L2003">
Buts, L., Bouckaert, J., De Gents, E., Loris, R., Oscarson, S., Lahmann, M., Messens, J., Brosens, E., Wyns, L. & De Greve, H. (2003). The fimbrial adhesin F17-G of enterotoxigenic Escherichia coli has an immunoglobulin-like lectin domain that binds N-acetylglucosamine. Mol. Microb. 49, 705-715.</ref><ref>Merckel, M. C., Tanskanen, J., Edelman, S., Westerlund-Wilkström, B., Korhonen, T. K. & Goldman, A. (2003). The structural basis of receptor-binding by Escherichia coli associaed with diarrhea and septicemia. J. Mol. Biol. 331, 897-905.</ref>. Related adhesins have been characterized in enteropathogenic ''E. coli'' ( FedF on F18 fimbriae<ref>Coddens, A., Diswall, M., Angstrom, J., Breimer, M. E., Goddeeris, B., Cox, E. & Teneberg, S. (2009). Recognition of blood group ABH type 1 determinants by the FedF adhesin of F18-fimbriated Escherichia coli. J Biol Chem 284, 9713-26.</ref> and CfaE on CFA/I pili<ref>Poole, S. T., McVeigh, A. L., Anantha, R. P., Lee, L. H., Akay, Y. M., Pontzer, E. A., Scott, D. A., Bullitt, E. & Savarino, S. J. (2007). Donor strand complementation governs intersubunit interaction of fimbriae of the alternate chaperone pathway. Mol Microbiol 63, 1372-84.</ref>) ) and uropathogenic ones (FimH on type 1 fimbriae<ref>Bouckaert, J., Berglund, J., Schembri, M., De Gents, E., Cools, L., Wuhrer, M., Hung, C.-S., Pinkner, J., Slättegard, R., Savialov, A., Choudhury, D., Langermann, S., Hultgren, S. J., Wyns, L., Klemm, P., Oscarson, S., Knight, S. D. & De Greve, H. (2005). Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin. Mol. Microb. 55, 441-455.</ref> and PapG on P-pili<ref>Dodson, K. W., Pinkner, J. S., Rose, T., Magnusson, G., Hultgren, S. J. & Waksman, G. (2001). Structural basis of the interaction of the pyelonephritic E. coli adhesin to ist human kideny receptor. Cell 105, 733-743.</ref>). Fimbrial adhesins from other organisms, such as CupB6 from ''Pseudomonas aeruginosa'' are also investigated. All share the immunoglobulin-like fold of the two structural components, despite lack of any sequence identity and diversity in carbohydrate specificity and binding site, and the corresponding pili are assembled by the chaperone-usher pathway<ref>De Greve, H., Wyns, L. & Bouckaert, J. (2007). Combining sites of bacterial fimbriae. Curr Opin Struct Biol 17, 506-12.</ref><ref>Sauer, F. G., Barnhart, M., Choudhury, D., Knight, S. D., Waksman, G. & Hultgren, S. J. (2000). Chaperone-assisted pilus assembly and bacterial attachment. Curr Opin Struct Biol 10, 548-56.</ref>. The paradigm is unique among TAD for his specificity toward GlcNAc. The binding site is located laterally and not at the tip of the pili, therefore the long and flexible F17 fimbriae could intrude between the microvilli of the epithelium, with the binding site of the lectin domain interacting laterally with GlcNAc-containing receptors. Five naturally occurring variants, differing in 1-18 amino acids of the adhesion domain have been identified<ref>De Kerpel, M., Van Molle, I., Brys, L., Wyns, L., De Greve, H. & Bouckaert, J. (2006). N-terminal truncation enables crystallization of the receptor-binding domain of the FedF bacterial adhesin. Acta Crystallogr Sect F Struct Biol Cryst Commun 62, 1278-82.</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
This is an emerging field of investigation and contributions arose from a small number of CFG Participating Investigators (PIs). These include: Esther Bullit, Eric Cox, Anne Imberty, Remy Loris, James Nataro

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about F17G/GafD, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===

The F17G adhesin is most specific for the disaccharide GlcNAcb1,3Gal that can be recognised as a terminal or internal sequence in bovine glycophorin <ref>Mouricout, M., Milhavet, M., Durié, C., Grange, P. Characterization of glycoprotein glycan receptors for Escherichia coli F17 fimbrial lectin, Microb. Pathog. (1995) 18, 297-306</ref>:
 
[[File:carbSynthe_0691_D000.jpg]]
 
The branched form gives a closely similar fluorescent signal:
 
[[File:carbSynthe_0897_D000.jpg]]
 
The presence of the b1,3 linkage of N-acetyl glucosamine to galactose enhances the affinity for F17G at least 2-fold, compared to the monosaccharide N-acetyl glucosamine, as validated using surface plasmon resonance measurements. Second best binders are the b1,4 and b1,6 galactose linked disaccharides, whereas chitobiose, that is also a characterized inhibitor of F17G-mediated bacterial adhesion, is clearly lagging behind. F17G can thus be ranked under glycan binding proteins that display high selectivity.

=== Cellular expression of GBP and ligands ===

F17G adhesins are expressed on enterotoxigenic E. coli infecting neonatal lambs, calves, and goat kids.
The F17G ligand GlcNAcb1,3Gal occurs universally but mostly internally in the sequence of poly-lactosaminyl glycans and blood group antigens.
These glycan structures are widely expressed on mamalian cell surfaces .

=== Biosynthesis of ligands ===
 
F17-fimbriated E. coli predominantly colonize neonatal animals, but also are a major causal agent (55%) of mastitis in bovines <ref>Lipman, L.J.A., de Nijs A., Gaastra W.. Isolation and identification of fimbriae and toxin production by Escherichia coli strains from cows with clinical mastitis, Vet. Microbiology 47 (1995) p. 1-7 </ref>. Congruent with the glycans recognized by F17G on the printed array versions 2.1 and 4.1, the N-acetyl glucosamine residue of GlcNAcb1,3Gal may be unsubstituted at the early life stage of calves, that are at the same time protected from bacterial infections by glycans secreted in the cow's milk.

=== Structure ===

The F17G adhesin is a two-domain adhesin (TDA) located at the F17 fimbrial tip. The determination of the crystal structure of the F17G lectin domain led to the discovery of the variable immunoglobulin-like structure
as a paradigm for bacterial fimbrial TDAs <ref name=" Buts, L2003">
</ref>. F17G has a shallow groove for carbohydrate recognition on its flank.

 
[[File:F17G_Igfold.jpg]]
 

=== Biological roles of GBP-ligand interaction ===

The F17G fimbrial lectin enhances intestinal colonization in the early life of ruminants. The long and flexible F17 fimbriae can penetrate deep between intestinal microvilli, where the fimbrial tip adhesin finds its glycan receptors. The subsequent secretion of heat stable and heat labile toxins can lead to severe diarrhea.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=fimbriae&maxresults=20 fimbriae] and [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=pili&maxresults=20 pili].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Not applicable, as the microarrays only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
F17G adhesins have been screened for their glycan specificity (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2_177_11182005 here]). To see all glycan array results for F17G adhesin, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=F17G&cat=coreh here].

== Related GBPs ==
FedF [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=FedF&maxresults=20 (CFG data)], CfaE [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CfaE&maxresults=20 (CFG data)], FimH [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=FimH&maxresults=20 (CFG data)], PapG, CupB6 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CupB6&maxresults=20 (CFG data)].

The specificity of some of the other fimbrial tip adhesins was determined by CFG glycan array analysis ([http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2358 ''P. gingivalis'' fimbriae], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2_178_11182005 ''E. coli'' FedF adhesin], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1106 ''E. coli'' CfaE adhesin from CFA/I pili])

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Alisdair Boraston, Julie Bouckaert, Anne Imberty

CBM47

2011-03-28T19:09:21Z

Carole Weaver: /* Glycogene microarray */

Enzymes that degrade host glycans are increasingly being found as virulence factors in pathogenic bacteria<ref>Shelburne, S. A., Davenport, M. T., Keith, D. B. & Musser, J. M. (2008). The role of complex carbohydrate catabolism in the pathogenesis of invasive streptococci. Trends Microbiol 16, 318-25.</ref><ref name="Hava 16">Hava, D. L. & Camilli, A. (2002). Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol Microbiol 45, 1389-406.</ref>. A common property of extracellular glycan degrading enzymes found in such bacteria is multi-modularity; these enzymes often comprise a large number of modules with a variety of functions. The most common class of ancillary module are carbohydrate-binding modules (CBMs)<ref>Boraston, A. B., Bolam, D. N., Gilbert, H. J. & Davies, G. J. (2004). Carbohydrate-binding modules: fine tuning polysaccharide recognition. Biochem J 382, 769-782.</ref>, which are alternatively referred to as lectin-domains. These modules are responsible for targeting carbohydrate-degrading enzymes to a glycan substrate or, when the enzymes are attached to the bacterial cell-surface, likely also function to adhere the bacterium to a glycan<ref>Ficko-Blean, E., Gregg, K. J., Adams, J. J., Hehemann, J. H., Czjzek, M., Smith, S. P. & Boraston, A. B. (2009). Portrait of an enzyme, a complete structural analysis of a multimodular {beta}-N-acetylglucosaminidase from Clostridium perfringens. J Biol Chem 284, 9876-84.</ref>. The presence of these lectin-domains in multi-modular proteins and their contribution of glycan binding function to catalytically active proteins distinguishes these modules from other bacterial glycan-binding proteins (GBPs). The CBM47 modules from the ''Streptococcus pneumoniae'' enzyme SpGH98 (or "fucolectin-related protein") are specific to the Lewisy antigen<ref name ="Boraston 2006">Boraston, A. B., Wang, D. & Burke, R. D. (2006). Blood group antigen recognition by a Streptococcus pneumoniae virulence factor. J Biol Chem 281, 35263-35271.</ref>, which is quite rare among all GBPs, and function to target this enzyme to this antigen when present on epithelial cells<ref>Higgins, M. A., Whitworth, G. E., El Warry, N., Randriantsoa, M., Samain, E., Burke, R. D., Vocadlo, D. J. & Boraston, A. B. (2009). Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases. J Biol Chem 284, 26161-73.</ref>. Recognition and destruction of this antigen appears to be a critical process in pneumococcal virulence<ref name="Hava 16"/><ref>Embry, A., Hinojosa, E. & Orihuela, C. J. (2007). Regions of Diversity 8, 9 and 13 contribute to Streptococcus pneumoniae virulence. BMC Microbiol 7, 80.</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
This is a very new area of investigation. CFG Participating Investigators (PIs) that have screened other CBMs or proteins containing CBMs include: Alisdair Boraston, Garry Taylor, Warren Wakarchuck

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about CBM47, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===
CBM47 is a fucose specific binding module that is related to ''Anguilla anguilla'' fucolectin. The high affinity ligand for CBM47 has been determined from glycan microarray screening on the CFG microarray[http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_34_08192004#]to be Fucα1-2Galβ1-4(Fucα1-3)GlcNAc [Lewis y]<ref name="Boraston 2006"/>.

[[File:Lewisy.jpg]]
 

=== Cellular expression of GBP and ligands ===

 
=== Biosynthesis of ligands ===

 
=== Structure ===

CBM47 originates from a multimodular ''S. pneumoniae'' that has an N-terminal, Lewisy degrading catalytic module. Indeed, three CBM47 modules are found in tandem.

[[File:modular.jpg]]

The high resolution X-ray structures of the N-terminal and C-terminal CBM47 modules have been determined and the N-terminal module in complex with the Lewisy tetrasaccharide<ref name="Boraston 2006"/>.

[[File:CBM47.jpg]]

 

=== Biological roles of GBP-ligand interaction ===

The primary role of CBM47, and indeed other CBMs found in carbohydrate-active enzymes, is to direct the entire enzyme to its glycan substrate. However, CBMs found in carbohydrate-active enzymes attached to microbial cell surfaces may also play a role in the adhesion of the bacterium to host glycan-bearing tissues.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CBM&maxresults=20 CFG database search results for CBM].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Not applicable, as the microarrays only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The specificity of several CBMs have been investigated by CFG glycan array analysis (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1831 here] for example). Isolated glycans for structural and quantitative binding studies have also been obtained from the CFG.

== Related GBPs ==

There are presently over 55 families of CBMs that are defined on the basis of amino acid sequence similarity; however, the majority of these CBMs families contain members specific for plant cell wall polysaccharides. CFG resources have been instrumental in studying the subset of CBMs that recognize complex glycans. In addition to CBM47 there are a number of CBMs belonging to family 32 and 51 that share the property of binding complex glycans. Though unrelated at the amino acid sequence level CBMs in families 32, 47, and 51 are structurally related and functionally related. It is important to note, however, that the diversity of complex glycan binding among these family members is considerable and only coming to light through glycan array screening. The availability of purfied glycans is facilitating structural and quantitative studies of glycan binding by CBMs in families 32, 47, and 51.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Alisdair Boraston, Anne Imberty

PA-IIL

2011-03-28T19:08:53Z

Carole Weaver: /* Glycogene microarray */

PA-IIL (Pseudomonas lectin II, LecB) is a cytoplasmic lectin produced in ''Pseudomonas aruginosa'' under the control of quorum sensing. It is a virulence factor involved in binding of human tissues such as epithelial lung of cystic fibrosis patients<ref name="Imberty 2004">Imberty, A., Wimmerova, M., Mitchell, E. P. & Gilboa-Garber, N. (2004). Structures of the lectins from Pseudomonas aeruginosa: Insights into molecular basis for host glycan recognition. Microb. Infect. 6, 222-229.</ref>. The role of PA-IIL in pathogenesis has been highlighted, since interfering with PA-IIL binding site reduces the mortality of ''P. aeruginosa'' induced-pneumonia in a murine model<ref name="Chemani 2009">Chemani, C., Imberty, A., de Bentzman, S., Pierre, P., Wimmerová, M., Guery, B. P. & Faure, K. (2009). Role of LecA and LecB lectins in Pseudomonas aeruginosa induced lung injury and effect of carbohydrates ligands. Infect. Immun. 77, 2065-2075.</ref>. The paradigm is unique among glycan-binding proteins (GBPs) in that it contains two cationic ions in the carbohydrate binding site that result in unusual high affinity for the carbohydrate target<ref>Loris, R., Tielker, D., Jaeger, K.-E. & Wyns, L. (2003). Structural basis of carbohydrate recognition by the lectin LecB from Pseudomonas aeruginosa. J. Mol. Biol. 331, 861-870.</ref><ref name="test 1">Mitchell, E., Houles, C., Sudakevitz, D., Wimmerova, M., Gautier, C., Pérez, S., Wu, A. M., Gilboa-Garber, N. & Imberty, A. (2002). Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. Nature Struct. Biol. 9, 918-921.</ref><ref>Mitchell, E. P., Sabin, C., Šnajdrová, L., Pokorná, M., Perret, S., Gautier, C., Hofr, C., Gilboa-Garber, N., Koča, J., Wimmerová, M. & Imberty, A. (2005). High affinity fucose binding of Pseudomonas aeruginosa lectin PA-IIL: 1.0 Å resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches. Proteins: Struct. Funct. Bioinfo. 58, 735-748.</ref>. The preferred glycan ligand of PA-IIL is the trisaccharide Lewis a<ref name="test 2">Perret, S., Sabin, C., Dumon, C., Pokorná, M., Gautier, C., Galanina, O., Ilia, S., Bovin, N., Nicaise, M., Desmadril, M., Gilboa-Garber, N., Wimmerova, M., Mitchell, E. P. & Imberty, A. (2005). Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa. Biochem. J. 389, 325-332.</ref>. PA-IIL-like lectins have been characterized in other opportunistic bacteria such as ''Ralstonia solanacearum''<ref>Sudakevitz, D., Kostlanova, N., Blatman-Jan, G., Mitchell, E. P., Lerrer, B., Wimmerova, M., Katcof, f. D. J., Imberty, A. & Gilboa-Garber, N. (2004). A new Ralstonia solanacearum high affinity mannose-binding lectin RS-IIL structurally resembling the Pseudomonas aeruginosa fucose-specific lectin PA-IIL. Mol. Microbiol. 52, 691-700.</ref>, ''Chromobacterium violaceum''<ref name="Pokorna 11">Pokorná, M., Cioci, G., Perret, S., Rebuffet, E., Kostlánová, N., Adam, J., Gilboa-Garber, N., Mitchell, E. P., Imberty, A. & Wimmerová, M. (2006). Unusual entropy driven affinity of Chromobacterium violaceum lectin CV-IIL towards fucose and mannose. Biochemistry 45, 7501-7510.</ref>, and ''Burkholderia cenocepacia''<ref>Lameignere, E., Malinovská, L., Sláviková, M., Duchaud, E., Mitchell, E. P., Varrot, A., Šedo, O., Imberty, A. & Wimmerová, M. (2008). Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia. Biochem. J. 411, 307-318.</ref><ref>Lameignere, E., Shiao, T. C., Roy, R., Wimmerová, M., Dubreuil, F., Varrot, A. & Imberty, A. (2010). Structural basis of the affinity for oligomannosides and analogs displayed by BC2L-A, a Burkholderia cenocepacia soluble lectin. Glycobiology 20, 87-98.</ref>, albeit with some variations in the fine specificity. Starting from the results obtained with the help of CFG tools, major efforts are devoided in collaboration with carbohydrate chemists in order to design anti-bacterial new glyco-derived compounds that bind to PA-IIL with high affinity<ref>Imberty, A., Chabre, Y. M. & Roy, R. (2008). Glycomimetics and glycodendrimers as high affinity microbial antiadhesins. Chemistry 14, 7490-7499.</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) have made major contribution to the understanding of the structure/specificity relationship of PA-IIL and PA-IIL-like proteins. These include: Anne Imberty, Remy Loris, Michaela Wimmerova

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about PA-IIL, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===

PA-IIL is a fucose-specific lectin that also recognises mannose residues. 
The high affinity ligand for PA-IIL has been deduced from glycan microarray screening on the CFG microarray[http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_221_02142006#] to be Galβ1-4(Fucα1-4)GlcNAc [Lewis a] <ref name="test 1"></ref><ref name="test 2"></ref><ref>Marotte K, Sabin C, Preville C, Moume-Pymbock M, Wimmerova M, Mitchell EP, Imberty A, Roy R. (2008) X-ray Structures and thermodynamics of the interaction of PA-IIL from Pseudomonas aeruginosa with disaccharide derivatives. ChemMedChem 10,1328-1338.</ref>

[[File:lewisa.jpg]]

=== Cellular expression of GBP and ligands ===
PA-IIL is produced in ''Pseudomonas aruginosa,'' and binds ligands in human tissues, including epithelial lung.
 
=== Biosynthesis of ligands ===

 
=== Structure ===
PA-IIL is a tetrameric lectin. Each monomer consists of a β-sandwich and contains two calcium ions that interact directly with the carbohydrate ligand. Crystal structures have been determined for PA-IIL alone, and its complex with fucose, with related monosaccharides, and with complex fucosylated oligosaccharides.

[[File:pa2ls.jpg]]
 
Information on crystal structures of PA-IIL and links to PDB are available at PA-IIL page of [http://www.cermav.cnrs.fr/cgi-bin/lectines/menu.cgi?Bacterial%20lectins+2-CA%20b-sandwich+Pseudomonas%20PA-IIL 3D-lectin database]

=== Biological roles of GBP-ligand interaction ===
PA-IIL, a virulence factor that binds human tissues such as epithelial lung of cystic fibrosis patients<ref name="Imberty 2004"/>, has roles in pathogenesis <ref name="Chemani 2009"/>.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=PA-IIL&maxresults=20 CFG database search results for PA-IIL].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Not applicable, as the CFG microarrays only contain probes for mouse and human glycogenes.
 

=== Knockout mouse lines ===
Not applicable.
 

=== Glycan array ===
The specificity of PA-IIl and related proteins was determined through glycan array analysis (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2_230_02102006 here] and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_221_02142006# here]).

== Related GBPs ==
CV-IIL [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=CV-IIL&maxresults=20 (CFG data)], RS-IIL [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=RS-IIL&cat=coreh (CFG data)], BC2L-A, BC2L-B [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BC2L-B&maxresults=20 (CFG data)], BC2L-C [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=BC2L-C&maxresults=20 (CFG data)].

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Anne Imberty and Micha Wimmerova

Ficolin M (Ficolin 1)

2011-03-28T19:07:51Z

Carole Weaver: /* Glycogene microarray */

The ficolins are a group of soluble animal proteins with roles in innate immunity<ref>Lu, J. and Le, Y. Ficolins and the fibrinogen-like domain. Immunobiology 199, 190-199 (1998)</ref><ref name="Teh 30">Teh, C., Le, Y., Lee, S. H. and Lu, J. M-ficolin is expressed on monocytes and is a lectin binding to N-acetyl-D-glucosamine and mediates monocyte adhesion and phagocytosis of Escherichia coli. Immunology 101, 225-232 (2000)</ref><ref>Matsushita, M. and Fujita, T. The role of ficolins in innate immunity. Immunobiology 205, 490-497 (2002)</ref><ref name="Endo 32">Endo, Y., Liu, Y. and Fujita, T. Structure and function of ficolins. Adv Exp Med Biol 586, 265-279 (2006) </ref><ref name="Zhang 33">Zhang, X. L. and Ali, M. A. Ficolins: structure, function and associated diseases. Adv Exp Med Biol 632, 105-115 (2008)</ref>. The classification of ficolins as lectins is somewhat controversial since the ligand binding domain in ficolins is specific for acetyl groups in both carbohydrates (e.g. GlcNAc, ManNAc, GalNAc) and non-carbohydrates (eg N-acetylglycine, N-acetylcysteine, acetylcholine)<ref name="Teh 30"/><ref name="Frederiksen 34">Frederiksen, P. D., Thiel, S., Larsen, C. B. and Jensenius, J. C. M-ficolin, an innate immune defence molecule, binds patterns of acetyl groups and activates complement. Scand J Immunol 62, 462-473 (2005)</ref>. Binding of sugars is not primarily dependent on the sugar ring, and sugars that do not contain acetyl groups are generally not ficolin ligands<ref name="Endo 32"/>. However, many of the bacterial surface molecules that appear to be natural ligands for the ficolins contain carbohydrate moieties, and ficolins have similar functional properties to lectins. They are certainly capable of binding acetylated sugars as evidenced by glycan array screening<ref name="Gout 35">Gout, E., Garlatti, V., Smith, D. F., Lacroix, M. M., Dumestre-Perard, C., Lunardi, T., Martin, L., Cesbron, J. Y., Arlaud, G. J., Gaboriaud, C. and Thielens, N. M. Carbohydrate recognition properties of human ficolins: Glycan array screening reveals the sialic acid binding specificity of M-ficolin. J Biol Chem (2009) </ref>. Ficolin M (Ficolin 1) and Ficolin L (Ficolin 2) are the most widely studied<ref name="Zhang 33"/><ref name="Frederiksen 34"/><ref name="Gout 35"/><ref name="Liu 2005">Liu, Y., Endo, Y., Iwaki, D., Nakata, M., Matsushita, M., Wada, I., Inoue, K., Munakata, M. and Fujita, T. Human M-ficolin is a secretory protein that activates the lectin complement pathway. J Immunol 175, 3150-3156 (2005)</ref><ref name="Tanio 2006">Tanio, M., Kondo, S., Sugio, S. and Kohno, T. Overexpression, purification and preliminary crystallographic analysis of human M-ficolin fibrinogen-like domain. Acta Crystallogr Sect F Struct Biol Cryst Commun 62, 652-655 (2006)</ref><ref name="Garlatti 2007">Garlatti, V., Martin, L., Gout, E., Reiser, J. B., Fujita, T., Arlaud, G. J., Thielens, N. M. and Gaboriaud, C. Structural basis for innate immune sensing by M-ficolin and its control by a pH-dependent conformational switch. J Biol Chem 282, 35814-35820 (2007)</ref><ref name="Frankenberger 39">Frankenberger, M., Schwaeble, W. and Ziegler-Heitbrock, L. Expression of M-Ficolin in human monocytes and macrophages. Mol Immunol 45, 1424-1430 (2008)</ref><ref name="Honore 2008">Honore, C., Rorvig, S., Munthe-Fog, L., Hummelshoj, T., Madsen, H. O., Borregaard, N. and Garred, P. The innate pattern recognition molecule Ficolin-1 is secreted by monocytes/macrophages and is circulating in human plasma. Mol Immunol 45, 2782-2789 (2008)</ref>. Ficolin L is a serum protein produced in the liver that through association with Mannose-binding protein-associated proteases (MASPs) triggers complement activation in response to binding to pathogen surfaces. It also serves as an opsinin triggering phagocytic uptake of pathogens by neutrophils. Polymorphisms in Ficolin L may have pathophysiological implications<ref>Messias-Reason, I. J., Schafranski, M. D., Kremsner, P. G. and Kun, J. F. Ficolin 2 (FCN2) functional polymorphisms and the risk of rheumatic fever and rheumatic heart disease. Clin Exp Immunol 157, 395-399 (2009)</ref>. Ficolin M is produced in the lung and has been examined structurally. Ficolin M is found in secretory granules in neutrophils and monocytes, recognizes pathogens, and also activates complement via MASPs<ref name="Frankenberger 39"/>.

See also: paradigm page for [[Ficolins/Mannose-binding protein]]
== CFG Participating Investigators contributing to the understanding of this paradigm ==
Investigators using CFG resources to study ficolins include: Raymond Dwek, Daniel Mitchell, Nicole Thielens

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about Ficolin M, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===

M-ficolin preferentially binds to 9-O-acetylated 2-6-linked sialic acid derivatives and to various glycans containing sialic acid engaged in α 2-3 linkage, including gangliosides<ref name="Gout 35"/>.

=== Cellular expression of GBP and ligands ===
M-ficolin has been localized at the surface of blood monocytes and in secretory granules of neutrophils, monocytes, and lung epithelial cells<ref name="Teh 30"/><ref name="Frederiksen 34"/><ref name="Liu 2005"/>. However, two recent studies have reported its detection in serum, with mean concentrations ranging from 0.06 to 1 μg/ml<ref name="Honore 2008"/><ref>Wittenborn, T., Thiel, S., Jensen, L., Nielsen, H. J., and Jensenius, J. C. Characteristics and biological variations of M-ficolin, a pattern recognition molecule, in plasma. J Innate Immun 2, 167-180 (2010)</ref>.

=== Biosynthesis of ligands ===
Pathways for ganglioside biosynthesis have been described ([http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/geMolecule.jsp GT Database]).
 O-acetylation at positions C-7,8,9 of sialic acid is catalyzed by O-acetyltransferase enzymes associated with the Golgi membrane in mammals<ref>Higa, H.H., Manzi, A. and Varki, A. O-acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-O-acetylated sialic acids. J Biol Chem 264, 19435-19442 (1989)</ref><ref>Butor, C., Higa, H.H. and Varki, A. Structural, immunological, and biosynthetic studies of a sialic acid-specific O-acetylesterase from rat liver. J Biol Chem 268, 10207-10213 (1993)</ref>. Sialic acid O-acetyltransferases have also been identified in bacteria, but they are not homologous to the vertebrate enzymes<ref>Lewis, A.L., Hensler, M.E., Varki, A. and Nizet, V. The group B streptococcal sialic acid O-acetyltransferase is encoded by neuD, a conserved component of bacterial sialic acid biosynthetic gene clusters. J Biol Chem 281, 11186-11192 (2006)</ref>.

=== Structure ===

The structure of the trimeric fibrinogen-like recognition domain of M-ficolin, alone and in complex with various acetylated ligands, has been solved by X-ray crystallography<ref name="Tanio 2006"/><ref name="Garlatti 2007"/>. A single ligand binding site was observed, located close to the calcium-binding site in the outer part of the trimer and homologous to the GlcNAc binding pocket of the invertebrate tachylectin TL5A <ref>Kairies, N., Beisel, H. G., Fuentes-Prior, P., Tsuda, R., Muta, T., Iwanaga, S., Bode, W., Huber, R., and Kawabata, S. I. Proc. Natl. Acad. Sci. U.S.A. 98, 13519–13524 (2001)</ref>. The essential role of Tyr 271 in the binding specificity for sialic acid was further demonstrated using site-directed mutagenesis<ref name="Gout 35"/>.

=== Biological roles of GBP-ligand interaction ===

Soluble M-ficolin has been shown to bind to ''Staphylococcus aureus'' through GlcNAc<ref name="Liu 2005"/>. It is tethered to monocytes and granulocytes through binding of its fibrinogen-like recognition domain to sialic acid on the cell surface<ref>Honore C, Rorvig S, Hummelshoj T, Skjoedt MO, Borregaard N, and Garred P. Tethering of Ficolin-1 to cell surfaces through recognition of sialic acid by the fibrinogen-like domain. J Leukoc Biol 88, 145-158 (2010)</ref>.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=ficolin&maxresults=20 CFG database search results for ficolin].

=== Glycan profiling ===
Glycan profiling has not been performed.
 

=== Glycogene microarray ===
No data available.
 

=== Knockout mouse lines ===

 
=== Glycan array ===
The soluble ficolins have been extensively examined using the CFG glycan array ([http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=ficolin&cat=coreh 28 screens]).
 Human M-ficolin
investigators have used CFG glycan arrays to study the ligand binding specificity of human recombinant wild-type M-ficolin at various concentrations (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1602 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2417 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2123 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2124 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2412 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2418 here], and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2419 here]), and of 4 variants with the following point mutations in the recognition domain: G221F (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2413 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2420 here], and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2421 here]), A256V (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2414 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2422 here], and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2423 here]), G221F/A256V (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2415 here]) and Y271F (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2416 here] and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2417 here]). The binding specificity of the rat homologue of M-ficolin, ficolin B, has also been investigated using CFG glycan arrays (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1708 here] and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1768 here]).

== Related GBPs ==
This family is characterized by the presence of a leader peptide, a short N-terminal segment, followed by a collagen-like region, and a C-terminal fibrinogen-like domain. Homologs are apparently absent in ''D. melanogaster'' and ''C. elegans''. Several human family members have been described but Ficolin L and M are the best characterized both biochemically and structurally. Ficolin L [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=ficolin+AND+L&maxresults=20 (CFG data)] and H [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=ficolin+AND+H&maxresults=20 (CFG data)] are made in the liver, while Ficolin M and H are produced by the lung. Two ficolins (A and B) are present in mouse. Ficolin B may be the ortholog of Ficolin M.
 A vertebrate membrane-bound chitin-binding protein, called FIBCD1 (Fibrinogen C domain containing 1) has been identified recently<ref>Schlosser A, Thomsen T, Moeller JB, Nielsen O, Tornoe I, Mollenhauer J, Moestrup SK, and Holmskov U. Characterization of FIBCD1 as an acetyl group-binding receptor that binds chitin. J Immunol 183, 3800-3809 (2009)</ref>. The ectodomain of FIBCD1 forms disulfide-linked tetramers assembled from a coiled-coil region, a polycationic region and a C-terminal fibrinogen-related domain. The acetyl-binding site of the fibrinogen-like recognition domain of FIBCD1 is homologous to that of TL5A and M-ficolin<ref> Thomsen T, Moeller JB, Schlosser A, Sorensen GL, Moestrup SK, Palaniyar N, Wallis R, Mollenhauer J, and Holmskov U. The recognition unit of FIBCD1 organizes into a non-covalently linked tetrameric structure and uses a hydrophobic funnel (S1) for acetyl group recognition. J Biol Chem 285, 1229-1028 (2010)</ref>.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: John Hanover, Nicole Thielens

Galectin-9

2011-03-25T23:50:19Z

Carole Weaver: /* Glycogene microarray */

Galectin-9 is the best-studied of the tandem-repeat galectins and the crystal structure of the N-terminal carbohydrate recognition domain (CRD) is known. In addition, Galectin-9...
* uniquely binds poly-N-acetyllactosamine sequences by recognizing internal N-acetyllactosamine repeats<ref>Nagae, M. et al. Structural analysis of the recognition mechanism of poly-N-acetyllactosamine by the human galectin-9 N-terminal carbohydrate recognition domain. Glycobiology 19, 112-117 (2009). </ref>
* binds distinct ligands from [[Galectin-1]]<ref>Bi, S., Earl, L.A., Jacobs, L. & Baum, L.G. Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways. J Biol Chem 283, 12248-12258 (2008).</ref>
* has three well-characterized linker domains between the CRDs, generated by alternative splicing, that regulate cellular localization and function of the protein
* is the only tandem-repeat galectin that has been administered in animal models of disease to assess therapeutic potential<ref>Baba, M. et al. Galectin-9 inhibits glomerular hypertrophy in db/db diabetic mice via cell-cycle-dependent mechanisms. J Am Soc Nephrol 16, 3222-3234 (2005). </ref><ref name="Seki 2008">Seki, M. et al. Galectin-9 suppresses the generation of Th17, promotes the induction of regulatory T cells, and regulates experimental autoimmune arthritis. Clin Immunol 127, 78-88 (2008).
</ref><ref>Tsuchiyama, Y. et al. Efficacy of galectins in the amelioration of nephrotoxic serum nephritis in Wistar Kyoto rats. Kidney Int 58, 1941-1952 (2000). </ref>
* null mice have increased susceptibility to autoimmune disease
* binds to a unique glycoprotein ligand Tim-3 expressed in Th1 and Th17 cells<ref name="Seki 2008" /><ref>Naka, E.L., Ponciano, V.C., Cenedeze, M.A., Pacheco-Silva, A. & Camara, N.O. Detection of the Tim-3 ligand, galectin-9, inside the allograft during a rejection episode. Int Immunopharmacol 9, 658-662 (2009).
</ref><ref>Niwa, H. et al. Stable form of galectin-9, a Tim-3 ligand, inhibits contact hypersensitivity and psoriatic reactions: a potent therapeutic tool for Th1- and/or Th17-mediated skin inflammation. Clin Immunol 132, 184-194 (2009).</ref><ref>Anderson, D.E. TIM-3 as a therapeutic target in human inflammatory diseases. Expert Opin Ther Targets 11, 1005-1009 (2007). </ref>

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of Galectin-9 include: Linda Baum, Richard Cummings, Gabriel Rabinovich, Sachiko Sato

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about Galectin-9, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Page for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Stlect_00120&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_1307&sideMenu=no mouse] Galectin-9 in the CFG database.
=== Carbohydrate ligands ===

 
=== Cellular expression of GBP and ligands ===

 
=== Biosynthesis of ligands ===

 
=== Structure ===

 
=== Biological roles of GBP-ligand interaction ===

 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-9&maxresults=20 CFG database search results for Galectin-9].

=== Glycan profiling ===

 
=== Glycogene microarray ===
Probes for human galectin-9 have been included in all versions of the CFG glycogene chip, and probes for mouse galectin-9 are included on versions 2, 3, and 4.
 

=== Knockout mouse lines ===

 
=== Glycan array ===
Investigators have used CFG carbohydrate compounds and glycan array screening to study ligand binding specificity of Galectin-9 (for example, click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2735 here]). To see all glycan array results for Galectin-9, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=galectin-9&cat=coreh here].

== Related GBPs ==
Galectin-4 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-4&maxresults=20 (CFG data)], galectin-6, galectin-8 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-8&maxresults=20 (CFG data)], and galectin-12 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-12&maxresults=20 (CFG data)].

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Linda Baum, Richard Cummings

Galectin-3

2011-03-25T23:49:38Z

Carole Weaver: /* Glycogene microarray */

Galectin-3...
* is the only member of the chimeric subfamily in mammals
* is a very well-studied glycan-binding protein (GBP)
* has a known crystal structure (C-terminal glycan-binding domain)
* has unique functions intra- and extra-cellularly, due to an unusual N-terminal domain that can participate in protein-protein interactions
* has a unique mode of multimerization
* is the only known anti-apoptotic galectin, and acts through intracellular action<ref>Saegusa J, Hsu DK, Liu W, Kuwabara I, Kuwabara Y, Yu L, Liu FT [http://www.ncbi.nlm.nih.gov/pubmed/18463681 Galectin-3 protects keratinocytes from UVB-induced apoptosis by enhancing AKT activation and suppressing ERK activation.] J Invest Dermatol. 2008 Oct;128(10):2403-11. PubMed PMID: 18463681; PubMed Central PMCID: PMC2768377.</ref>
* null mice have distinct phenotypes, including alterations in inflammatory and wound-healing responses, and cyst formation in disease<ref>Chiu MG, Johnson TM, Woolf AS, Dahm-Vicker EM, Long DA, Guay-Woodford L,
Hillman KA, Bawumia S, Venner K, Hughes RC, Poirier F, Winyard PJ. [http://www.ncbi.nlm.nih.gov/pubmed/17148658 Galectin-3 associates with the primary cilium and modulates cyst growth in congenital polycystic kidney disease.] Am J Pathol. 2006 Dec;169(6):1925-38.</ref>
* has unique functions in innate immune response to microbial pathogens
* has been administered in animal models of disease to assess therapeutic potential
* binds distinct cell surface glycoprotein ligands in lymphocytes compared to galectin-1
* expression is involved in growth modulation<ref>Baptiste TA, James A, Saria M, Ochieng J. [http://www.ncbi.nlm.nih.gov/pubmed/17184769 Mechano-transduction mediated secretion and uptake of galectin-3 in breast carcinoma cells: implications in the extracellular functions of the lectin.] Exp Cell Res. 2007 Feb 15;313(4):652-64. Epub 2006 Nov 16. PubMed PMID: 17184769; PubMed Central PMCID: PMC1885467.</ref>
 
Galectin-3 is the only member of the galectin family with an extended N-terminal region composed of tandem repeats of short amino-acid segments (a total of approximately 120 amino acids) connected to a C-terminal CRD. Like other galectins, galectin-3 lacks a signal sequence required for secretion through the classical secretory pathway, but the protein is released into the extracellular space. 
Galectin-3 can oligomerize in the presence of multivalent carbohydrate ligands and is capable of crosslinking glycans on the cell surface, thereby initiating transmembrane signaling events and affecting various cellular functions (reviewed in <ref name="Liu 2005">Liu FT, Rabinovich GA. [http://www.ncbi.nlm.nih.gov/pubmed/15630413 Galectins as modulators of tumour progression.] Nat Rev Cancer. 2005 Jan;5(1):29-41. Review. PubMed PMID: 15630413.</ref><ref>Almkvist J, Karlsson A. [http://www.ncbi.nlm.nih.gov/pubmed/14758082 Galectins as inflammatory mediators.] Glycoconj J.
2004;19(7-9):575-81. Review. PubMed PMID: 14758082.</ref><ref>Ochieng J, Furtak V, Lukyanov P. [http://www.ncbi.nlm.nih.gov/pubmed/14758076 Extracellular functions of galectin-3.] Glycoconj J. 2004;19(7-9):527-35. Review. PubMed PMID: 14758076.</ref>). This ability to self-associate is dependent on the N-terminal region of the protein.
 Compared to other galectins, intracellular functions of galectin-3 have been more extensively documented (reviewed in <ref>Liu FT, Patterson RJ, Wang JL. [http://www.ncbi.nlm.nih.gov/pubmed/12223274 Intracellular functions of galectins.] Biochim Biophys Acta. 2002 Sep 19;1572(2-3):263-73. Review. PubMed PMID: 12223274.</ref>). In some cases, intracellular proteins with which the protein interacts and which possibly mediate these functions have been identified. Galectin-3 can be phosphorylated at serines 6 & 12<ref>Huflejt ME, Turck CW, Lindstedt R, Barondes SH, Leffler H. [http://www.ncbi.nlm.nih.gov/pubmed/8253806 L-29, a soluble lactose-binding lectin, is phosphorylated on serine 6 and serine 12 in vivo and by casein kinase I.] J Biol Chem. 1993 Dec 15;268(35):26712-8. PubMed PMID:8253806.</ref>, and tyrosines 79 & 118 by c-Abl<ref>Balan V, Nangia-Makker P, Jang YS, Wang Y, Raz A. [http://www.ncbi.nlm.nih.gov/pubmed/20600357 Galectin-3: A novel substrate for c-Abl kinase.] Biochim Biophys Acta. 2010 Jun 30. PubMed PMID: 20600357</ref><ref>Li X, Ma Q, Wang J, Liu X, Yang Y, Zhao H, Wang Y, Jin Y, Zeng J, Li J, Song L, Li X, Li P, Qian X, Cao C. [http://www.ncbi.nlm.nih.gov/pubmed/20150913 c-Abl and Arg tyrosine kinases regulate lysosomal degradation of the oncoprotein Galectin-3.] Cell Death Differ. 2010 Aug;17(8):1277-87. Epub 2010 Feb 12. PubMed PMID: 20150913.</ref>.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of Galectin-3 include: Pablo Argüeso, Linda Baum, Susan Bellis, Roger Chammas, Richard Cummings, James Dennis, Margaret, Huflejt, Fu-Tong Liu, Joshiah Ochieng, Noorjahan Panjawani, Mauro Perretti, Avram Raz, James Rini, Maria Roque-Barreira, Sachiko Sato, Tariq Sethi, Irma van Die, Gerardo Vasta, John Wang, Paul Winyard

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about Galectin-3, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Pages for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Stlect_00118&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_1306&sideMenu=no mouse] Galectin-3 in the CFG database.
=== Carbohydrate ligands ===

 
=== Cellular expression of GBP and ligands===
Galectin-3 is constitutively expressed in epithelial and myeloid cells, and regulated by processes that include cell proliferation, inflammation, and tumor initiation and progression[cite]. Gene expression in various cells and tissues have been performed with CFG [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=galectin-3&cat=coree Core IgE].
 
=== Biosynthesis of ligands ===
The immunomodulatory activity of galectin 3 is mediated by binding to poly N-acetyllactosamine chains attached to the T cell receptor, resulting in a decrease in the lateral mobility of the receptor, which suppresses its activation. Attachment of the poly N-acetyllactosamine chains is dependent on the establishment of a 1-6 branch on the core oligosaccharide, through the action of GlcNAc transferase V (Mgat5) ([http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_hum_553&sideMenu=true&pageType=general Human][http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_mou_597&sideMenu=true&pageType=general Mouse]). Extension of the chain requires the action of UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyltransferase 1 ([http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_hum_536&sideMenu=true&pageType=general Human][http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_mou_571&sideMenu=true&pageType=general Mouse]) and galactosyltransferase and β1-4galatosyltransferase 1 ([http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_hum_436&sideMenu=true&pageType=general Human][http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_mou_460&sideMenu=true&pageType=general Mouse]).
 

=== Structure ===
The structures of the CRD of galectin-3 from X-ray crystallographic<ref>Seetharaman, J., Kanigsberg, A., Slaaby, R., Leffler, H., Barondes, S.H., and Rini, J.M. [http://www.ncbi.nlm.nih.gov/pubmed/9582341 X-ray crystal structure of the human galectin-3 carbohydrate recognition domain at 2.1 angstrom resolution.] J. Biol. Chem. 1998; 273: 13047–13052. PMID: 9582341.</ref> and NMR<ref>Umemoto K, Leffler H, Venot A, Valafar H, Prestegard JH. [http://www.ncbi.nlm.nih.gov/pubmed/12667058 Conformational differences in liganded and unliganded states of Galectin-3.] Biochem. 2003; 8;42(13):3688-3695. PMID: 12667058.</ref> analyses have been described. The proline-rich N-terminal domain is required for oligomerization of galectin-3<ref>Hsu DK, Zuberi RI, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/1629216 Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin.] J Biol Chem. 1992; 267(20):14167-14174. PMID: 1629216.</ref> and demonstrates significant interaction with the CRD as initially suggested by observations that a monoclonal antibody recognizing an epitope in the N-terminus was capable of inhibiting glycan binding in the C-terminal CRD<ref>Liu FT, Hsu DK, Zuberi RI, Hill PN, Shenhav A, Kuwabara I, Chen SS. [http://www.ncbi.nlm.nih.gov/pubmed/8634249 Modulation of functional properties of galectin-3 by monoclonal antibodies binding to the non-lectin domains.] Biochemistry. 1996; 35(19):6073-6079. PMID: 8634249.</ref>, and revealed by NMR and EM studies<ref>Birdsall B, Feeney J, Burdett ID, Bawumia S, Barboni EA, Hughes RC. [http://www.ncbi.nlm.nih.gov/pubmed/11294654 NMR solution studies of hamster galectin-3 and electron microscopic visualization of surface-adsorbed complexes: evidence for interactions between the N- and C-terminal domains.] Biochem. 2001; 40:4859-4866. PMID: 11294654.</ref>.
 
=== Biological roles of GBP-ligand interaction ===

'''Regulation of cellular responses.''' 
Galectin-3 induces various kinds of biological responses in a variety cell types in vitro by engaging glycoproteins or glycolipids on the cell surfaces (reviewed in <ref>Rabinovich GA, Liu FT, Hirashima M, Anderson A. An emerging role for galectins in tuning the immune response: lessons from experimental models of inflammatory disease, autoimmunity and cancer. Scand J Immunol. 2007 Aug-Sep;66(2-3):143-58. Review. PubMed PMID:17635792 http://www.ncbi.nlm.nih.gov/pubmed/17635792</ref><ref>Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/15775687 Regulatory roles of galectins in the immune response.] Int Arch Allergy Immunol. 2005 Apr;136(4):385-400. Review. PubMed PMID: 15775687.</ref>). 

Galectin-3 can form lattices with selected cell surface glycans, in which galectin-3 oligomers bind to glycans on different glycoproteins displayed on the cell surface. Through this mechanism, galectin-3 modulates the properties and responses of the glycoproteins, such as their lateral mobility on the cell surface, rate of endocytosis, and transmission of signals at the cell surface (reviewed in <ref>Lajoie P, Goetz JG, Dennis JW, Nabi IR. [http://www.ncbi.nlm.nih.gov/pubmed/19398762 Lattices, rafts, and scaffolds: domainregulation of receptor signaling at the plasma membrane.] J Cell Biol. 2009 May 4;185(3):381-5. Epub 2009 Apr 27. Review. PubMed PMID: 19398762; PubMed Central PMCID: PMC2700393.</ref><ref>Grigorian A, Torossian S, Demetriou M. [http://www.ncbi.nlm.nih.gov/pubmed/19594640 T-cell growth, cell surface organization, and the galectin-glycoprotein lattice.] Immunol Rev. 2009 Jul;230(1):232-46. Review. PubMed PMID: 19594640.</ref><ref>Dennis JW, Nabi IR, Demetriou M. [http://www.ncbi.nlm.nih.gov/pubmed/20064370 Metabolism, cell surface organization, and disease.] Cell. 2009 Dec 24;139(7):1229-41. Review. PubMed PMID: 20064370.</ref>). 

Endogenous galectin-3 regulates cellular responses by functioning inside the cells, including pre-mRNA splicing, where galectin-3 functions as a component of spliceosomes<ref>Haudek KC, Spronk KJ, Voss PG, Patterson RJ, Wang JL, Arnoys EJ. [http://www.ncbi.nlm.nih.gov/pubmed/19616076 Dynamics of galectin-3 in the nucleus and cytoplasm.] Biochim Biophys Acta. 2010 Feb;1800(2):181-9. Epub 2009 Jul 16. Review. PubMed PMID: 19616076; PubMed Central PMCID: PMC2815258.</ref>, and regulation of expression of certain genes, including those for cyclin D1, thyroid-specific TTF-1 transcription factor, MUC2 mucin, and c-Jun N-terminal kinase (reviewed in <ref>Nakahara S, Raz A. [http://www.ncbi.nlm.nih.gov/pubmed/17726578 Regulation of cancer-related gene expression by galectin-3 and the molecular mechanism of its nuclear import pathway.] Cancer Metastasis Rev. 2007 Dec;26(3-4):605-10. Review. PubMed PMID: 17726578.</ref><ref>Yang RY, Rabinovich GA, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/18549522 Galectins: structure, function and therapeutic potential.] Expert Rev Mol Med. 2008 Jun 13;10:e17. Review. PubMed PMID: 18549522.</ref>). 

Endogenous Galectin-3 inhibits apoptosis in various cell types by functioning inside the cells (reviewed in <ref>Hsu DK, Yang RY, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/17132510 Galectins in apoptosis.] Methods Enzymol.2006;417:256-73. Review. PubMed PMID: 17132510.</ref><ref>Hsu DK, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/14758074 Regulation of cellular homeostasis by galectins.] Glycoconj J. 2004;19(7-9):507-15. Review. PubMed PMID: 14758074.</ref>). 

Endogenous Galectin-3 controls intracellular trafficking of glycoproteins<ref>Delacour D, Koch A, Jacob R. [http://www.ncbi.nlm.nih.gov/pubmed/19650851 The role of galectins in protein trafficking.] Traffic. 2009 Oct;10(10):1405-13. Epub 2009 Jun 26. Review. PubMed PMID:19650851.</ref><ref>Stechly L, Morelle W, Dessein AF, André S, Grard G, Trinel D, Dejonghe MJ,Leteurtre E, Drobecq H, Trugnan G, Gabius HJ, Huet G. [http://www.ncbi.nlm.nih.gov/pubmed/19192249 Galectin-4-regulated delivery of glycoproteins to the brush border membrane of enterocyte-like cells.] Traffic. 2009 Apr;10(4):438-50. Epub 2009 Jan 24. PubMed PMID: 19192249.</ref>, which may be linked to its ability to translocate into the lumen of transport vesicles. Intracellular galectin-3 is associated with centrosomes in epithelial cells transiently during the process of epithelial polarization and may thus regulate epithelial polarization in enterocytes<ref>Delacour D, Koch A, Ackermann W, Eude-Le Parco I, Elsässer HP, Poirier F,Jacob R. [http://www.ncbi.nlm.nih.gov/pubmed/18211959 Loss of galectin-3 impairs membrane polarisation of mouse enterocytes in vivo.] J Cell Sci. 2008 Feb 15;121(Pt 4):458-65. Epub 2008 Jan 22. PubMed PMID:18211959.</ref><ref>Koch A, Poirier F, Jacob R, Delacour D. [http://www.ncbi.nlm.nih.gov/pubmed/19923323 Galectin-3, a novel centrosome-associated protein, required for epithelial morphogenesis.] Mol Biol Cell. 2010 Jan;21(2):219-31. Epub 2009 Nov 18. PubMed PMID: 19923323; PubMed Central PMCID: PMC2808235.</ref>. Galectin-3 contributes to maintenance of the barrier function of ocular surface epithelial cells<ref>Argüeso P, Guzman-Aranguez A, Mantelli F, Cao Z, Ricciuto J, Panjwani N. [http://www.ncbi.nlm.nih.gov/pubmed/19556244 Association of cell surface mucins with galectin-3 contributes to the ocular surface epithelial barrier.] J Biol Chem. 2009 Aug 21;284(34):23037-45. Epub 2009 Jun 25. PubMed PMID: 19556244; PubMed Central PMCID: PMC2755710.</ref>. 

'''Immunity and inflammation.''' 

''Functions demonstrated in vitro.'' 
T and B cells 
Endogenous galectin-3
:* regulates differentiation of B cells into plasma cells and memory B cells<ref>Acosta-Rodríguez EV, Montes CL, Motrán CC, Zuniga EI, Liu FT, Rabinovich GA, Gruppi A. [http://www.ncbi.nlm.nih.gov/pubmed/14688359 Galectin-3 mediates IL-4-induced survival and differentiation of B cells: functional cross-talk and implications during Trypanosoma cruzi infection.] J Immunol. 2004 Jan 1;172(1):493-502. PubMed PMID: 14688359.</ref>
:* is anti-apoptotic in B cell lines<ref>Hoyer KK, Pang M, Gui D, Shintaku IP, Kuwabara I, Liu FT, Said JW, Baum LG, Teitell MA. [http://www.ncbi.nlm.nih.gov/pubmed/14982843 An anti-apoptotic role for galectin-3 in diffuse large B-cell lymphomas.] Am J Pathol. 2004 Mar;164(3):893-902. PubMed PMID: 14982843; PubMed Central PMCID: PMC1614710.</ref> 

In T cells, purified galectin-3
:* induces IL-2 production<ref>Hsu DK, Hammes SR, Kuwabara I, Greene WC, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/8623933 Human T lymphotropic virus-I infection of human T lymphocytes induces expression of the beta-galactoside-binding lectin, galectin-3.] Am J Pathol. 1996
May;148(5):1661-70. PubMed PMID: 8623933; PubMed Central PMCID: PMC1861566.</ref> and calcium influx<ref>Dong S, Hughes RC. [http://www.ncbi.nlm.nih.gov/pubmed/8898087 Galectin-3 stimulates uptake of extracellular Ca2+ in human Jurkat T-cells.] FEBS Lett. 1996 Oct 21;395(2-3):165-9. PubMed PMID: 8898087.</ref> in Jurkat T cells
:* induces apoptosis in human T leukemic cell lines, human peripheral blood mononuclear cells, and mouse activated T cells<ref>Fukumori T, Takenaka Y, Yoshii T, Kim HR, Hogan V, Inohara H, Kagawa S, Raz A. [http://www.ncbi.nlm.nih.gov/pubmed/14678989 CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis.] Cancer Res. 2003 Dec 1;63(23):8302-11. PubMed PMID: 14678989.</ref><ref name="Stillman 2006">Stillman BN, Hsu DK, Pang M, Brewer CF, Johnson P, Liu FT, Baum LG. [http://www.ncbi.nlm.nih.gov/pubmed/16393961 Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death.] J Immunol. 2006 Jan 15;176(2):778-89. PubMed PMID: 16393961.</ref>, normal human T cells<ref name="Stowell 2008">Stowell SR, Qian Y, Karmakar S, Koyama NS, Dias-Baruffi M, Leffler H, McEver RP, Cummings RD. [http://www.ncbi.nlm.nih.gov/pubmed/18292532 Differential roles of galectin-1 and galectin-3 in regulating leukocyte viability and cytokine secretion.] J Immunol. 2008 Mar 1;180(5):3091-102. PubMed PMID: 18292532.</ref>, and a human tumor infiltrating T cell line<ref>Peng W, Wang HY, Miyahara Y, Peng G, Wang RF. [http://www.ncbi.nlm.nih.gov/pubmed/18757439 Tumor-associated galectin-3 modulates the function of tumor-reactive T cells.] Cancer Res. 2008 Sep 1;68(17):7228-36. PubMed PMID: 18757439.</ref>. In some T cell lines, such as MOLT-4 cells, galectin-3 induces phosphatidylserine exposure, an early event in apoptosis, but not cell death<ref name="Stowell 2008"/>
:* induces apoptosis in both Th1 and Th2 cells<ref>Toscano MA, Bianco GA, Ilarregui JM, Croci DO, Correale J, Hernandez JD, Zwirner NW, Poirier F, Riley EM, Baum LG, Rabinovich GA. [http://www.ncbi.nlm.nih.gov/pubmed/17589510 Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death.] Nat Immunol. 2007 Aug;8(8):825-34. Epub 2007 Jun 24. PubMed PMID: 17589510.</ref>
:* induces apoptosis in CD4-CD8- human thymocytes<ref name="Stillman 2006"/>
:* attenuates interaction of thymocytes with thymic nurse cells<ref>Silva-Monteiro E, Reis Lorenzato L, Kenji Nihei O, Junqueira M, Rabinovich GA, Hsu DK, Liu FT, Savino W, Chammas R, Villa-Verde DM. [http://www.ncbi.nlm.nih.gov/pubmed/17255323 Altered expression of galectin-3 induces cortical thymocyte depletion and premature exit of immature thymocytes during Trypanosoma cruzi infection.] Am J Pathol. 2007 Feb;170(2):546-56. PubMed PMID: 17255323; PubMed Central PMCID: PMC1851869.</ref> 

Endogenous Galectin-3 has anti-apoptotic activity in the human T cell line Jurkat<ref>Yang RY, Hsu DK, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/8692888 Expression of galectin-3 modulates T-cell growth and
apoptosis.] Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6737-42. PubMed PMID:8692888; PubMed Central PMCID: PMC39096.</ref>. 

Galectin-3 has also been documented in the following T cell functions:
:* binds to Mgat5-modified T cell receptor (TCR) and suppresses T cell activation induced by TCR engagement; this is associated with a decrease in lateral mobility of TCR<ref>Demetriou M, Granovsky M, Quaggin S, Dennis JW. [http://www.ncbi.nlm.nih.gov/pubmed/11217864 Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation.] Nature. 2001 Feb 8;409(6821):733-9. PubMed PMID: 11217864.</ref>
:* attenuates association of CD8 and TCR on CD8+ tumor-infiltrating lymphocytes, thus causing anergy<ref>Demotte N, Stroobant V, Courtoy PJ, Van Der Smissen P, Colau D, Luescher IF, Hivroz C, Nicaise J, Squifflet JL, Mourad M, Godelaine D, Boon T, van der Bruggen P. [http://www.ncbi.nlm.nih.gov/pubmed/18342010 Restoring the association of the T cell receptor with CD8 reverses anergy in human tumor-infiltrating lymphocytes.] Immunity. 2008 Mar;28(3):414-24. PubMed PMID: 18342010.</ref>
:* negatively regulates TCR-mediated CD4+ T cell activation at the immunological synapse, by intracellular action<ref>Chen HY, Fermin A, Vardhana S, Weng IC, Lo KF, Chang EY, Maverakis E, Yang RY, Hsu DK, Dustin ML, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/19706535 Galectin-3 negatively regulates TCR-mediated CD4+ T-cell activation at the immunological synapse.] Proc Natl Acad Sci U S A. 2009 Aug 25;106(34):14496-501. Epub 2009 Aug 12. PubMed PMID: 19706535; PubMed Central PMCID: PMC2732795.</ref>

Dendritic cells 
Endogenous Galectin-3
:* suppresses the production of IL-12 by dendritic cells<ref name="Bernardes 2006">Bernardes ES, Silva NM, Ruas LP, Mineo JR, Loyola AM, Hsu DK, Liu FT, Chammas R, Roque-Barreira MC. [http://www.ncbi.nlm.nih.gov/pubmed/16723706 Toxoplasma gondii infection reveals a novel regulatory role for galectin-3 in the interface of innate and adaptive immunity.] Am J Pathol. 2006 Jun;168(6):1910-20. PubMed PMID: 16723706; PubMed Central PMCID: PMC1606628.</ref> and may suppress Th1 responses<ref name="Saegusa 2009">Saegusa J, Hsu DK, Chen HY, Yu L, Fermin A, Fung MA, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/19179612 Galectin-3 is critical for the development of the allergic inflammatory response in a mouse model of atopic dermatitis.] Am J Pathol. 2009 Mar;174(3):922-31. Epub 2009 Jan 29. PubMed PMID: 19179612; PubMed Central PMCID: PMC2665752.</ref>
:* promotes Th2 polarization in the setting of antigen presentation to T cells by dendritic cells<ref name="Saegusa 2009"/>. Another study suggests that galectin-3 suppresses the antigen-presenting function of dendritic cells<ref name="Breuilh 2007">Breuilh L, Vanhoutte F, Fontaine J, van Stijn CM, Tillie-Leblond I, Capron M, Faveeuw C, Jouault T, van Die I, Gosset P, Trottein F. [http://www.ncbi.nlm.nih.gov/pubmed/17785480 Galectin-3 modulates immune and inflammatory responses during helminthic infection: impact of galectin-3 deficiency on the functions of dendritic cells.] Infect Immun. 2007 Nov;75(11):5148-57. Epub 2007 Sep 4. PubMed PMID: 17785480; PubMed Central PMCID: PMC2168304.</ref>)
:* promotes dendritic cell trafficking by functioning intracellularly<ref name="Hsu 2009">Hsu DK, Chernyavsky AI, Chen HY, Yu L, Grando SA, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/18843294 Endogenous galectin-3 is localized in membrane lipid rafts and regulates migration of dendritic cells.] J Invest Dermatol. 2009 Mar;129(3):573-83. Epub 2008 Oct 9. PubMed PMID: 18843294; PubMed Central PMCID: PMC2645233.</ref>
Galectin-3 promotes adhesion of mouse dendritic cells<ref>[Vray B, Camby I, Vercruysse V, Mijatovic T, Bovin NV, Ricciardi-Castagnoli P,
Kaltner H, Salmon I, Gabius HJ, Kiss R. [http://www.ncbi.nlm.nih.gov/pubmed/15044384 Up-regulation of galectin-3 and its ligands by Trypanosoma cruzi infection with modulation of adhesion and migration of murine dendritic cells.] Glycobiology. 2004 Jul;14(7):647-57. Epub 2004 Mar 24.PubMed PMID: 15044384</ref>. 

Neutrophils 
Galectin-3 acts on these cells in the following manner:
:* induces oxidative burst<ref>Yamaoka A, Kuwabara I, Frigeri LG, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/7897228 A human lectin, galectin-3 (epsilon bp/Mac-2), stimulates superoxide production by neutrophils.] J Immunol. 1995 Apr 1;154(7):3479-87. PubMed PMID: 7897228.</ref><ref>Karlsson A, Follin P, Leffler H, Dahlgren C. [http://www.ncbi.nlm.nih.gov/pubmed/9558402 Galectin-3 activates the NADPH-oxidase in exudated but not peripheral blood neutrophils.] Blood. 1998 May 1;91(9):3430-8. PubMed PMID: 9558402.</ref><ref>Almkvist J, Fäldt J, Dahlgren C, Leffler H, Karlsson A. [http://www.ncbi.nlm.nih.gov/pubmed/11159975 Lipopolysaccharide-induced gelatinase granule mobilization primes neutrophils for activation by galectin-3 and formylmethionyl-Leu-Phe.] Infect Immun. 2001 Feb;69(2):832-7. PubMed PMID: 11159975; PubMed Central PMCID: PMC97959.</ref> and L-selectin shedding as well as IL-8 production<ref name="Farnworth 2008">Farnworth SL, Henderson NC, Mackinnon AC, Atkinson KM, Wilkinson T, Dhaliwal K, Hayashi K, Simpson AJ, Rossi AG, Haslett C, Sethi T. [http://www.ncbi.nlm.nih.gov/pubmed/18202191 Galectin-3 reduces the severity of pneumococcal pneumonia by augmenting neutrophil function.] Am J Pathol. 2008 Feb;172(2):395-405. Epub 2008 Jan 17. PubMed PMID: 18202191; PubMed Central PMCID: PMC2312371.</ref>
:* promotes neutrophil adhesion to the extracellular protein laminin<ref>Kuwabara I, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/8621934 Galectin-3 promotes adhesion of human neutrophils to laminin.] J Immunol. 1996 May 15;156(10):3939-44. PubMed PMID: 8621934.</ref> and endothelial cells<ref>Sato S, Ouellet N, Pelletier I, Simard M, Rancourt A, Bergeron MG. [http://www.ncbi.nlm.nih.gov/pubmed/11823514 Role of galectin-3 as an adhesion molecule for neutrophil extravasation during streptococcal pneumonia.] J Immunol. 2002 Feb 15;168(4):1813-22. PubMed PMID: 11823514.</ref>
:* induces phosphatidylserine exposure in the absence of cell death<ref name="Stowell 2008"/>, and induces apoptosis<ref>Fernández GC, Ilarregui JM, Rubel CJ, Toscano MA, Gómez SA, Beigier Bompadre M, Isturiz MA, Rabinovich GA, Palermo MS. [http://www.ncbi.nlm.nih.gov/pubmed/15604089 Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways.] Glycobiology. 2005 May;15(5):519-27. Epub 2004 Dec 15. PubMed PMID: 15604089.</ref>
Endogenous galectin-3 protects neutrophils from apoptosis<ref name="Farnworth 2008"/>. 

Macrophages 
Endogenous galectin-3
:* is anti-apoptotic in macrophages treated with LPS and IFN-γ<ref name="Colnot 1998">Colnot C, Ripoche MA, Milon G, Montagutelli X, Crocker PR, Poirier F. [http://www.ncbi.nlm.nih.gov/pubmed/9767409 Maintenance of granulocyte numbers during acute peritonitis is defective in galectin-3-null mutant mice.] Immunology. 1998 Jul;94(3):290-6. PubMed PMID: 9767409; PubMed Central PMCID: PMC1364244.</ref>. It plays a critical role in the phagocytic function of macrophages in ingesting opsonized sheep red blood cells and apoptotic thymocytes. 
:* plays a critical role in alternative macrophage activation<ref>MacKinnon AC, Farnworth SL, Hodkinson PS, Henderson NC, Atkinson KM, Leffler H, Nilsson UJ, Haslett C, Forbes SJ, Sethi T. [http://www.ncbi.nlm.nih.gov/pubmed/18250477 Regulation of alternative macrophage activation by galectin-3.] J Immunol. 2008 Feb 15;180(4):2650-8. PubMed PMID: 18250477.</ref>

Recombinant galectin-3
:* triggers human peripheral blood monocytes to produce superoxide anion<ref>Liu FT, Hsu DK, Zuberi RI, Kuwabara I, Chi EY, Henderson WR Jr. [http://www.ncbi.nlm.nih.gov/pubmed/7573347 Expression and function of galectin-3, a beta-galactoside-binding lectin, in human monocytes and macrophages.] Am J Pathol. 1995 Oct;147(4):1016-28. PubMed PMID: 7573347; PubMed Central PMCID: PMC1871012.</ref> and potentiates LPS-induced IL-1 production<ref>Jeng KC, Frigeri LG, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/7890309 An endogenous lectin, galectin-3 (epsilon BP/Mac-2), potentiates IL-1 production by human monocytes.] Immunol Lett. 1994 Oct;42(3):113-6. PubMed PMID: 7890309.</ref>
:* functions as a chemoattractant for monocytes and macrophages<ref>Sano H, Hsu DK, Yu L, Apgar JR, Kuwabara I, Yamanaka T, Hirashima M, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/10925302 Human galectin-3 is a novel chemoattractant for monocytes and macrophages.] J Immunol. 2000 Aug 15;165(4):2156-64. PubMed PMID: 10925302.</ref>
:* is an opsonin and enhances the macrophage clearance of apoptotic neutrophils<ref>Karlsson A, Christenson K, Matlak M, Björstad A, Brown KL, Telemo E, Salomonsson E, Leffler H, Bylund J. [http://www.ncbi.nlm.nih.gov/pubmed/18849325 Galectin-3 functions as an opsonin and enhances the macrophage clearance of apoptotic neutrophils.] Glycobiology. 2009 Jan;19(1):16-20. Epub 2008 Oct 10. PubMed PMID: 18849325.</ref>
:* activates microglia (tissue macrophages of the central nervous system) to phagocytose degenerated myelin mediated by complement receptor-3 and scavenger receptor<ref>Rotshenker S. [http://www.ncbi.nlm.nih.gov/pubmed/19253007 The role of Galectin-3/MAC-2 in the activation of the innate-immune function of phagocytosis in microglia in injury and disease.] J Mol Neurosci. 2009 Sep;39(1-2):99-103. Epub 2009 Feb 28. Review. PubMed PMID:19253007.</ref> 
:* binds to a major xenoantigen, α-Gal [Galα(1,3)Galβ(1,4)GlcNAc], expressed on porcine endothelial cells<ref>Jin R, Greenwald A, Peterson MD, Waddell TK. [http://www.ncbi.nlm.nih.gov/pubmed/16818789 Human monocytes recognize porcine endothelium via the interaction of galectin 3 and alpha-GAL.] J Immunol. 2006 Jul 15;177(2):1289-95. PubMed PMID: 16818789.</ref> and mediates adhesion of human monocytes to porcine endothelial cells
:* suppresses LPS-induced production of inflammatory cytokines by macrophages, including IL-6, IL-12, and TNF-α<ref name="Li 2008">Li Y, Komai-Koma M, Gilchrist DS, Hsu DK, Liu FT, Springall T, Xu D. [http://www.ncbi.nlm.nih.gov/pubmed/18684969 Galectin-3 is a negative regulator of lipopolysaccharide-mediated inflammation.] J Immunol. 2008 Aug 15;181(4):2781-9. PubMed PMID: 18684969.</ref> 

Mast cells 
Galectin-3 induces mediator release from both IgE-sensitized and nonsensitized mast cells<ref>Frigeri LG, Zuberi RI, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/8347574 Epsilon BP, a beta-galactoside-binding animal lectin, recognizes IgE receptor (Fc epsilon RI) and activates mast cells.] Biochemistry. 1993 Aug 3;32(30):7644-9. PubMed PMID: 8347574. </ref><ref>Zuberi RI, Frigeri LG, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/8200029 Activation of rat basophilic leukemia cells by epsilon BP, an IgE-binding endogenous lectin.] Cell Immunol. 1994 Jun;156(1):1-12. PubMed PMID: 8200029.</ref>, but apoptosis following prolonged treatment (18-44 h)<ref>Suzuki Y, Inoue T, Yoshimaru T, Ra C. [http://www.ncbi.nlm.nih.gov/pubmed/18302939 Galectin-3 but not galectin-1 induces mast cell death by oxidative stress and mitochondrial permeability transition.] Biochim Biophys Acta. 2008 May;1783(5):924-34. Epub 2008 Feb 12. PubMed PMID: 18302939.</ref>. Endogenous Galectin-3 is a positive regulator of mast cell mediator release and cytokine production<ref>Chen HY, Sharma BB, Yu L, Zuberi R, Weng IC, Kawakami Y, Kawakami T, Hsu DK, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/17015681 Role of galectin-3 in mast cell functions: galectin-3-deficient mast cells exhibit impaired mediator release and defective JNK expression.] J Immunol. 2006 Oct 15;177(8):4991-7. PubMed PMID: 17015681.</ref>. 

Eosinophils 
Recombinant galectin-3
:* suppresses IL-5 production by human eosinophils<ref>Cortegano I, del Pozo V, Cárdaba B, de Andrés B, Gallardo S, del Amo A, Arrieta I, Jurado A, Palomino P, Liu FT, Lahoz C. [http://www.ncbi.nlm.nih.gov/pubmed/9647247 Galectin-3 down-regulates IL-5 gene expression on different cell types.] J Immunol. 1998 Jul 1;161(1):385-9. PubMed PMID: 9647247.</ref>
:* mediates rolling and adhesion of eosinophils on immobilized VCAM-1 under conditions of flow<ref>Rao SP, Wang Z, Zuberi RI, Sikora L, Bahaie NS, Zuraw BL, Liu FT, Sriramarao P. [http://www.ncbi.nlm.nih.gov/pubmed/18025226 Galectin-3 functions as an adhesion molecule to support eosinophil rolling and adhesion under conditions of flow.] J Immunol. 2007 Dec 1;179(11):7800-7. PubMed PMID: 18025226.</ref>. 

''Functions demonstrated in vivo.'' 

A number of biological functions have been identified by using ''Lgals3-/-'' mice. With respect to acute inflammation and allergic inflammation galectin-3:
:# has a proinflammatory role in acute inflammation induced by intraperitoneal injection of thioglycollate broth, in terms of the neutrophil response,<ref name="Colnot 1998"/> and macrophage response<ref>Hsu DK, Yang RY, Pan Z, Yu L, Salomon DR, Fung-Leung WP, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/10702423 Targeted disruption of the galectin-3 gene results in attenuated peritoneal inflammatory responses.] Am J Pathol. 2000 Mar;156(3):1073-83. PubMed PMID: 10702423; PubMed Central PMCID: PMC1876862.</ref>
:# promotes allergic airway inflammation, airway hyperresponsiveness, and a Th2 response in a mouse model of asthma in which mice are sensitized with ovalbumin systemically and challenged with the same antigen through the airways<ref>Zuberi RI, Hsu DK, Kalayci O, Chen HY, Sheldon HK, Yu L, Apgar JR, Kawakami T, Lilly CM, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/15579447 Critical role for galectin-3 in airway inflammation and bronchial hyperresponsiveness in a murine model of asthma.] Am J Pathol. 2004 Dec;165(6):2045-53. PubMed PMID: 15579447; PubMed Central PMCID: PMC1618718.</ref>
:# promotes allergic skin inflammation and a systemic Th2 response in a model of atopic dermatitis, in which mice are repeatedly sensitized with ovalbumin epicutaneously<ref name="Saegusa 2009"/>
:# promotes allergic contact hypersensitivity, in which mice are sensitized with the hapten oxazalone, and then challenged with the same hapten at another skin site<ref name="Hsu 2009"/> (38)
However, rats and mice treated by intranasal delivery of cDNA encoding Galectin-3 showed reduced eosinophil infiltration following airway antigen challenge<ref>del Pozo V, Rojo M, Rubio ML, Cortegano I, Cárdaba B, Gallardo S, Ortega M, Civantos E, López E, Martín-Mosquero C, Peces-Barba G, Palomino P, González-Mangado N, Lahoz C. [http://www.ncbi.nlm.nih.gov/pubmed/12204873 Gene therapy with galectin-3 inhibits bronchial obstruction and inflammation in antigen challenged rats through interleukin-5 gene downregulation.] Am J Respir Crit Care Med. 2002 Sep 1;166(5):732-7. PubMed PMID: 12204873.</ref><ref>López E, del Pozo V, Miguel T, Sastre B, Seoane C, Civantos E, Llanes E, Baeza ML, Palomino P, Cárdaba B, Gallardo S, Manzarbeitia F, Zubeldia JM, Lahoz C. [http://www.ncbi.nlm.nih.gov/pubmed/16424226 Inhibition of chronic airway inflammation and remodeling by galectin-3 gene therapy in a murine model.] J Immunol. 2006 Feb 1;176(3):1943-50. PubMed PMID: 16424226.</ref>. 

With regard to autoimmunity, galectin-3
:* contributes to the disease severity in a mouse model of autoimmune encephalomyelitis (EAE) induced by immunization with a myelin oligodendrocyte glycoprotein peptide<ref>Jiang HR, Al Rasebi Z, Mensah-Brown E, Shahin A, Xu D, Goodyear CS, Fukada SY, Liu FT, Liew FY, Lukic ML. [http://www.ncbi.nlm.nih.gov/pubmed/19124760 Galectin-3 deficiency reduces the severity of experimental autoimmune encephalomyelitis.] J Immunol. 2009 Jan 15;182(2):1167-73. PubMed PMID: 19124760.</ref>
:* suppresses the development of glomerulopathy in mice rendered diabetic with streptozotocin, associated with lower accumulation of advanced glycation end products (AGE) in the kidneys<ref>Pugliese G, Pricci F, Iacobini C, Leto G, Amadio L, Barsotti P, Frigeri L, Hsu DK, Vlassara H, Liu FT, Di Mario U. [http://www.ncbi.nlm.nih.gov/pubmed/11689472 Accelerated diabetic glomerulopathy in galectin-3/AGE receptor 3 knockout mice.] FASEB J. 2001 Nov;15(13):2471-9. PubMed PMID: 11689472.</ref>
:* may serve as an AGE receptor and protects from AGE-induced tissue injury<ref>Iacobini C, Menini S, Oddi G, Ricci C, Amadio L, Pricci F, Olivieri A, Sorcini M, Di Mario U, Pesce C, Pugliese G. [http://www.ncbi.nlm.nih.gov/pubmed/15361471 Galectin-3/AGE receptor 3 knockout mice show accelerated AGE-induced glomerular injury: evidence for a protective role of galectin-3 as an AGE receptor.] FASEB J. 2004 Nov;18(14):1773-5. Epub 2004 Sep 10. PubMed PMID: 15361471</ref> and age-dependent changes<ref>Iacobini C, Oddi G, Menini S, Amadio L, Ricci C, Di Pippo C, Sorcini M, Pricci
F, Pugliese F, Pugliese G. [http://www.ncbi.nlm.nih.gov/pubmed/15870382 Development of age-dependent glomerular lesions in galectin-3/AGE-receptor-3 knockout mice.] Am J Physiol Renal Physiol. 2005 Sep;289(3):F611-21. Epub 2005 May 3. PubMed PMID: 15870382.</ref>
:* contributes to development of diabetes induced by multiple low doses of streptozotocin<ref>Mensah-Brown EP, Al Rabesi Z, Shahin A, Al Shamsi M, Arsenijevic N, Hsu DK, Liu FT, Lukic ML. [http://www.ncbi.nlm.nih.gov/pubmed/18845486 Targeted disruption of the galectin-3 gene results in decreased susceptibility to multiple low dose streptozotocin-induced diabetes in mice.] Clin Immunol. 2009 Jan;130(1):83-8. Epub 2008 Oct 8. PubMed PMID: 18845486.</ref>; this may be related to its upregulation of TNF-&alpha: and nitric oxide production by macrophages
:* contributes to ischemia and neovascularization in retina in a mouse model of oxygen-induced proliferative retinopathy after perfusion of preformed AGEs<ref>Stitt AW, McGoldrick C, Rice-McCaldin A, McCance DR, Glenn JV, Hsu DK, Liu FT, Thorpe SR, Gardiner TA. [http://www.ncbi.nlm.nih.gov/pubmed/15734857 Impaired retinal angiogenesis in diabetes: role of advanced glycation end products and galectin-3.] Diabetes. 2005 Mar;54(3):785-94. PubMed PMID: 15734857.</ref>
:* is expressed in foam cells and macrophages in atherosclerotic lesions<ref>Nachtigal M, Al-Assaad Z, Mayer EP, Kim K, Monsigny M. [http://www.ncbi.nlm.nih.gov/pubmed/9588889 Galectin-3 expression in human atherosclerotic lesions.] Am J Pathol. 1998 May;152(5):1199-208. PubMed PMID: 9588889; PubMed Central PMCID: PMC1858580.</ref> and contributes to the development of atherosclerosis in apolipoprotein (Apo)E-deficient mice<ref>Nachtigal M, Ghaffar A, Mayer EP. [http://www.ncbi.nlm.nih.gov/pubmed/18156214 Galectin-3 gene inactivation reduces atherosclerotic lesions and adventitial inflammation in ApoE-deficient mice.] Am J Pathol. 2008 Jan;172(1):247-55. Epub 2007 Dec 21. PubMed PMID: 18156214; PubMed Central PMCID: PMC2189631.</ref> 

''Infectious processes.'' 

The roles of galectin-3 in a large number of mouse models of infectious disease have been studied in ''Lgals3-/-'' mice, as follows:
:# suppresses LPS-induced shock accompanied by lower inflammatory cytokine and nitric oxide production, possibly a result of its ability to bind to this endotoxin. However, it enhances sensitivity to ''Salmonella'' infection<ref name="Li 2008"/>
:# contributes to recruitment of neutrophils to lungs of mice infected with ''S. pneumoniae'' and has a protective role in development of pneumonia after the infection, possibly by augmenting the function of neutrophils<ref>Nieminen J, St-Pierre C, Bhaumik P, Poirier F, Sato S. [http://www.ncbi.nlm.nih.gov/pubmed/18250456 Role of galectin-3 in leukocyte recruitment in a murine model of lung infection by Streptococcus pneumoniae.] J Immunol. 2008 Feb 15;180(4):2466-73. PubMed PMID: 18250456.</ref>
:# contributes to inflammatory responses in intestines, liver, and brain (but not in lungs) and a lower systemic Th1-polarized response in mice infected by ''Toxoplasma gondii''<ref name="Bernardes 2006"/>; galectin-3 suppresses parasite burden in the brain
:# promotes development of T and B responses in the spleen, as well formation of liver granulomas, but suppresses the Th1-polarized response in mice infected with ''Schistosoma mansoni''<ref name="Breuilh 2007"/>
:# contributes to sensitivity in lethal effects of ''Rhodococcus equi'', a facultative intracellular bacterium of macrophages<ref>Ferraz LC, Bernardes ES, Oliveira AF, Ruas LP, Fermino ML, Soares SG, Loyola AM, Oliver C, Jamur MC, Hsu DK, Liu FT, Chammas R, Roque-Barreira MC. [http://www.ncbi.nlm.nih.gov/pubmed/18825751 Lack of galectin-3 alters the balance of innate immune cytokines and confers resistance to Rhodococcus equi infection.] Eur J Immunol. 2008 Oct;38(10):2762-75. PubMed PMID: 18825751.</ref>. It suppresses inflammatory responses, including production of the Th1 cytokines IL-12 and IFN-γ, as well as IL-1β 
:# promotes resistance of mice to infection by “Paracoccidioides brasiliensis” and favors a Th1-polarized immune response<ref>Ruas LP, Bernardes ES, Fermino ML, de Oliveira LL, Hsu DK, Liu FT, Chammas R, Roque-Barreira MC. [http://www.ncbi.nlm.nih.gov/pubmed/19229338 Lack of galectin-3 drives response to Paracoccidioides brasiliensis toward a Th2-biased immunity.] PLoS One. 2009;4(2):e4519. Epub 2009 Feb 20. PubMed PMID: 19229338; PubMed Central PMCID: PMC2641003.</ref> 

Interestingly, recombinant Galectin-3 is able to induce cell death in the yeast Candida albicans in vitro<ref>Kohatsu L, Hsu DK, Jegalian AG, Liu FT, Baum LG. [http://www.ncbi.nlm.nih.gov/pubmed/16982911 Galectin-3 induces death of Candida species expressing specific beta-1,2-linked mannans.] J Immunol. 2006 Oct 1;177(7):4718-26. PubMed PMID: 16982911.</ref>. 

'''Tumor development/progression.''' 
Galectin-3 expression is altered in a variety of tumors in comparison to normal tissues<ref>Danguy A, Camby I, Kiss R. [http://www.ncbi.nlm.nih.gov/pubmed/12223276 Galectins and cancer.] Biochim Biophys Acta. 2002 Sep 19;1572(2-3):285-93. Review. PubMed PMID: 12223276.</ref>. The diagnostic utility of Galectin-3 expression in thyroid cancer has been extensively demonstrated (e.g., <ref>Chiu CG, Strugnell SS, Griffith OL, Jones SJ, Gown AM, Walker B, Nabi IR, Wiseman SM. [http://www.ncbi.nlm.nih.gov/pubmed/20363921 Diagnostic utility of galectin-3 in thyroid cancer.] Am J Pathol. 2010 May;176(5):2067-81. Epub 2010 Apr 2. PubMed PMID: 20363921; PubMed Central PMCID: PMC2861072.</ref><ref>Carpi A, Mechanick JI, Saussez S, Nicolini A. [http://www.ncbi.nlm.nih.gov/pubmed/20578236 Thyroid tumor marker genomics and proteomics: diagnostic and clinical implications.] J Cell Physiol. 2010 Sep;224(3):612-9. PubMed PMID: 20578236.</ref>). The role of Galectin-3 in tumor growth, progression, and metastasis has been comprehensively documented (reviewed in <ref name="Liu 2005"/>). There is evidence that Galectin-3 expression is necessary for the initiation of the transformed phenotype of tumors, possibly related to its ability to interact with oncogenic K-Ras<ref>Shalom-Feuerstein R, Plowman SJ, Rotblat B, Ariotti N, Tian T, Hancock JF, Kloog Y. [http://www.ncbi.nlm.nih.gov/pubmed/18701484 K-ras nanoclustering is subverted by overexpression of the scaffold protein galectin-3.] Cancer Res. 2008 Aug 15;68(16):6608-16. PubMed PMID: 18701484; PubMed Central PMCID: PMC2587079.</ref>. 

The most extensively studied function of Galectin-3 is its inhibition of apoptosis in a range of tumor cell types exposed to diverse apoptotic stimuli (reviewed in <ref>Yang RY, Rabinovich GA, Liu FT. [http://www.ncbi.nlm.nih.gov/pubmed/18549522 Galectins: structure, function and therapeutic potential.] Expert Rev Mol Med. 2008 Jun 13;10:e17. Review. PubMed PMID: 18549522.</ref>). The mechanism by which Galectin-3 inhibits apoptosis in tumor cells has been extensively studied <ref name="Liu 2005"/><ref>Nakahara S, Oka N, Raz A. [http://www.ncbi.nlm.nih.gov/pubmed/15843888 On the role of galectin-3 in cancer apoptosis.] Apoptosis. 2005; 10:267-75. PubMed PMID: 15843888.</ref>). Apoptosis induced by the tumor suppressor p53 involves repression of Galectin-3<ref>Cecchinelli B, et al. [http://www.ncbi.nlm.nih.gov/pubmed/16738336 Repression of the antiapoptotic molecule galectin-3 by homeodomain-interacting protein kinase 2-activated p53 is required for p53-induced apoptosis.] Mol Cell Biol. 2006; 26:4746-57. PubMed PMID: 16738336; PMCID: PubMed Central PMC1489111</ref>. 

Endogenous galectin-3 promotes tumor cell growth (reviewed in <ref name="Liu 2005"/>), one mechanism may involve interaction with transcription factors<ref>Paron I, et al. [http://www.ncbi.nlm.nih.gov/pubmed/12615069 Nuclear localization of Galectin-3 in transformed thyroid cells: a role in transcriptional regulation.] Biochem Biophys Res Commun. 2003; 302:545-53. PubMed PMID: 12615069.</ref>, another may be facilitation of the signaling of K-Ras to Raf and PI3 kinase<ref>Ashery U, et al. [http://www.ncbi.nlm.nih.gov/pubmed/16691442 Spatiotemporal organization of Ras signaling: rasosomes and the galectin switch.] Cell Mol Neurobiol. 2006; 26:471-95. PubMed PMID: 16691442.</ref>. Endogenous galectin-3 also regulates tumor progression by influencing cell cycling; its binds to β-catenin and stimulates the expression of cyclin D and c-Myc<ref>Shimura T, Takenaka Y, Tsutsumi S, Hogan V, Kikuchi A, Raz A. [http://www.ncbi.nlm.nih.gov/pubmed/15374939 Galectin-3, a novel binding partner of beta-catenin.] Cancer Res. 2004; 64:6363-7. PubMed PMID: 15374939.</ref>. 

Galectin-3 can affect tumor metastasis by exerting its effect in the tumor microenvironment, including angiogenesis and fibrosis<ref name="Liu 2005"/>. Galectin-3 plays a role in activation of myofibroblasts in the liver and contributes to liver fibrosis induced by carbon tetrachloride<ref>Henderson NC, et al. [http://www.ncbi.nlm.nih.gov/pubmed/16549783 Galectin-3 regulates myofibroblast activation and hepatic fibrosis.] Proc Natl Acad Sci U S A. 2006; 103:5060-5. PubMed PMID: 16549783; PubMed Central PMCID: PMC1458794.</ref>. 

In a human melanoma tumor model in immunodeficient mice, administration of galectin-3 results in suppressing the tumor killing effect of tumor-reactive T cells<ref>Peng W, Wang HY, Miyahara Y, Peng G, Wang RF. [http://www.ncbi.nlm.nih.gov/pubmed/18757439 Tumor-associated galectin-3 modulates the function of tumor-reactive T cells.] Cancer Res. 2008 Sep 1;68(17):7228-36. PubMed PMID: 18757439.</ref>. Tumor-associated galectin-3 may also contribute to tumor immune escape by rendering tumor-infiltrating cytolytic lymphocytes anergic<ref>Demotte N, Stroobant V, Courtoy PJ, Van Der Smissen P, Colau D, Luescher IF, Hivroz C, Nicaise J, Squifflet JL, Mourad M, Godelaine D, Boon T, van der Bruggen P. [http://www.ncbi.nlm.nih.gov/pubmed/18342010 Restoring the association of the T cell receptor with CD8 reverses anergy in human tumor-infiltrating lymphocytes.] Immunity. 2008 Mar;28(3):414-24. PubMed PMID: 18342010.</ref>. 

Galectin-3 affects the motility of tumor cells and influences their invasiveness in vitro. However, both positive and negative effects have been reported<ref>Le Marer N, Hughes RC. [http://www.ncbi.nlm.nih.gov/pubmed/8647922 Effects of the carbohydrate-binding protein galectin-3 on the invasiveness of human breast carcinoma cells.] J Cell Physiol. 1996; 168:51-8. PubMed PMID: 8647922.</ref><ref>Moisa A, et al. [http://www.ncbi.nlm.nih.gov/pubmed/17695496 Growth/adhesion-regulatory tissue lectin galectin-3: stromal presence but not cytoplasmic/nuclear expression in tumor cells as a negative prognostic factor in breast cancer.] Anticancer Res. 2007; 27:2131-9. PubMed PMID: 17695496.</ref>. Endogenous galectin-3 can also contribute to cell motility and in vitro invasiveness<ref>Matarrese P, et al. [http://www.ncbi.nlm.nih.gov/pubmed/10699929 Galectin-3 overexpression protects from apoptosis by improving cell adhesion properties.] Int J Cancer. 2000; 85:545-54. PubMed PMID: 10699929.</ref><ref>O'Driscoll L, Linehan R, Liang YH, Joyce H, Oglesby I, Clynes M. [http://www.ncbi.nlm.nih.gov/pubmed/12530054 Galectin-3 expression alters adhesion, motility and invasion in a lung cell line (DLKP), in vitro.] Anticancer Res. 2002; 22:3117-25. PubMed PMID: 12530054.</ref><ref>Shimura T, et al. [http://www.ncbi.nlm.nih.gov/pubmed/15867344 Implication of galectin-3 in Wnt signaling.] Cancer Res. 2005; 65:3535-7. PubMed PMID: 15867344.</ref>. Galectin-3 has angiogenic activity, which may be related to its ability to induce migration of endothelial cells<ref>Nangia-Makker P, et al. [http://www.ncbi.nlm.nih.gov/pubmed/10702407 Galectin-3 induces endothelial cell morphogenesis and angiogenesis.] Am J Pathol. 2000; 156:899-909. PubMed PMID: 10702407; PubMed Central PMCID: PMC1876842.</ref>. 

Studies with animal models have provided evidence for the role of galectins in tumor metastasis in vivo (reviewed in <ref name="Liu 2005"/>). For example, liver metastases of human adenocarcinoma xenotransplants in SCID mice are inhibitable by anti-galectin-3 antibody. Breast carcinoma cells overexpressing transgenic galectin-3 have higher metastatic potential. In an orthotopic nude mouse model of human breast cancer, tumor metastasis is inhibitable by C-terminal domain fragment of galectin-3 (galectin-3C)<ref>John CM, Leffler H, Kahl-Knutsson B, Svensson I, Jarvis GA. [http://www.ncbi.nlm.nih.gov/pubmed/12796408 Truncated galectin-3 inhibits tumor growth and metastasis in orthotopic nude mouse model of human breast cancer.] Clin Cancer Res. 2003; 9:2374-83. PubMed PMID: 12796408.</ref>. 

Galectin-3 contributes to chemotherapeutic resistance of thyroid cancer cells in vitro, the progression of disease in prostate cancer<ref>Wang Y, et al. [http://www.ncbi.nlm.nih.gov/pubmed/19286570 Regulation of prostate cancer progression by galectin-3.] Am J Pathol. 2009; 174:1515-23. PubMed PMID: 19286570; PubMed Central PMCID: PMC2671381.</ref> and development of carcinogen-induced lung tumorigenesis<ref>Abdel-Aziz HO, et al. [http://www.ncbi.nlm.nih.gov/pubmed/18204863 Targeted disruption of the galectin-3 gene results in decreased susceptibility to NNK-induced lung tumorigenesis: an oligonucleotide microarray study.] J Cancer Res Clin Oncol. 2008; 134:777-88. PubMed PMID: 18204863.</ref> in mouse models. However, the absence of galectin-3 may not affect the evolution of cancers<ref>Eude-Le Parco I, et al. [http://www.ncbi.nlm.nih.gov/pubmed/18849326 Genetic assessment of the importance of galectin-3 in cancer initiation, progression, and dissemination in mice.] Glycobiology. 2009; 19:68-75. PubMed PMID: 18849326.</ref>. Galectin-3-targeting small molecule inhibitors enhancs apoptosis induced by chemo- and radio-therapy in papillary thyroid cancer in vitro<ref>Lin CI, et al. [http://www.ncbi.nlm.nih.gov/pubmed/19825987 Galectin-3 targeted therapy with a small molecule inhibitor activates apoptosis and enhances both chemosensitivity and radiosensitivity in papillary thyroid cancer.] Mol Cancer Res. 2009; 7:1655-62. PubMed PMID: 19825987.</ref>. GCS-100, a galectin-3 antagonist, induces myeloma cell death in vitro<ref>Streetly MJ, Maharaj L, Joel S, Schey SA, Gribben JG, Cotter FE. [http://www.ncbi.nlm.nih.gov/pubmed/20190189 GCS-100, a novel galectin-3 antagonist, modulates MCL-1, NOXA, and cell cycle to induce myeloma cell death.] Blood. 2010; 115:3939-48. PubMed PMID: 20190189.</ref>). 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-3&maxresults=20 CFG database search results for Galectin-3].

=== Glycan profiling ===
Glycan recognition analyses have been performed by [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=galectin-3&cat=coreh Core H] of the CFG.
 
=== Glycogene microarray ===
Gene expression analyses have been performed on several cell types and tissues at [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=galectin-3&cat=coree Core E] of the CFG. Probes for human galectin-3 have been included in all versions of the CFG glycogene chip, and probes for mouse galectin-3 are included on versions 2, 3, and 4.
 

=== Knockout mouse lines ===
Galectin-3 knockout mice were [https://www.functionalglycomics.org/glycomics/publicdata/phenotyping.jsp phenotyped by Core G] of the CFG and continue to be used by investigators to study the biological functions of Galectin-3.

=== Glycan array ===
Investigators have used CFG carbohydrate compounds and glycan arrays to study ligand binding specificity of Galectin-3 (for example, click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v2_10_02132003 here]). To see all glycan array results for Galectin-3, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=galectin-3&cat=coreh here].

== Related GBPs ==
Structure unique among galectins in mammals; homologues in vertebrates & invertebrates.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Linda Baum, Richard Cummings, Michael Demetriou, Daniel Hsu, Fu-Tong Liu

Galectin-1

2011-03-25T23:48:40Z

Carole Weaver: /* Glycogene microarray */

Galectin-1 is the best-studied of the prototypic galectins. The crystal structure of Galectin-1 is known, and was the first crystal structure identified for a prototypic galectin.
 
In addition, Galectin-1...
* was the first prototypic galectin for which a function was identified.
* binds novel N- and O-glycan determinants that are involved in cell signaling<ref name="Leppanen 2005" /><ref>Earl LA, Bi S, Baum LG. N- and O-glycans modulate galectin-1 binding, CD45 signaling, and T cell death. ''J Biol Chem'' 285, 2232-2244 (2010).</ref><ref>Song X, ''et al''. Novel fluorescent glycan microarray strategy reveals ligands for galectins. ''Chem Biol'' 16, 36-47 (2009).</ref><ref name="Cooper 2008">Cooper D, Norling LV, Perretti M. Novel insights into the inhibitory effects of Galectin-1 on neutrophil recruitment under flow. ''J Leukoc Biol'' 83, 1459-1466 (2008).</ref><ref>Stillman BN, ''et al''. Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death.'' J Immunol'' 176, 778-789 (2006).</ref>.
* was the first prototypic galectin that was genetically ablated in mice; galectin-1 knockout mice have distinct phenotypes, including aberrant T lymphocyte expansion and increased susceptibility to autoimmune disease <ref>Rabinovich GA, Toscano MA. Turning "sweet" on immunity: galectin-glycan interactions in immune tolerance and inflammation. ''Nat Rev Immunol'' 9, 338-352 (2009). </ref>.
* is the only prototypic galectin that has been administered in animal models of disease to assess therapeutic potential <ref>Rabinovich GA, Daly G, Dreja H, Tailor H, Riera CM, Hirabayashi J, Chernajovsky Y. Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T cell apoptosis. ''J Exp Med'' 190, 385-398 (1999)</ref>
* selectively regulates Th1, Th2 and Th17 cell survival<ref>Toscano MA, Bianco GA, Ilarregui JM, Croci DO, Correale J, Hernandez JD, Zwirner NW, Poirier F, Riley EM, Baum LG, Rabinovich GA. Differential glycosylation of Th1, Th2 and Th17 effector cells selectively regulates susceptibility to cell death. ''Nat Immunol'' 8, 825-834 (2007).</ref>
* has novel dynamics and functions regarding it oxidized versus reduced status, as well as its dimerization status<ref>Stowell SR, ''et al''. Ligand reduces galectin-1 sensitivity to oxidative inactivation by enhancing dimer formation. ''J Biol Chem'' 284, 4989-4999 (2009).</ref><ref name="Leppanen 2005">Leppanen A, Stowell S, Blixt O, Cummings RD. Dimeric galectin-1 binds with high affinity to alpha2,3-sialylated and non-sialylated terminal N-acetyllactosamine units on surface-bound extended glycans. ''J Biol Chem'' 280, 5549-5562 (2005). </ref>.
* is involved in lymphocyte trafficking and leukocyte recruitment<ref name="Cooper 2008" /><ref>Norling LV, Sampaio AL, Cooper D, Perretti M. Inhibitory control of endothelial galectin-1 on in vitro and in vivo lymphocyte trafficking. ''Faseb J'' 22, 682-690 (2008). </ref>.
* promotes the differentiation of tolerogenic dendritic cells and plays a pivotal role in fetomaternal tolerance <ref>Ilarregui JM, Croci DO, Bianco GA, Toscano MA, Salatino M, Vermeulen ME, Geffner JR, Rabinovich GA.''Nat Immunol'' 10, 981-991 (2009).</ref><ref>Blois SM, ''et al''. A pivotal role for galectin-1 in fetomaternal tolerance. ''Nat Med'' 13,1450-1457 (2007).</ref>
* contributes to tumor cell evasion of immune responses.<ref>Rubinstein N, Alvarez M, Zwirner NW, Toscano MA, Ilarregui JM, Bravo A, Mordoh J, Fainboim L, Podhajcer OL, Rabinovich GA. ''Cancer Cell'' 5, 241-251 (2004).</ref>
* demonstrates novel distributions in muscle cells versus non-muscle cells<ref>Dias-Baruffi M, ''et al''. Differential expression of immunomodulatory galectin-1 in peripheral leukocytes and adult tissues and its cytosolic organization in striated muscle. ''Glycobiology'' '''In Press'''. (2010).</ref>.
* has ligands that are modulated by their differential sialylation, which is also associated with glycoprotein positioning in membranes<ref>Cha SK, ''et al''. Removal of sialic acid involving Klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1. ''Proc Natl Acad Sci U S A'' 105, 9805-9810 (2008).</ref>.

 
== CFG Participating Investigators contributing to the understanding of this paradigm ==

CFG Participating Investigators (PIs) contributing to the understanding of Galectin-1 include: Linda Baum, C. Fred Brewer, Richard Cummings, Anne Dell, Ten Feizi, M.G. Finn, Thomas Gerken, Benhur Lee, J. Michael Pierce, Mauro Perretti, Gabriel Rabinovich, James Rini, Sachiko Sato, Gerald Schwarting, Pamela Stanley, Victor Thijssen, Gerardo Vasta, John Wang

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about Galectin-1, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Pages for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Stlect_00116&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_1304&sideMenu=no mouse] Galectin-1 in the CFG database.
=== Carbohydrate ligands ===

The ligand of galectin-1 has been shown to be Galβ1-4GlcNAc (or LacNAc).

=== Cellular expression of GBP and ligands ===

Galectin-1 is expressed in many cell types including muscle, epithelial and endothelial cells. Within the immune system this GBP is considerably up-regulated in activated T lymphocytes, macrophages, uterine NK cells and regulatory T cells.

=== Biosynthesis of ligands ===
The apoptotic activity of galectin 3 is mediated at least in part by binding to (poly) N-acetyllactosamine chains on the surface of galectin-1-sensitive thymocytes in the thymus. Core 2 β-1,6-N-acetylglucosaminyltransferase ([http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/viewGlycoEnzyme.jsp?gbpId=gt_hum_541&sideMenu=true&pageType=general Core 2 GnT]) creates a branched structure on O-glycans that can be elongated to present multiple lactosamine sequences.<ref name=”Galvan2000”> Galvan M, Tsuboi S, Fukuda M, Baum LG. (2000) Expression of a specific glycosyltransferase enzyme regulates T cell death mediated by galectin-1. J. Biol. Chem. 275, 16730-16737</ref><ref name=”Earl2010”> Earl LA, Bi S, Baum LG. (2010) N- and O-glycans modulate galectin-1 binding, CD45 signaling, and T cell death. J. Biol. Chem. 285, 2232-2244</ref>
 

=== Structure ===

Galectin-1 can be found as a monomer as well as a non-covalent homodimer composed of subunits of 14.5 kDa, each containing an identical CRD. Crystals of galectin-1 dimers bound to complex biantennary N-glycans have been analyzed. Infinite chains of lectin dimers (cyan) are cross-linked through N-acetyllactosamine units located at the ends antennae (green/yellow) biantennary N-glycans.<ref>Bourne et al. Crosslinking of mammalian lectin (galectin-1) by complex biantennary saccharides. Nat Struct Biol. 12:863-70 (1994).</ref>
 [[File:Galect1Bourne1994.jpg]]

=== Biological roles of GBP-ligand interaction ===

Galectin-1 is involved in immunoregulation, cytokine secretion, host-pathogen interactions, cell adhesion and migration and tumor-immune escape.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-1&maxresults=20 CFG database search results for Galectin-1].

=== Glycan profiling ===

Glycan profiling of cells known to express Galectin-1 has been done by the CFG analytical core (e.g. [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=t-lymphocytes&cat=corec T-lymphocytes]).

=== Glycogene microarray ===
Probes for human galectin-1 have been included in all versions of the CFG glycogene chip, and probes for mouse galectin-1 are included on versions 2, 3, and 4.
 

=== Knockout mouse lines ===
CFG-generated [http://www.functionalglycomics.org/static/consortium/resources/resourcecoref6.shtml Galectin-1 knockout mice] have been used to study the biological functions of this paradigm GBP. The [http://www.functionalglycomics.org/glycomics/publicdata/phenotyping.jsp phenotype] of Galectin-1 knockout mice was analyzed by the CFG.

=== Glycan array ===
Investigators have made extensive use of carbohydrate compounds and glycan microarrays to study ligand binding specificity of galectin-1 ([http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2745 example]). See all glycan array results for galectin-1 [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=galectin-1&cat=coreh here].

== Related GBPs ==
Galectin-2 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-2&maxresults=20 (CFG data)], galectin-5, galectin-7 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-7&maxresults=20 (CFG data)], galectin-10 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-10&maxresults=20 (CFG data)], galectin-11, galectin-13, and galectin-14 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=galectin-14&maxresults=20 (CFG data)].

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Linda Baum, Yves Bourne, Richard Cummings, Ten Feizi, Gabriel Rabinovich

Ficolins/Mannose-binding protein

2011-03-25T23:47:09Z

Carole Weaver: /* Glycogene microarray */

The ficolins share a common organization and function with the collectins: serum mannose-binding and the pulmonary surfactant proteins A and D. All of these proteins are soluble mediators of innate immunity and consist of globular sugar-binding domains attached to collagenous stalks, which can invoke innate immune responses either through complement fixation or interaction with receptors on the surfaces of macrophages. Amongst these proteins, the ficolins have been most extensively investigated with CFG resources, while mannose-binding protein is the best characterized. The ficolins have fibrinogen-like sugar-binding domains, rather than C-type carbohydrate-recognition domains, but conceptually fall within the same group.

See also: paradigm page for [[Ficolin M (Ficolin 1)]]
== CFG Participating Investigators contributing to the understanding of this paradigm ==
Participating Investigators have generated and characterized knockout mice, defined the sugar-binding properties and undertaken structural analysis for members of this glycan-binding protein (GBP) group.
* PIs working on ficolins include: Raymond Dwek, Daniel Mitchell, Nicole Thielens
* PIs investigating other paradigms in this GBP group include: Kurt Drickamer, Ten Feizi, Toshisuke Kawasaki, Laura Kiessling, Reiko Lee, Yuan Lee, Jamie Marth, Kenneth Ng, Michel Nussenzweig, Pauline Rudd, Maureen Taylor, Bill Weis
* Non-PIs with who have used CFG resources to study ficolins include: David Stephens

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about ficolins and mannose-binding protein, their carbohydrate ligands, and how they interact with ligands to mediate cell communication. Further information can be found in the GBP Molecule Pages for [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_hum_Ctlect_227&sideMenu=no human] and [http://www.functionalglycomics.org/glycomics/molecule/jsp/viewGbpMolecule.jsp?gbpId=cbp_mou_Ctlect_169&sideMenu=no mouse] mannose-binding protein in the CFG database.
=== Carbohydrate ligands ===

L-ficolin preferentially recognizes disulfated LacNAc and tri- and tetrasaccharides containing a terminal LacNAc or GlcNAc unit, provided that the linkage with the following carbohydrate is not of the β1-3 type<ref>Gout E, Garlatti V, Smith DF, Lacroix M M, Dumestre-Perard C, Lunardi T, Martin L, Cesbron JY, Arlaud GJ, Gaboriaud C, Thielens NM (2010) Carbohydrate recognition properties of human ficolins: Glycan array screening reveals the sialic acid binding specificity of M-ficolin. J Biol Chem 285:6612-22</ref><ref>Krarup A, Mitchell DA, Sim RB (2008) Recognition of acetylated oligosaccharides by human L-ficolin. Immunol Lett 118:152-6</ref>. H-ficolin does not bind to any of the glycans.

Mannose-binding protein, also known as mannan-binding lectin (MBL), binds to terminal mannose, fucose and GlcNAc residues on the outer surfaces of bacterial and fungal cell walls. MBL belongs to a family of soluble immune proteins known as the collectins that consist of N-terminal collagen tail regions and C-terminal C-type lectin domains. Other family members include lung surfactant protein A (SP-A) that preferentially binds to galactose, mannose and fucose residues on microbial glycolipids <ref>Childs RA, Wright JR, Ross GF, Yuen CT, Lawson AM, Chai W, Drickamer K, Feizi T (1992) Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids. J Biol Chem 267:9972-9</ref>, and lung surfactant protein D (SP-D) that has been shown to interact with mannoside and glucoside moieties. <ref>Shrive AK, Martin C, Burns I, Paterson JM, Martin JD, Townsend JP, Waters P, Clark HW, Kishore U, Reid KB, Greenhough TJ (2009) Structural characterisation of ligand-binding determinants in human lung surfactant protein D: influence of Asp325. J Mol Biol 394:776-88.</ref>

Ficolins and mannose-binding protein share the ability to associate with mannan-binding lectin-associated serine protease-2.

=== Cellular expression of GBP and ligands ===

Mannose-binding protein is produced mostly by hepatocytes and secreted into the circulation. SP-A and SP-D are produced mostly by alveolar cells and secreted to the pulmonary surfactant that lines the lung.
 L- and H-ficolins are serum proteins that are essentially synthesized in the liver. H-ficolin is also synthesized by bile duct epithelial cells, by lung ciliated bronchial and type II alveolar epithelial cells, and by glioma cells <ref> Akaiwa M, Yae Y, Sugimoto R, Suzuki SO, Iwaki T, Izuhara K, Hamasaki N (1999) Hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile. Histochem Cytochem 47:777-86</ref><ref> Kuraya M, Matsushita M, Endo Y, Thiel S, Fujita T (2003) Expression of H-ficolin/Hakata antigen, mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 by human glioma cell line T98G. Int Immunol 2003:15:109-17</ref>.

L-ficolin recognizes ligands on several strains of opportunistic capsulated bacteria and ''Salmonella typhimurium'' whereas H-ficolin specifically recognizes ''Aerococcus viridans''.

=== Biosynthesis of ligands ===
Glycans on viruses 
High mannose oligosaccharides on viral envelope proteins that are ligands for mannose-binding protein result from incomplete processing of glycans in the pathway for biosynthesis of complex N-linked glycans ([http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/geMolecule.jsp?slideNumber=default GT Database]).
 
Glycans on bacteria 
The biosynthesis pathways for the bacterial lipopolysaccharides have been extensively studied and the gene families responsible for the expression of different glycan sequences have been characterized.<ref name”Raetz2002”>Raetz CR and Whitfield C (2002) Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635-700</ref>
 
Glycans on fungi 
The polysaccharide beta(1,3)-D-glucan is a component of the cell wall of many fungi. The linear polymer is synthesized from UDP-glucose (UDGG) by the multisubunit enzyme UDP-glucose beta(1,3)-D-glucan beta(3)-D-glucosyltransferase. This enzymatic complex contains two catalytic and one regulatory subunits that were first identified in "S. cerevisiae".<ref>Douglas CM, Foor F, Marrinan JA, Morin N, Nielsen JB, Dahl AM, Mazur P, Baginsky W, Li W, el-Sherbeini M, Clemas JA, Mandala SM, Frommer BR, Kurz MB (1994) The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase. Proc Natl Acad Sci U S A 91:12907-11</ref><ref>Mazur P, Morin N, Baginsky W, el-Sherbeini M, Clemas JA, Nielsen JB, Foor F. (1995) Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase. Mol Cell Biol. 15:5671-81</ref><ref>Qadota H, Python CP, Inoue SB, Arisawa M, Anraku Y, Zheng Y, Watanabe T, Levin DE, Ohya Y (1996) Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-glucan synthase. Science 272:279-81</ref>

=== Structure ===

The 3-D structures of the trimeric fibrinogen-like recognition domains of L- and H-ficolins have been solved by X-ray crystallography, revealing similar three-lobed clover-like assemblies, whereas different recognition mechanisms have been deciphered from the structure of complexes with various ligands<ref> Garlatti V, Belloy N, Martin L, Lacroix M, Matsushita M, Endo Y, Fujita T, Fontecilla-Camps JC, Arlaud GJ, Thielens NM, Gaboriaud C (2007) Structural insights into the innate immune recognition specificities of L- and H-ficolins. EMBO J 26:623-33</ref>. An external ligand binding site able to accommodate neutral carbohydrates such as galactose and D-fucose has been identified for H-ficolin. In contrast, L-ficolin exhibited three additional binding sites which define a continuous recognition surface able to bind acetylated and neutral carbohydrates in the context of extended polysaccharides such as 1,3-β-D-glucan.

=== Biological roles of GBP-ligand interaction ===

Ficolins share with mannan-binding lectin the ability to associate with mannan-binding lectin-
associated serine protease-2, thus triggering activation of the lectin complement pathway upon binding to suitable targets and enhancing their phagocytosis. L-ficolin recognizes several strains of opportunistic capsulated bacteria and ''Salmonella typhimurium'' whereas H-ficolin specifically recognizes ''Aerococcus viridans''. In addition to pathogenic microorganisms, L- ficolin binds specifically to apoptotic HL60, U937 and Jurkat T cells, whereas binding of H-ficolin is restricted to apoptotic Jurkat T cells <ref>Kuraya M, Ming Z, Liu X, Matsushita M, Fujita T (2005) Specific binding of L-ficolin and H-ficolin to apoptotic cells leads to complement activation. Immunobiology 209:689-97</ref><ref>Honoré C, Hummelshoj T, Hansen BE, Madsen HO, Eggleton P, Garred P(2007) The innate immune component ficolin 3 (Hakata antigen) mediates the clearance of late apoptotic cells. Arthritis Rheum 56:1598-1607</ref><ref> Jensen ML, Honoré C, Hummelshøj T, Hansen BE, Madsen HO, Garred P(2007) Ficolin-2 recognizes DNA and participates in the clearance of dying host cells. Mol Immunol 44:856-65</ref>.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=ficolin&maxresults=20 ficolin] and [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=mannose-binding&maxresults=20 mannose-binding protein].

=== Glycan profiling ===
Because L and H ficolin and mannose-binding protein bind ligands on bacteria and other micro-organisms, profiling of mammalian glycans is not relevant for these proteins.
 

=== Glycogene microarray ===
Probes for mouse and human MBP have been included in all versions of the CFG glycogene chip.
 

=== Knockout mouse lines ===

 
=== Glycan array ===
The binding specificities of several of the ficolins have been analyzed and other members of the group were screened on the CFG glycan array.
 Investigators have used CFG glycan arrays to study ligand binding specificity of human L-ficolin (see examples [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2.1_609_10172006 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2.1_611_10172006 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1604 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2121 here], and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2122 here]) and of its rat homologue ficolin A (see example [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1707 here]).
Several analyses with human H-ficolin, which has no homologue in rodents, yielded inconclusive results (see examples [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2.1_610_10172006 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1511 here], [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1603 here],
[http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1950 here], and [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2120 here]). See all glycan array results for ficolin [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=ficolin&cat=coreh here].

== Related GBPs ==
Serum mannose-binding protein ([http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=Mannose-binding%20AND%20protein&cat=coreh CFG data]; MBP, also designated mannose-binding lectin, MBL [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Mannose-binding+AND+lectin&maxresults=20 (CFG data)]); and the pulmonary surfactant proteins SP-C and SP-D

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Kurt Drickamer, Nicole Thielens, Daniel Mitchell, Yvette van Kooyk

Calreticulin

2011-03-25T23:13:26Z

Carole Weaver: /* Knockout mouse lines */

Calreticulin and calnexin are components of the quality control system that promotes correct folding of proteins that enter the secretory pathway and targets misfolded proteins for degradation. This family of GBPs is widespread in evolution<ref name="Ellgaard 2003a">Ellgaard, L. and Frickel, E. M. Calnexin, calreticulin, and ERp57: teammates in glycoprotein folding. Cell Biochem Biophys 39, 223-247 (2003)</ref><ref name="Jorgensen 2003">Jorgensen, M. M., Bross, P. and Gregersen, N. Protein quality control in the endoplasmic reticulum. APMIS Suppl 86-91 (2003)</ref><ref name="Ellgaard 2003">Ellgaard, L. and Helenius, A. Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4, 181-191 (2003)</ref><ref name="Helenius 2004">Helenius, A. and Aebi, M. Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73, 1019-1049 (2004)</ref><ref>Molinari, M., Eriksson, K. K., Calanca, V., Galli, C., Cresswell, P., Michalak, M. and Helenius, A. Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control. Mol Cell 13, 125-135 (2004)</ref><ref>Deprez, P., Gautschi, M. and Helenius, A. More than one glycan is needed for ER glucosidase II to allow entry of glycoproteins into the calnexin/calreticulin cycle. Mol Cell 19, 183-195 (2005)</ref><ref>Wu, J. C., Liang, Z. Q. and Qin, Z. H. Quality control system of the endoplasmic reticulum and related diseases. Acta Biochim Biophys Sin (Shanghai) 38, 219-226 (2006) </ref><ref>Caramelo, J. J. and Parodi, A. J. Getting in and out from calnexin/calreticulin cycles. J Biol Chem 283, 10221-10225 (2008) </ref><ref name="Michalak 2009">Michalak, M., Groenendyk, J., Szabo, E., Gold, L. I. and Opas, M. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem J 417, 651-666 (2009)</ref>. Other ER chaperones are also glycan-binding proteins<ref name="Ellgaard 2003a"/>, but calreticulin and calnexin are the best studied examples of glycan-binding proteins involved in intracellular glycoprotein quality control.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) working on calreticulin include: John Hanover, Jamie Rossjohn, Bingdong Sha
* Non-PIs researchers actively pursuing the ER lectins include: Ari Helenius
* Non-PIs focused on ER stress include: Mark Lehrman, Kelly Moreman

== Progress toward understanding this GBP paradigm ==
This section documents what is currently known about calreticulin, its carbohydrate ligand(s), and how they interact to mediate cell communication.
=== Carbohydrate ligands ===
Ligands for calreticulin are glycoproteins bearing Glc1Man9GlcNAc2.
 

=== Cellular expression of GBP and ligands ===
Calnexin and calreticulin are ubiquitously expressed in the ER of all mammalian cell types.
 

=== Biosynthesis of ligands ===
N-linked glycans generated by the core N-linked biosynthesis pathway ([http://www.functionalglycomics.org/glycomics/molecule/jsp/glycoEnzyme/geMolecule.jsp?slideNumber=default GT Database]), are trimmed by the action of ER glucosidases I and II to create the calreticulin ligand Glc1Man9GlcNAc2. The third and final glucose residue is removed by ER glucosidase II, thus destroying the ligand for calreticulin. If the glycoprotein remains incorrectly folded, UDP-glucose:glycoprotein glucosyltransferase adds back the glucose, regenerating the ligand for calreticulin. Cycles of deglucosylation and re-glucosylation continue until the protein is correctly folded.<ref name="Parodi2000">Parodi, A.J. (2000) Role of N-oligosaccharides endoplasmic reticulum processing reactions in glycoprotein folding and degradation. Biochem. J., 348, 1-13</ref>
 

=== Structure ===
Calreticulin consists of a globular carbohydrate-recognition domain, which has a beta-sandwich fold, and an extended arm, which consists of repeated polypeptide segments that bring unfolded enzymes into proximity with ERp57, a member of the protein disulphide isomerase family, to assist in correct folding. <ref name=”Kato2007”>Kato, K. and Kamiya, Y. (2007). Structural views of glycoprotein-fate determination in cells, Glycobiology 17, 1031–1044</ref> The crystal structure of the carbohydrate-recognition domain with a bound glycan has been determined.<ref name=”Kozlov2010”>Kozlov, G, Pocanschi, CL, Rosenauer, A, Bastros-Aristizabal, S, Gorelik, Williams, DB and Gehring, K (2010) Structural basis of carbohydrate recognition by calreticulin. Journal of Biological Chemistry 285, 38612-38620</ref> The structure of the related protein calnexin has also been determined.<ref name=”Schrag2001>Schrag, J. D., Bergeron, J. J. M., Li, Y., Borisova, S., Hahn, M., Thomas, D. Y., and Cygler, M.(2001). The structure of calnexin, an ER chaperone involved in quality control of protein folding, Mol. Cell 8, 633–644</ref>
 

=== Biological roles of GBP-ligand interaction ===
Calreticulin promotes correct folding of secretory glycoprotein. Both calreticulin and calnexin are resident in the ER. Calreticulin possesses a C-terminal ER-retention signal to localize it to the ER lumen, whereas calnexin is anchored to the ER membrane by a transmembrane domain.<ref name="Jorgensen 2003" /><ref name="Ellgaard 2003"/><ref name="Helenius 2004" /><ref name="Michalak 2009" /><ref>Gelebart, P., Opas, M. and Michalak, M. Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum. Int J Biochem Cell Biol 37, 260-266 (2005)</ref><ref name="Wearsch 2008">Wearsch, P. A. and Cresswell, P. The quality control of MHC class I peptide loading. Curr Opin Cell Biol 20, 624-631 (2008)</ref>. Incorrectly folded glycoproteins are tagged with a single glucose residue that binds to calnexin or calreticulin. The process acts in a cycle, in which glucose is continually removed and then re-attached if the glycoprotein is incorrectly folded.<ref name=”Aebi1009”>Aebi, M., Bernasconi, R., Clerc, S., and Molinari, M. (2009). N-glycan structures: recognition and processing in the ER. Trends Biochem Sci 35, 74–82</ref><ref name=”Lederkremer2009”>Lederkremer, G.Z. (2009). Glycoprotein folding, quality control and ER-associated degradation. Cur. Opin. Struct. Biol. 19, 515–523</ref>
 
Calreticulin has also been suggested to be a component of the peptide loading complex where it interacts with other ER resident proteins to produce class-I major histocompatibility complex (MHC-1) molecules<ref name="Wearsch 2008" /><ref>Raghavan, M., Del Cid, N., Rizvi, S. M. and Peters, L. R. MHC class I assembly: out and about. Trends Immunol 29, 436-443 (2008) </ref><ref>Howe, C., Garstka, M., Al-Balushi, M., Ghanem, E., Antoniou, A. N., Fritzsche, S., Jankevicius, G., Kontouli, N., Schneeweiss, C., Williams, A., Elliott, T. and Springer, S. Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules. EMBO J 28, 3730-3744 (2009)</ref>
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=calreticulin&maxresults=20 CFG database search results for calreticulin].

=== Glycan profiling ===
No data available.
 

=== Glycogene microarray ===
No data available.
 

=== Knockout mouse lines ===
The CFG did not undertake creation of knockout mice for calreticulin because generation of such mice was already underway. The knockout is embryonic lethal, as a result of abnormalities in cardiac development, which may be related to the additional role of calreticulin in controlling Ca2+ levels in cells.<ref name=”Mesaeili1999”>Mesaeli, N, Nakamura, K, Zvaritch, E, Dickie, P, Dziak, E, Krause, K-M, Opas, M, MacLennan, DH and Michalak, M. (1999) Calreticulin Is essential for cardiac development. J. Cell Biol. 144, 857-868</ref>
 

=== Glycan array ===
Human recombinant calreticulin was screened on the CFG glycan array (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v1_133_06212005 here]).

== Related GBPs ==
Calnexin

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Kurt Drickamer, John Hanover

Influenza hemagglutinin H3

2011-03-22T18:21:40Z

Carole Weaver: /* Related GBPs */

'''Influenza hemagglutinin (H3 serotype)''' was the first glycoprotein structure to be solved at atomic resolution, by Ian Wilson, John Skehel and Don Wiley in 1981. The collaboration between the Skehel and Wiley labs provided great insight into hemagglutinin function, and it remains the prototype for understanding receptor recognition, antigenic variation, and the extraordinary conformational changes associated with target membrane insertion and ultimately fusion of viral with cell membrane to allow the viral genome to enter the cell and replicate.

In the 1980s, the Paulson lab made the seminal discovery that human and avian viruses with the H3 serotype have different receptor specificities; that human viruses bind to Neu5Acα2-6Gal, while avian viruses bind Neu5Acα2-3Gal. In two very elegant experiments, they were able to switch these specificities by applying selective pressure, and showed that a single amino acid change (L226Q) was all that was required for early H3N2 viruses to switch between human and avian specificities.
These results showed how easy it can be for avian viruses to cross the species barrier into humans. Seasonal influenza viruses with the H3 serotype continue to circulate in the human
population, and subtleties in their receptor specificities appear to be playing a role in how clinical isolates can be recovered in laboratory hosts. CFG investigators are using tools, such as the glycam microarray, provided by the CFG to analyze the detailed receptor specificity of the circulating H3N2 as well as other influenza viruses and their interaction with laboratory hosts to better understand this phenomenon, which has direct consequences on production of vaccines.

Although the influenza H3 hemagglutinin has been chosen as the paradigm, since so much is known, there are 16 subtypes of influenza HA (H1-H16), defined by lack of antigenic cross-reactivity. There is typically only about 20% amino acid sequence identity between HAs of different subtypes. There are interesting and important differences in how easily a particular strain within the subtype can change its binding specificity between avian-like and human-like receptors, leading to the failure so far of H5N1 to be established in the human population, whereas swine-origin H1N1 showed high transmissibility between humans from the time it was first isolated.

To understand the transmission of influenza viruses and how new pandemics begin, it will be important to study a variety of HA subtypes and strains. but for other subtypes the precise rules may differ. Hoever, much progress had been made in the CFG with participating investiigators of understanding the receptor specificity and transmissability of H1 and H2 subypes. Fortunately, the H5N1 avian virus has still not acquired the ability to transmit between humans, as the rules seem more complex compared to H1, H2 and H3, despite at least 15 years of opportunity. The CFG has facilitated considerable advances in our knowledge of the role of sialic acid binding in influenza host specificity and tropism for the upper or lower respiratory tract, and these studies need to be continued until we understand how influenza viruses enter the human population to cause each new pandemic, and the role of receptor specificity in pathogenicity.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of H3 include: Gillian Air, Rafi Ahmed, Nicolai Bovin, Ruben Donis, Chwan-Chuen King, Vladimir Lugovtsev, Christopher Olsen, Peter Palese, James Paulson, Andrew Pekosz, Daniel Perez, Peter P.J.M. Rottier, Charles Russell, Ram Sasisekharan, Dorothy Scott, David Smith, James Stevens, Stephen Mark Tompkins, Reinhard Vlasak, Qinghua Wang, Ian Wilson

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
Ligands for H3 hemagglutinin are sialylated glycans. The H3 hemagglutinin of human viruses (subtype H3N2) binds to N-acetylneuraminic acid linked α2-6 to galactose, sometimes N-acetylgalactosamine. Glycan array analyses indicate that human influenza viruses such as those carrying the H3 HA bind only to structures with NeuAcα2-6 and avian isolates bind only to structures containing NeuAcα2-3. Recent human H3 HAs have shown variation in their specificity of binding downstream sugars.<ref>Gulati S, Smith DF, Air GM. Deletions of neuraminidase and resistance to oseltamivir may be a consequence of restricted receptor specificity in recent H3N2 influenza viruses. Virology J 2009;6(22).</ref>
 
[[File:H3binding2.png]]

=== Cellular expression of GBP and ligands ===
HA is expressed on the surface of influenza virus infected cells before being budded out into progeny virions. H3N2 viruses infect the respiratory tract of humans and birds; in birds they may also infect the gut epithelia. H3N2 viruses infect very few continuous cell lines. Madin-Darby canine kidney cells are most commonly used. Non-permissive cell lines may take up virus efficiently, replicate RNA and express HA on the cell surface but do not bud new virus particles <ref>Kumari K, Gulati S, Smith DF, Gulati U, Cummings RD, Air GM. Receptor binding specificity of recent human H3N2 influenza viruses. Virol J 2007;4(42):1-12.</ref>.

=== Biosynthesis of ligands ===
 
=== Structure ===
The crystal structure of H3 HA was determined by Wilson, Wiley & Skehel in 1981. This has served as a model for more recent HA structure determinations such as H1 HA <ref>Xu R, Ekiert DC, Krause JC, Hai R, Crowe JE, Wilson IA. Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science 2010 Apr 16;328(5976):357-60.</ref>. 
The image of the HA trimer was made with PyMol (Delano Scientific) from PDB file 5HMG. The three subunits are colored green, blue and magenta. For each, the darker shade is the HA1 polypeptide and the lighter shade is HA2.
 
[[File:5HMGlow.png]]
 

=== Biological roles of GBP-ligand interaction ===
Sialylated glycans on the surface of cells lining the respiratory tract serve to capture virus to initiate infection. Glycan array analyses have confirmed that human influenza viruses such as those carrying the H3 HA bind only to structures with NeuAcα2-6 and avian isolates bind only to structures containing NeuAcα2-3. The role of this GBP-glycan interaction in initiating endocytosis and replication is still unclear.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=hemagglutinin&maxresults=20 CFG database search results for "hemagglutinin"].

=== Glycan profiling ===
Virologists have used lectin binding to try to determine where the influenza virus receptors specific for human or avian HAs are located in the human respiratory tract, with mixed results <ref>Nicholls JM, Chan RW, Russell RJ, Air GM, Peiris JS. Evolving complexities of influenza virus and its receptors. Trends Microbiol 2008 2008 Apr;16(4):149-57.</ref>. A complete profile of human trachea as well as lung is needed.

=== Glycogene microarray ===
There are no glycogene array results with the H3 HA, but related paradigm H1 HA has been used by Dr Linda Sherman to assess the role of protein glycosylation in the decision between deletion vs. anergy in immune tolerance. The antigen used was a peptide of A/PR/8/34 (H1N1) HA, 518-IYSTVASSL-526. CFG Request #1155

=== Knockout mouse lines ===
Unfortunately the mouse is a very poor model of influenza infection. Some viruses with H3 HA infect mice quite readily, but do not cause a human-like disease. This means that studies of infection and transmission of H3N2 influenza viruses in SiaT knockout mice are difficult to translate to the human disease. However, studies were done using a mouse-adapted virus <ref> Glaser L, Conenello G, Paulson J, Palese P. Effective replication of human influenza viruses in mice lacking a major alpha2,6 sialyltransferase. Virus Res. 2007 Jun;126(1-2):9-18.</ref>

=== Glycan array ===
The majority of PI-initiated requests for CFG resources to study influenza have been requests for analysis of receptor specificity on the glycan array (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v1_260_12072005 here] for example), and the remainder have been requests for compounds to conduct ''in vitro'' assays in investigators' laboratories. In addition, the CFG glycan array library has been used print custom sialic acid glycan arrays for the U.S. Centers for Disease Control (CDC) for analysis of the receptor specificity of emerging viruses, with data deposited to the CFG database. Glycan Array analyses of H3 HAs have been run for the following PI's: 
Compans (Resource Request #1781; A/Aichi/1/68, A/Udorn/72 and A/Wyoming/3/03),
Steinhauer (#1777; A/Aichi/68 and mutants),
Olsen (#1796, A/swine/Mn/593/99 and A/swine/Ontario/130/97),
Rottier (#1797, A/Finland),
Air (#1660, 1380, 1033, 948, 175; A/Oklahoma/483/2008, A/OK/309/06, A/Oklahoma/323/2003, A/OK/370/05, A/OK/369/05, A/OK/1992/05, A/Wyoming/3/03, A/Philippines/82),
Chen (#1468; A/Victoria/75),
Donis (#138; A/canine/Florida/2004, A/equine/MA/2003),
Paulson (#451; duck/Ukraine/63, A/Moscow/10/99)<ref>Stevens, J., Blixt, O., Chen, L. M., Donis, R. O., Paulson, J. C., and Wilson, I. A. (2008). Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J Mol Biol 381(5), 1382-94.</ref><ref>
Stevens, J., Blixt, O., Glaser, L., Taubenberger, J. K., Palese, P., Paulson, J. C., and Wilson, I. A. (2006). Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J Mol Biol 355(5), 1143-55.</ref><ref>
Stevens, J., Blixt, O., Paulson, J. C., and Wilson, I. A. (2006). Glycan microarray technologies: tools to survey host specificity of influenza viruses. Nat Rev Microbiol 4(11), 857-64.</ref><ref>
Stevens, J., Blixt, O., Tumpey, T. M., Taubenberger, J. K., Paulson, J. C., and Wilson, I. A. (2006). Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312(5772), 404-10.</ref>
 

== Related GBPs ==
Influenza virus HAs of other serotype H1, H2, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 and type B. Type A subtypes H1, H2, H5, H6, H7, and H9 are all being actively investigated by CFG investigators for their potential to jump to humans and type B for its failure to spread in non-human species. CFG data for many of these subtypes are available in the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=hemagglutinin&maxresults=20 CFG database search results for "hemagglutinin."]

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Gillian Air, James Paulson, Ian Wilson

Influenza hemagglutinin H3

2011-03-22T18:14:32Z

Carole Weaver: /* Knockout mouse lines */

'''Influenza hemagglutinin (H3 serotype)''' was the first glycoprotein structure to be solved at atomic resolution, by Ian Wilson, John Skehel and Don Wiley in 1981. The collaboration between the Skehel and Wiley labs provided great insight into hemagglutinin function, and it remains the prototype for understanding receptor recognition, antigenic variation, and the extraordinary conformational changes associated with target membrane insertion and ultimately fusion of viral with cell membrane to allow the viral genome to enter the cell and replicate.

In the 1980s, the Paulson lab made the seminal discovery that human and avian viruses with the H3 serotype have different receptor specificities; that human viruses bind to Neu5Acα2-6Gal, while avian viruses bind Neu5Acα2-3Gal. In two very elegant experiments, they were able to switch these specificities by applying selective pressure, and showed that a single amino acid change (L226Q) was all that was required for early H3N2 viruses to switch between human and avian specificities.
These results showed how easy it can be for avian viruses to cross the species barrier into humans. Seasonal influenza viruses with the H3 serotype continue to circulate in the human
population, and subtleties in their receptor specificities appear to be playing a role in how clinical isolates can be recovered in laboratory hosts. CFG investigators are using tools, such as the glycam microarray, provided by the CFG to analyze the detailed receptor specificity of the circulating H3N2 as well as other influenza viruses and their interaction with laboratory hosts to better understand this phenomenon, which has direct consequences on production of vaccines.

Although the influenza H3 hemagglutinin has been chosen as the paradigm, since so much is known, there are 16 subtypes of influenza HA (H1-H16), defined by lack of antigenic cross-reactivity. There is typically only about 20% amino acid sequence identity between HAs of different subtypes. There are interesting and important differences in how easily a particular strain within the subtype can change its binding specificity between avian-like and human-like receptors, leading to the failure so far of H5N1 to be established in the human population, whereas swine-origin H1N1 showed high transmissibility between humans from the time it was first isolated.

To understand the transmission of influenza viruses and how new pandemics begin, it will be important to study a variety of HA subtypes and strains. but for other subtypes the precise rules may differ. Hoever, much progress had been made in the CFG with participating investiigators of understanding the receptor specificity and transmissability of H1 and H2 subypes. Fortunately, the H5N1 avian virus has still not acquired the ability to transmit between humans, as the rules seem more complex compared to H1, H2 and H3, despite at least 15 years of opportunity. The CFG has facilitated considerable advances in our knowledge of the role of sialic acid binding in influenza host specificity and tropism for the upper or lower respiratory tract, and these studies need to be continued until we understand how influenza viruses enter the human population to cause each new pandemic, and the role of receptor specificity in pathogenicity.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of H3 include: Gillian Air, Rafi Ahmed, Nicolai Bovin, Ruben Donis, Chwan-Chuen King, Vladimir Lugovtsev, Christopher Olsen, Peter Palese, James Paulson, Andrew Pekosz, Daniel Perez, Peter P.J.M. Rottier, Charles Russell, Ram Sasisekharan, Dorothy Scott, David Smith, James Stevens, Stephen Mark Tompkins, Reinhard Vlasak, Qinghua Wang, Ian Wilson

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
Ligands for H3 hemagglutinin are sialylated glycans. The H3 hemagglutinin of human viruses (subtype H3N2) binds to N-acetylneuraminic acid linked α2-6 to galactose, sometimes N-acetylgalactosamine. Glycan array analyses indicate that human influenza viruses such as those carrying the H3 HA bind only to structures with NeuAcα2-6 and avian isolates bind only to structures containing NeuAcα2-3. Recent human H3 HAs have shown variation in their specificity of binding downstream sugars.<ref>Gulati S, Smith DF, Air GM. Deletions of neuraminidase and resistance to oseltamivir may be a consequence of restricted receptor specificity in recent H3N2 influenza viruses. Virology J 2009;6(22).</ref>
 
[[File:H3binding2.png]]

=== Cellular expression of GBP and ligands ===
HA is expressed on the surface of influenza virus infected cells before being budded out into progeny virions. H3N2 viruses infect the respiratory tract of humans and birds; in birds they may also infect the gut epithelia. H3N2 viruses infect very few continuous cell lines. Madin-Darby canine kidney cells are most commonly used. Non-permissive cell lines may take up virus efficiently, replicate RNA and express HA on the cell surface but do not bud new virus particles <ref>Kumari K, Gulati S, Smith DF, Gulati U, Cummings RD, Air GM. Receptor binding specificity of recent human H3N2 influenza viruses. Virol J 2007;4(42):1-12.</ref>.

=== Biosynthesis of ligands ===
 
=== Structure ===
The crystal structure of H3 HA was determined by Wilson, Wiley & Skehel in 1981. This has served as a model for more recent HA structure determinations such as H1 HA <ref>Xu R, Ekiert DC, Krause JC, Hai R, Crowe JE, Wilson IA. Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science 2010 Apr 16;328(5976):357-60.</ref>. 
The image of the HA trimer was made with PyMol (Delano Scientific) from PDB file 5HMG. The three subunits are colored green, blue and magenta. For each, the darker shade is the HA1 polypeptide and the lighter shade is HA2.
 
[[File:5HMGlow.png]]
 

=== Biological roles of GBP-ligand interaction ===
Sialylated glycans on the surface of cells lining the respiratory tract serve to capture virus to initiate infection. Glycan array analyses have confirmed that human influenza viruses such as those carrying the H3 HA bind only to structures with NeuAcα2-6 and avian isolates bind only to structures containing NeuAcα2-3. The role of this GBP-glycan interaction in initiating endocytosis and replication is still unclear.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=hemagglutinin&maxresults=20 CFG database search results for "hemagglutinin"].

=== Glycan profiling ===
Virologists have used lectin binding to try to determine where the influenza virus receptors specific for human or avian HAs are located in the human respiratory tract, with mixed results <ref>Nicholls JM, Chan RW, Russell RJ, Air GM, Peiris JS. Evolving complexities of influenza virus and its receptors. Trends Microbiol 2008 2008 Apr;16(4):149-57.</ref>. A complete profile of human trachea as well as lung is needed.

=== Glycogene microarray ===
There are no glycogene array results with the H3 HA, but related paradigm H1 HA has been used by Dr Linda Sherman to assess the role of protein glycosylation in the decision between deletion vs. anergy in immune tolerance. The antigen used was a peptide of A/PR/8/34 (H1N1) HA, 518-IYSTVASSL-526. CFG Request #1155

=== Knockout mouse lines ===
Unfortunately the mouse is a very poor model of influenza infection. Some viruses with H3 HA infect mice quite readily, but do not cause a human-like disease. This means that studies of infection and transmission of H3N2 influenza viruses in SiaT knockout mice are difficult to translate to the human disease. However, studies were done using a mouse-adapted virus <ref> Glaser L, Conenello G, Paulson J, Palese P. Effective replication of human influenza viruses in mice lacking a major alpha2,6 sialyltransferase. Virus Res. 2007 Jun;126(1-2):9-18.</ref>

=== Glycan array ===
The majority of PI-initiated requests for CFG resources to study influenza have been requests for analysis of receptor specificity on the glycan array (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v1_260_12072005 here] for example), and the remainder have been requests for compounds to conduct ''in vitro'' assays in investigators' laboratories. In addition, the CFG glycan array library has been used print custom sialic acid glycan arrays for the U.S. Centers for Disease Control (CDC) for analysis of the receptor specificity of emerging viruses, with data deposited to the CFG database. Glycan Array analyses of H3 HAs have been run for the following PI's: 
Compans (Resource Request #1781; A/Aichi/1/68, A/Udorn/72 and A/Wyoming/3/03),
Steinhauer (#1777; A/Aichi/68 and mutants),
Olsen (#1796, A/swine/Mn/593/99 and A/swine/Ontario/130/97),
Rottier (#1797, A/Finland),
Air (#1660, 1380, 1033, 948, 175; A/Oklahoma/483/2008, A/OK/309/06, A/Oklahoma/323/2003, A/OK/370/05, A/OK/369/05, A/OK/1992/05, A/Wyoming/3/03, A/Philippines/82),
Chen (#1468; A/Victoria/75),
Donis (#138; A/canine/Florida/2004, A/equine/MA/2003),
Paulson (#451; duck/Ukraine/63, A/Moscow/10/99)<ref>Stevens, J., Blixt, O., Chen, L. M., Donis, R. O., Paulson, J. C., and Wilson, I. A. (2008). Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J Mol Biol 381(5), 1382-94.</ref><ref>
Stevens, J., Blixt, O., Glaser, L., Taubenberger, J. K., Palese, P., Paulson, J. C., and Wilson, I. A. (2006). Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J Mol Biol 355(5), 1143-55.</ref><ref>
Stevens, J., Blixt, O., Paulson, J. C., and Wilson, I. A. (2006). Glycan microarray technologies: tools to survey host specificity of influenza viruses. Nat Rev Microbiol 4(11), 857-64.</ref><ref>
Stevens, J., Blixt, O., Tumpey, T. M., Taubenberger, J. K., Paulson, J. C., and Wilson, I. A. (2006). Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312(5772), 404-10.</ref>
 

== Related GBPs ==
Influenza virus HAs of other serotype H1, H2, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 and type B. Type A subtypes H1, H2, H5, H6, H7, and H9 are all being actively investigated by CFG investigators for their potential to jump to humans and type B for its failure to spread in non-human species.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Gillian Air, James Paulson, Ian Wilson

Parainfluenza virus type 3 hemagglutinin-neuraminidase

2011-03-22T18:14:00Z

Carole Weaver:

Human parainfluenza viruses (HPIVs) are a group of respiratory viruses associated with human respiratory diseases including bronchitis, bronchiolitis, and pneumonia<ref>Moscona, A. 2005. Entry of parainfluenza virus into cells as a target for interrupting childhood respiratory disease. J Clin Invest 115(7):1688-98.</ref><ref>Sato, M. and P.F. 2008. Current status of vaccines for parainfluenza virus infections. Pediatr Infect Dis J 27(10 Suppl):S123-5.</ref><ref>Johnstone, J., S.R. Majumdar, J.D. Fox, and T.J. Marrie. 2008. Viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation. Chest 134(6):1141-8.</ref>. Paramyxoviruses, including HPIVs, possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor-cleaving as well as receptor-binding activity where the two activities reside on the same glycoprotein unlike influenza which carries hemagglutinin and neuraminidase activities as individual glycoproteins. HN is also essential for activating the fusion protein (F) to mediate merger of the viral envelope with the host cell membrane <ref>Lamb, R. 1993. Paramyxovirus fusion: A hypothesis for changes. Virology 197:1-11.</ref><ref>Iorio, R.M., V.R. Melanson, and P.J. Mahon. 2009. Glycoprotein interactions in paramyxovirus fusion. Future Virol 4(4):335-351.</ref>. For the parainfluenza viruses as well as other HN-containing paramyxoviruses, this single molecule carries out three different but critical activities at specific points in the process of viral entry. The first step in infection by HPIV is binding to the lung cells’ surface via interaction of the viral receptor-binding molecule with sialic acid-containing receptor molecules on the cell surface <ref>Suzuki, T., A. Portner, R.A. Scroggs, M. Uchikawa, N. Koyama, et al. 2001. Receptor specificities of human respiroviruses. J Virol 75(10):4604-13.</ref><ref>Moscona, A., M. Porotto, S. Palmer, C. Tai, L. Aschenbrenner, et al. 2010. A Recombinant Sialidase Fusion Protein Effectively Inhibits Human Parainfluenza Viral Infection In Vitro and In Vivo. J Infect Dis.</ref>.

Structures of three paramyxovirus HNs have been determined; they are Newcastle Disease virus (NDV), HPIV type 3, and HPIV type 5 (formerly called SV5). Determination of the HN structure of hPIV3 (globular domain) show an enzyme active site very similar to that of influenza neuraminidase and PIV5 HN and this appears to also be a binding site. A second site at the dimer interface has been crystallographically determined only for Newcastle Disease virus (NDV) HN, but a rising number of reports postulate the presence of such a second site for other paramyxoviruses <ref>Zaitsev, V., M. von Itzstein, D. Groves, M. Kiefel, T. Takimoto, et al. 2004. Second sialic acid binding site in newcastle disease virus hemagglutinin-neuraminidase: implications for fusion. J Virol 78(7):3733-41.</ref><ref>Lawrence, M.C., N.A. Borg, V.A. Streltsov, P.A. Pilling, V.C. Epa, et al. 2004. Structure of the Haemagglutinin-neuraminidase from Human Parainfluenza Virus Type III. J Mol Biol 335(5):1343-57.</ref><ref>Yuan, P., T.B. Thompson, B.A. Wurzburg, R.G. Paterson, R.A. Lamb, et al. 2005. Structural studies of the parainfluenza virus 5 hemagglutinin-neuraminidase tetramer in complex with its receptor, sialyllactose. Structure 13(5):803-15.</ref><ref>Lamb, R.A., R.G. Paterson, and T.S. Jardetzky. 2005. Paramyxovirus membrane fusion: lessons from the F and HN atomic structures. Virology 344(1):30-7.</ref><ref>Bousse, T. and T. Takimoto. 2006. Mutation at residue 523 creates a second receptor binding site on human parainfluenza virus type 1 hemagglutinin-neuraminidase protein. J Virol 80(18):9009-16.</ref><ref>Porotto, M., M. Fornabaio, G. Kellogg, and A. Moscona. 2007. A second receptor binding site on the human parainfluenza 3 hemagglutinin-neuraminidase contributes to activation of the fusion mechanism. J Virol 81(7):3216-3228.</ref>. Interestingly for NDV HN, functional analysis of the two sites indicated that engagement of the first site activates the second <ref>Porotto, M., M. Fornabaio, O. Greengard, M.T. Murrell, G.E. Kellogg, et al. 2006. Paramyxovirus receptor-binding molecules: engagement of one site on the hemagglutinin-neuraminidase protein modulates activity at the second site. J Virol 80(3):1204-13.</ref><ref>Ryan, C., V. Zaitsev, D.J. Tindal, J.C. Dyason, R.J. Thomson, et al. 2006. Structural analysis of a designed inhibitor complexed with the hemagglutinin-neuraminidase of Newcastle disease virus. Glycoconj J 23(1-2):135-41.</ref>. Further studies on the relationship between the sialic acid binding and cleavage activity of wildtype and mutant HPIV HNs are ongoing among CFG PIs. A region next to the transmembrane domain of HN (stalk region) is still elusive to crystal determination, however several studies showed the importance of this domain in fusion promotion <ref>Melanson, V.R. and R.M. Iorio. 2004. Amino acid substitutions in the F-specific domain in the stalk of the newcastle disease virus HN protein modulate fusion and interfere with its interaction with the F protein. J Virol 78(23):13053-61.</ref><ref>Melanson, V.R. and R.M. Iorio. 2006. Addition of N-glycans in the stalk of the Newcastle disease virus HN protein blocks its interaction with the F protein and prevents fusion. J Virol 80(2):623-33.</ref><ref>Porotto, M., M. Murrell, O. Greengard, and A. Moscona. 2003. Triggering of human parainfluenza virus 3 fusion protein(F) by the hemagglutinin-neuraminidase (HN): an HN mutation diminishing the rate of F activation and fusion. J Virol 77(6):3647-3654.</ref><ref>Bishop, K.A., A.C. Hickey, D. Khetawat, J.R. Patch, K.N. Bossart, et al. 2008. Residues in the stalk domain of the hendra virus g glycoprotein modulate conformational changes associated with receptor binding. J Virol 82(22):11398-409.</ref>.

To further emphasize the importance of understanding GBP paradigms, CFG PIs have shown that HPIV3 infection in cultured monolayer cells greatly differs from infection in human airway epithelial (HAE) cell cultures or in animal models <ref>Zhang, L., M.E. Peeples, R.C. Boucher, P.L. Collins, and R.J. Pickles. 2002. Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J Virol 76(11):5654-66.</ref><ref>Mellow, T.E., P.C. Murphy, J.L. Carson, T.L. Noah, L. Zhang, et al. 2004. The effect of respiratory synctial virus on chemokine release by differentiated airway epithelium. Exp Lung Res 30(1):43-57.</ref><ref>Zhang, L., A. Bukreyev, C.I. Thompson, B. Watson, M.E. Peeples, et al. 2005. Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium. J Virol 79(2):1113-24.</ref><ref>Thompson, C.I., W.S. Barclay, M.C. Zambon, and R.J. Pickles. 2006. Infection of human airway epithelium by human and avian strains of influenza A virus. J Virol 80(16):8060-8.</ref>. HPIV3 with a single amino acid mutation in the HN glycoprotein with better than wildtype growth in cell culture had a disadvantage in an ex vivo or in vivo system, revealing a gap in our understanding of the biology of these viruses in their natural host <ref>Palermo, L., M. Porotto, C. Yokoyama, S. Palmer, B. Mungall, et al. 2009. Human parainfluenza virus infection of the airway epithelium: the viral hemagglutinin-neuraminidase regulates fusion protein activation and modulates infectivity. J Virol 83(13):6900-6908.</ref>. This suggests that even slight variations in receptor types may influence HPIV infectivity. Recently a series of studies using glycoarray analysis started to navigate the complexity of the interaction between these viruses and glycomolecules <ref>Amonsen, M., D.F. Smith, R.D. Cummings, and G.M. Air. 2007. Human parainfluenza viruses hPIV1 and hPIV3 bind oligosaccharides with alpha2-3-linked sialic acids that are distinct from those bound by H5 avian influenza virus hemagglutinin. J Virol 81(15):8341-5.</ref>. The three functions of HN depend upon interaction with glycomolecules, therefore understanding whether glycomolecules are preferentially bound, cleave, or activate the fusion process will unravel the biology of these viruses and will help in developing targeted antivirals.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of parainfluenza virus type 3 HN include: Gillian Air, Theodore Jardetsky, Matteo Porotto, Charles Russell

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

Parainfluenza virus type 3 hemagglutinin-neuraminidase binds sialylated glycans. The sialic acid is linked α2-3 to galactose. The minimal binding motif is a pentasaccharide if there are no modifications, but smaller units bind if there is sulfation or fucosylation, as shown in the figure below <ref>Amonsen, M., D.F. Smith, R.D. Cummings, and G.M. Air. 2007. Human parainfluenza viruses hPIV1 and hPIV3 bind oligosaccharides with alpha2-3-linked sialic acids that are distinct from those bound by H5 avian influenza virus hemagglutinin. J Virol 81(15):8341-5.</ref>
 [[File: PIV3glycans.png]]
[[Media:Example.ogg]]

=== Cellular expression of GBP and ligands ===

Parainfluenza virus type 3 hemagglutinin-neuraminidase is expressed by HPIV paramyxoviruses that bind to sialic acid-containing receptor molecules on the surface of host lung cells.

=== Biosynthesis of ligands ===

 
=== Structure ===
The crystal structure of a hPIV3 HN has been determined in dimer form <ref>Lawrence, M.C., N.A. Borg, V.A. Streltsov, P.A. Pilling, V.C. Epa, et al. 2004. Structure of the Haemagglutinin-neuraminidase from Human Parainfluenza Virus Type III. J Mol Biol 335(5):1343-57.</ref> and serves as the model for glycan binding and neuraminidase studies.
 
The subunits are colored green and blue. A molecule of inhibitor 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid is bound to the active site of each subunit (stick model: C, O and N atoms are gray, red and blue respectively). The figure was made using PyMol (Delano Scientific) from PDB file 1V3D.
 
[[file: 1V3D.png]]
 

=== Biological roles of GBP-ligand interaction ===
hPIV HN plays important roles in several distinct steps associated with viral entry, which causes human respiratory infections.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=parainfluenza&maxresults=20 CFG database search results for "parainfluenza"].

=== Glycan profiling ===

 
=== Glycogene microarray ===

 
=== Knockout mouse lines ===

 
=== Glycan array ===
There have been many resource requests for glycan array screening of paramyxovirus hemagglutinin-neuraminidase (for example, click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2490 here]). To see all glycan array results for parainfluenza hemagglutinin-neuraminidase, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=parainfluenza&cat=coreh here].

== Related GBPs ==
* Other paramyxovirus HNs: some appear to have one site that carries out both activities; others appear to have separate sites. For glycan array results of other paramyxovirus HNs, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=paramyxovirus&cat=coreh here].
* Human parainfluenza types 1, 2, 4 and 5
* Newcastle Disease virus
* Mumps virus

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Gillian Air, James Paulson, Matteo Porotto

[[Category:Introduction]]

Cyanovirin-N (CVN)

2011-03-22T17:54:43Z

Carole Weaver: /* Related GBPs */

'''Microbial Antiviral Proteins with GBP Activity''' 

Antiviral compounds made by eukaryotes that recognize unique glycan determinants represent a new paradigm in terms of understanding the innate immune system of primitive organisms and how that might relate to mammalian innate immune systems. Many mammalian GBPs act as innate immune defenders, including the C-type lectins [[Ficolins/Mannose-binding protein]] and other collectins. Interestingly, many of these bind glycan determinants often relatively rich in mannose and/or fucose. One promising anti-HIV-1 drug in development is cyanovirin-N, initially isolated from an extract of the cyanobacterium Nostoc ellipsosoprum<ref name="Boyd1997">Boyd, M.R., Gustafson, K.R., McMahon, J.B., Shoemaker, R.H., O'Keefe, B.R., Mori, T., Gulakowski, R.J., Wu, L., Rivera, M.I., Laurencot, C.M., Currens, M.J., Cardellina, J.H., 2nd, Buckheit, R.W., Jr., Nara, P.L., Pannell, L.K., Sowder, R.C., 2nd and Henderson, L.E. 1997. Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob Agents Chemother, 41, 1521-1530</ref>. While CVN was originally thought to be an orphan lectin with little homology to any other known protein family<ref name="Bewley1998">Bewley, C.A., Gustafson, K.R., Boyd, M.R., Covell, D.G., Bax, A., Clore, G.M., and Gronenborn, A.M. 1998. Solution structure of cyanovirin-N, a potent HIV-inactivating protein. Nat Struct Biol 5, 571-578</ref>, a family of CVN homologs, termed CVNHs, has been described<ref>Percudani, R., Montanini, B. and Ottonello, S. 2005. The anti-HIV cyanovirin-N domain is evolutionarily conserved and occurs as a protein module in eukaryotes. Proteins, 60, 670-678</ref>. Members of this family are found in multicellular ascomycetous fungi and in ferns and share a 3-D fold<ref>Koharudin, L.M., Viscomi, A.R., Jee, J.G., Ottonello, S. and Gronenborn, A.M. 2008. The evolutionarily conserved family of cyanovirin-N homologs: structures and carbohydrate specificity. Structure, 16, 570-584</ref>. A CVNH of the toxin-producing cyanobacterium Microcystis aeruginosa also binds high mannose-type glycans and is involved in cell–cell attachment of Microcystis<ref>Kehr, J.C., Zilliges, Y., Springer, A., Disney, M.D., Ratner, D.D., Bouchier, C., Seeberger, P.H., de Marsac, N.T. and Dittmann, E. 2006. A mannan binding lectin is involved in cell-cell attachment in a toxic strain of Microcystis aeruginosa. Mol Microbiol, 59, 893-906</ref>.

Defining the glycan binding specificity and mode of action for virucidal lectins may help to develop new therapeutic approaches directed at combating viral infections.

'''Cyanovirin-N''' (''Nostoc ellipsosporum'' - a cyanobacterium) 

Cyanovirin-N (CVN) was chosen as a paradigm because of its relevance to human disease and as an example of a new virucidal found in nature. CVN was originally isolated from the cyanobacterium Nostoc ellipsosporum, in a screening program for anti-HIV activities<ref name="Boyd1997"/>. CVN is a small protein of 101 amino acids with two internal tandem repeats of ~50 amino acid. Its structure established a novel fold with no significant similarity to any other known protein<ref name="Bewley1998"/>. It exhibits pseudo-symmetry and comprises two domains, each possessing an independent glycan binding site<ref>Bewley, C.A., Kiyonaka, S., and Hamachi, I. (2002). Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9). J Mol Biol 322, 881-889</ref><ref>Barrientos, L.G., Matei, E., Lasala, F., Delgado, R., and Gronenborn, A.M. (2006). Dissecting carbohydrate-Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition. Protein Eng Des Sel 19, 525-535</ref>. The protein can also exist as a domain-swapped dimer<ref>Yang, F., Bewley, C.A., Louis, J.M., Gustafson, K.R., Boyd, M.R., Gronenborn, A.M., Clore, G.M., and Wlodawer, A. (1999). Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping. J. Mol. Biol. 288, 403-412</ref><ref>Barrientos, L.G., Louis, J.M., Botos, I., Mori, T., Han, Z., O'Keefe, B.R., Boyd, M.R., Wlodawer, A., and Gronenborn, A.M. (2002). The domain-swapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of X-ray and NMR structures. Structure 10, 673-686</ref>. CVN inhibits HIV entry into cells by interacting with the high mannose-type N-glycans on the envelope glycoprotein gp120 of HIV-1. CVN also binds to the glycoproteins of other enveloped viruses, such as SIV, Ebola, influenza and hepatitis C. Thus, CVN represents a new paradigm of microbial GBPs, wherein a unique glycan binding domain comprised of approximately 50 amino acids, exhibits specificity toward α1-2-linked mannose residues.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) who have contributed to studies of this paradigmatic protein include: Simone Ottonello (University of Parma, Italy) and Angela M. Gronenborn (University of Pittsburgh, USA).

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
The physiological ligand for Cyanovirin-N is not precisely known. However, based on extensive data on carbohydrate binding studies on this protein by NMR, X-ray, and glycan microarray screening on the CFG microarray, it is expected that CV-N would recognize any glycans that contain highly enriched alpha (1->2) linkage - mannoses.

=== Cellular expression of GBP and ligands ===
The Cyanovirin-N protein is expressed by Cyanobacterium (blue-green alga) Nostoc ellipsosporum. It binds ligands on glycoproteins of enveloped viruses including HIV, SIV, Ebola, influenza and hepatitis C.

=== Biosynthesis of ligands ===
 
=== Structure ===
The structures of Cyanovirin-N can be found at http://www.pdb.org/ [http://www.pdb.org/pdb/results/results.do?outformat=&qrid=620C4AC9&tabtoshow=Current].

Below are a few representations for the available structures of CV-N determined so far:

The solution structure of wild type CV-N as a monomer (PDB:2EZM).

[[File:CVN1.png]]

The crystal structure of P51G mutant of CV-N as a swapped dimer (PDB:1L5B).

[[File:CVN2.png]]

The crystal structure of swapped-dimeric CV-N in complex with hexamannose (PDB:3GXY).

[[File:CVN3.png]]

 

=== Biological roles of GBP-ligand interaction ===
In vitro, low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus<ref name = "Boyd1997"/>.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=Cyanovirin-N&maxresults=20 CFG database search results for "cyanovirin-N"].

=== Glycan profiling ===

 

=== Glycogene microarray ===
Analysis has not been conducted.

=== Knockout mouse lines ===
Not applicable.

=== Glycan array ===
The CFG has contributed glycans for various glycan specificity studies.
Glycan specificity analysis has been conducted for [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_3001 Cyanovirin-N] using the CFG glycan microarray as shown below. To see all glycan array results for Cyanovirin-N, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=cyanovirin-N&cat=coreh here].

[[File:CVNglycan.png]]

== Related GBPs ==
A large family of CVNHs has been found in both eukaryotic fungi and cyanobacteria (see refs 3, 4 and 5 below). Click here for [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=CVNH&cat=coreh CFG data] on CVNHs.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Angela Gronenborn

Candida glabrata EPA7

2011-03-22T17:50:08Z

Carole Weaver: /* Related GBPs */

'''Fungal adhesins with lectin properties''' 
Cell adhesion proteins on fungal cell surfaces mediate interactions both with other cells of the same type and with the external environment<ref>Douglas, L.M., Li, L., Yang, Y. and Dranginis, A.M. 2007. Expression and characterization of the flocculin Flo11/Muc1, a Saccharomyces cerevisiae mannoprotein with homotypic properties of adhesion. Eukaryot Cell, 6, 2214-2221.</ref><ref>Dranginis, A.M., Rauceo, J.M., Coronado, J.E. and Lipke, P.N. 2007. A biochemical guide to yeast adhesins: glycoproteins for social and antisocial occasions. Microbiol Mol Biol Rev, 71, 282-294.</ref>. These interactions impact critical processes including mating, pathogenesis, and biofilm formation. Fungal adhesins are typically GPI-anchored proteins that have been covalently linked to the cell wall, such that their N-terminal ligand binding domains extend from the cell surface. They frequently occur as families of related proteins<ref>Tronchin, G., Pihet, M., Lopes-Bezerra, L.M. and Bouchara, J.P. 2008. Adherence mechanisms in human pathogenic fungi. Med Mycol, 46, 749-772. </ref>. Members of two such groups, the flocculation/agglutination genes of the model yeast ''Saccharomyces cerevisiae''<ref>Kobayashi, O., Hayashi, N., Kuroki, R. and Sone, H. 1998. Region of FLO1 proteins responsible for sugar recognition. J Bacteriol, 180, 6503-6510.</ref> and the related EPA genes<ref>Kaur, R., Domergue, R., Zupancic, M.L. and Cormack, B.P. 2005. A yeast by any other name: Candida glabrata and its interaction with the host. Curr Opin Microbiol, 8, 378-384.</ref> of the pathogenic fungus ''Candida glabrata'', are lectins. Several of the 23 identified EPA genes have been functionally shown to mediate binding of ''C. glabrata'' to host cells<ref name="Castano 2005">Castano, I., Pan, S.J., Zupancic, M., Hennequin, C., Dujon, B. and Cormack, B.P. 2005. Telomere length control and transcriptional regulation of subtelomeric adhesins in Candida glabrata. Mol Microbiol, 55, 1246-1258.</ref><ref name="Domergue 2005">Domergue, R., Castano, I., de las Penas, A., Zupancic, M., Lockatell, V., Hebel, J.R. et al. 2005. Nicotinic acid limiation regulates silencing of Candida albicans adhesins during UTI. Science, 308, 866-870.</ref>, an essential step in infection and virulence. Defining the specificity of these proteins and their biological roles will elucidate the interactions between host and pathogen, and potentially indicate ways in which to inhibit them for the benefit of the host.

'''''Candida glabrata'' EPA7''' 
The EPA family was chosen as a paradigm because of its relevance to a fungal pathogen that affects human health that can be studied in mouse models of infection. EPA7 was chosen to represent this group because it has been demonstrated to function as an adhesin<ref name="Castano 2005"/> and is one of the EPA proteins that has been studied in the most detail. The N-terminal binding domain of this protein, expressed on the surface of S''. cerevisiae'', has been analyzed on the CFG glycan array. These studies demonstrated EPA7 binding specificity for β1,3- and β1,4-linked galactosides
<ref name=" Zupancic, M.L., 2008">
Zupancic, M.L., Frieman, M., Smith, D., Alvarez, R.A., Cummings, R.D. and Cormack, B.P. 2008. Glycan microarray analysis of Candida glabrata adhesin ligand specificity. Mol Microbiol, 58, 547-559.</ref><ref>de Groot, P.W.J. and Klis, F.M. 2008. The conserved PA14 domain of cell wall-associated fungal adhesins governs their glycan-binding specificity. Mol Microbiol, 68, 535-537. </ref>. This work represents a significant step forward in the area of lectin-like fungal adhesins; in general the specificity of these important proteins remains unexplored.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) who have contributed to studies of this paradigmatic protein include: Brendan Cormack, Rick Cummings
* PIs using CFG resources to study related ''S. cerevisiae'' proteins include: Lars-Oliver Essen (several flocculins), Peter Lipke (alpha agglutinin and ''Candida albicans'' Als adhesins)

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===

Carbohydrate ligands of Epa7 have been examined by glycan array analysis in work from the Cormack group <ref name=" Zupancic, M.L., 2008"></ref>and found to bind almost exclusively to non-reducing terminal β1-4 or β1-3-linked galactose residues. The Epa1 family member has similar specificity while Epa6 binds to most ligands with terminal galactose residues. The related ''S. cerevisiae'' flocculins bind α-mannose or α-glucose residues.

=== Cellular expression of GBP and ligands ===

EPA7 is expressed by the pathogenic fungus ''Candida glabrata''. The EPA gene family is subject to an interesting mechanism of regulation mediated by telomeric silencing. This silencing is relieved in low niacin, leading to increased protein expression. Because urine provides a low niacin growth environment, these adhesins are upregulated in precisely the niche where ''C. glabrata'' must adhere to cause urinary tract infections. 
Consistent with the site of ''C. glabrata'' infection, Epa6 and Epa7 mediate binding to uroepithelial cells in vitro <ref name="Castano 2005"></ref><ref name="Domergue 2005">Domergue, R., Castano, I., de las Penas, A., Zupancic, M., Lockatell, V., Hebel, J.R. et al. 2005. Nicotinic acid limiation regulates silencing of Candida albicans adhesins during UTI. Science, 308, 866-870.</ref>. It has also been noted <ref name=" Zupancic, M.L., 2008"></ref> that "Epa1, Epa6, and Epa7 recognize the T antigen Gal-β1-3GalNAc-α-R, one of the major mucin-type O-glycans found in the colonic epithelium", another site colonized by ''C. glabrata''. 

=== Biosynthesis of ligands ===
 

=== Structure ===

Epa proteins are C-type lectins, but no detailed structural information for these proteins is yet available.

=== Biological roles of GBP-ligand interaction ===

Epa7 and other Epa family members, as summarized above, participate in adhesion of the pathogenic yeast ''Candida glabrata'' cells to host cells, leading to infection. Related proteins participate in cell:cell interactions between yeast that can be important for critical processes such as mating or biofilm formation.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=EPA7&maxresults=20 CFG database search results for "EPA7"].

=== Glycan profiling ===

Glycan profiling of host cell glycans has not been used in connection with this paradigm.

=== Glycogene microarray ===

 Mammalian glycogene profiling has not been performed for this paradigm of a fungal protein. Although yeast binding to mammalian cells could conceivably trigger changes in glycogene expression, the critical issue for this adhesin is which glycans are present on the mammalian cell surface upon initial contact. These structures will mediate the cell:cell interactions that are important for establishment of infection.

=== Knockout mouse lines ===

 CFG knockout mouse lines have not been used for studies pertaining to this paradigm. Examining ''C. glabrata'' infection using wild type and ''epa'' mutant strains in mice with defects in terminal galactosylation could potentially be of interest.

=== Glycan array ===
The specificity of Epa7 and related proteins was determined through CFG glycan array analysis<ref name=" Zupancic, M.L., 2008"></ref> (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_1056 here] to see results). To see glycan array results for other EPA family members, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=EPA&cat=coreh here]. Flocculins from ''S. cerevisiae'' have also been examined by CFG glycan array analysis (click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=flocculin&cat=coreh here]), and glycan array studies of flocculins from ''P. Pastoris'' and of additional ''Candida'' Epa domains have been approved.

== Related GBPs ==
* 22 additional EPA family members in ''C. glabrata''
* Related proteins in ''S. cerevisiae''
* EPA7-like glycan-binding domain also occurs in predicted proteins of ''Ashbya gossypii'' and ''Kluyveromyces lactis''.
(Click here for [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=EPA&maxresults=20 CFG data] on EPA family members)

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Richard Cummings, Tamara Doering, Peter Lipke

Parvovirus Minute Virus of Mice (MVM)

2011-03-21T20:05:03Z

Carole Weaver: /* Related GBPs */

The '''Parvoviridae''' is a family of small non-enveloped ssDNA viruses with a broad range of natural vertebrate and invertebrate hosts, including humans, monkeys, dogs, cats, mice, and insects. Pathogenic members cause severe disease in the young and immunocompromised adults. As examples, the newly discovered human bocavirus causes respiratory tract infections and gastroenteritis in young children and human parvovirus B19, which causes a mild rash in children infects, can cause acute severe or chronic anemia. Severe anemia due to B19 infection of an unborn baby can result in miscarriage in ~5% of pregnant women who are not immune to the virus. Non-pathogenic members, such as the Adeno-associated viruses (AAVs), are being developed for therapeutic gene deliver applications.

The ssDNA parvovirus genome, ~5000 bases, is packaged into a T=1 capsid that is ~260 Å in diameter<ref>Chapman, M.S. and M. Agbandje-McKenna, Atomic structure if viral particles., in Parvoviruses, J.R. Kerr, et al., Editors. 2006, Edward Aenold Ltd. New York: New York. p. 107-123.</ref>. The capsid is assembled from 60 copies (in total) of the common C-terminal region (~520 aa) of two to four overlapping capsid viral proteins (VPs), depending on the family member, with the larger proteins having N-terminal extensions which are not required for capsid assembly, but perform other functions in the life cycle, including a PLA2 domain required for endosomal escape during cellular trafficking. Thus these capsids are assembled from essentially one protein which is responsible for performing the multitude of functions required for successful host infection, including host cell receptor attachment, an essential first step in the life cycle<ref>Agbandje-McKenna, M. and M.S. Chapman, Correlating structure with function in the viral capsid, in Parvoviruses, J.R. Kerr, et al., Editors. 2006, Edward Arnold, New York: New York.</ref>. While the cell surface receptors are not known for the majority of the Parvoviridae, recognition of cell surface glycans, in the context of proteins or lipids, have been reported to be an important first step in infection. This glycan recognition also plays a role in (I) tissue tropism and pathogenicity differences between highly homologous strains for several pathogenic members and (II) tissue tropism and transduction efficiency in viral gene delivery vectors. Thus characterizing the interaction(s) of these viruses with their receptors is important for understanding the capsid determinants of tissue tropism and pathogenicity, and for manipulating the capsids for improved efficacy in corrective gene delivery applications. Minute Virus of Mice (MVM), is assembled from three VPs, VP1, VP2, and VP3, with the entire sequence of the smallest VP3 contained within VP2 which is in turn contained with VP1; VP1 has a unique N-terminal PLA2 region. VP3, which is only generated from VP2 following genome packaging, forms the majority of the capsid protein content, at ~90%, and contains the receptor recognition site. Significantly, this receptor-recognition site is only created in the assembled capsid with amino acid contributions from icosahedral symmetry related VPs<ref name="Kontou 2005">Kontou, M., L. Govindasamy, H.-J. Nam, N. Bryant, A. L. Llamas-Saiz, C. Foces-Foces, E. Hernando, M.-P Rubio, R. McKenna, J. M. Almendral., M. Agbandje-McKenna. 2005. Structural determinants of tissue tropism and in vivo pathogenicity for the parvovirus minute virus of mice. J. Virol., 79:10931-10943.</ref>.

The parvoviruses provide an excellent example of genomic economy in the coding of a “single” viral protein which assembles a multifunctional capsid, including a receptor recognition site. '''MVM''' has proved to be an ideal model for studying the capsid determinant of tissue tropism, pathogenicity, and host range adaption dictated by glycan receptor interaction(s) for this family. Interaction with cell surface sialic acid, in the context of a glycoprotein, is an essential first step in cellular recognition and tropism by MVM<ref name="Cotmore 1987">Cotmore, S. F., and P. Tattersall. 1987. The autonomously replicating parvoviruses of vertebrates. Adv. Virus Res. 33:91–174. </ref><ref name="Lopez-Bueno 2006">López-Bueno, A, M-P. Rubio, N. Bryant, R. McKenna, M. Agbandje-McKenna, J. M. Almendral. 2006. Host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence. J. Virol., 80: 1563-1573. </ref>. Two homologous MVM strains (MVMp, the prototype non-pathogenic strain and MVMi, the immunosuppressive pathogenic strain) that are 97% identical have pronounced differences in tissue tropism and in vivo pathogenesis<ref>Brownstein, D. G., A. L. Smith, R. O. Jacoby, E. A. Johnson, G. Hansen, and P. Tattersall. 1991. Pathogenesis of infection with a virulent allotropic variant of minute virus of mice and regulation by host genotype. Lab. Investig. 65:357–363.</ref><ref>Brownstein, D. G., A. L. Smith, E. A. Johnson, D. J. Pintel, L. K. Naeger, and P. Tattersall. 1992. The pathogenesis of infection with minute virus of mice depends on expression of the small nonstructural protein NS2 and on the genotype of the allotropic determinants VP1 and VP2. J. Virol. 66:3118– 3124. </ref><ref>Ramı´rez, J. C., A. Fiaren, and J. M. Almendral. 1996. Parvovirus minute virus of mice strain I multiplication and pathogenesis in the newborn mouse brain are restricted to proliferative areas and to migratory cerebral young neurons. J. Virol. 70:8109–8116.</ref><ref>Segovia, J. C., J. A. Bueren, and J. M. Almendral. 1995. Myeloid depression follows infection of susceptible newborn mice with the parvovirus minute virus of mice (strain i). J. Virol. 69:3229–3232.</ref><ref>Segovia, J. C., J. M. Gallego, J. A. Bueren, and J. M. Almendral. 1999. Severe leukopenia and dysregulated erythropoiesis in SCID mice persistently infected with the parvovirus minute virus of mice. J. Virol. 73:1774–1784. </ref>. Their phenotypes are associated with one or two VP amino acid differences resulting in local structural variations that alter MVM – infectious sialic acid receptor interactions and utilization<ref name="Nam 2006">Nam, H.-J., B. Gurda-Whitaker, W. Y. Gan, S. Ilaria, R. McKenna, P. Mehta, R. A. Alvarez, M. Agbandje-McKenna. 2006. Identification of the sialic acid structures recognized by minuterole of binding affinity in virulence adaptation. J. Bio. Chem., 281:25670-25677.</ref><ref name="Lopez-Bueno 2006"/>. Studies of the MVMp, in which virulent mutations were observed, also provided information on capsid adaptations, associated with altered receptor affinity, which confer a pathogenic phenotype<ref name="Nam 2006"/>. In addition, MVM has served as a model for studying capsid adaptations, involving the sialic acid receptor recognition site, which enables the infection of a new host<ref>Etingov I, R. Itah, M. Mincberg, A. Keren-Naus, H.-J. Nam, M. Agbandje-McKenna, C. Davis C. 2008. An extension of the Minute Virus of Mice tissue tropism. Virology, 379:245-255.</ref>. While it is known that a limited number of amino acids differences can also dictate tropism and pathogenicity disparities between homologous strains of other Parvoviridae members, unlike MVM, these have not been extensively studied with respect to the contribution of glycans interactions in dictating these differences.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
* CFG Participating Investigators (PIs) contributing to the understanding of MVM include: Mavis McKenna
* CFG PIs contributing to the understanding of AAV capsid include: Aravind Asokan, Regine Heilbronn

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
MVM interacts with cell surface sialic acid, in the context of a glycoprotein<ref name="Cotmore 1987"/><ref name="Lopez-Bueno 2006"/>. This glycan is also recognized by several members of the Parvoviridae, including H1 (rat parvovirus) and members of the AAV[13] (and unpublished data).
 

=== Cellular expression of GBP and ligands ===
MVM is a member of the Parvoviridae, a group of small non-enveloped ssDNA family of viruses with a T=1 icosahedral capsid. Members of this family infect a broad range of vertebrate and invertebrate hosts, including humans, dogs, cats, mice, and insects and have tropism for different tissues and organs In vitro MVM strains grow in mouse fibroblasts, T lymphocytes, and hematopoietic precursors. In vivo MVM replicates in many organs, including the kidneys, liver, and the brain (for MVM strain i)
 

=== Biosynthesis of ligands ===
Sialylated glycoproteins recognized by MVM are synthesized by host cells.
 

=== Structure ===

MVM is assembled from three VPs, VP1, VP2, and VP3, with the entire sequence of the smallest VP3 contained within VP2 which is in turn contained with VP1; VP1 has a unique N-terminal PLA2 region. VP3, which is only generated from VP2 following genome packaging, forms the majority of the capsid protein content, at ~90%, and contains the receptor recognition site. Significantly, this receptor-recognition site is only created in the assembled capsid with amino acid contributions from icosahedral symmetry related VPs<ref name="Kontou 2005"/>.
 [[File:MVMMonoCapsid.jpg]]
 
The glycan structures below have been observed to interact with MVM capsids using the CFG glycan array
 
[[File:structuresGD3SiaLN.jpg]]
 

=== Biological roles of GBP-ligand interaction ===
Glycan recognition by members of the Parvoviridae family dictates tissue tropism and is involved in tissue tropism and pathogenicity differences between highly homologous strains for several pathogenic members.
 

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=parvovirus&maxresults=20 CFG database search results for "parvovirus"].

Three CFG resources have been used in the characterization of MVM glycan interactions and ''in vitro'' tissue tropism.
=== Glycan profiling ===
TThe CFG performed glycan profiling of three different cell lines, A9 fibroblasts, EL4T lymphocytes, and NB324K cells used for in vitro studies of MVM, in order to correlate the glycan screening results with the glycans present on the cells that are permissive to the MVM strains (unpublished data).

=== Glycogene microarray ===
N/A
 

=== Knockout mouse lines ===
N/A
 

=== Glycan array ===
The glycan recognition properties of the two MVM strains (click [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_GLYCAN_v3_35_08102004 here]), as well as virulent mutants arising from ''in vivo'' studies of MVMp, were further defined using the CFG glycan array<ref name="Nam 2006"/> (and unpublished data). These capsids were the first intact virus capsids to be screened by the CFG. This was done using plate arrays versions 2 and 3<ref name="Nam 2006"/>. The glycans identified in the array screening were then provided by the CFG for X-ray crystallographic studies to map the receptor binding site on the virus capsids (unpublished data). Mutations of capsid surface amino acids residues at the mapped binding site results in reduced sialic acid affinity<ref name="Lopez-Bueno 2006"/><ref name="Nam 2006"/>. CFG PIs have also used the CFG glycan array to screen AAV capsids. To see all glycan array results for MVM, click [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=MVM&cat=coreh here].

== Related GBPs ==
Other parvovirus GBPs have been studied by the CFG, including H1 (rat parvovirus) and several AAV serotypes: AAV1 [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV1&maxresults=20 (CFG data)], AAV2, AAV4-AAV9<ref name="Wu 2006">Wu Z, E. Miller E, M. Agbandje-McKenna, R. J. Samulski. 2006. {alpha}2,3 and {alpha}2,6 N Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6. J. Virol., 80:9093-9103. </ref> (and unpublished data; CFG data: [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV5&maxresults=20 AAV5,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV6&maxresults=20 AAV6,] [http://www.functionalglycomics.org/glycomics/search/jsp/result.jsp?query=AAV7&cat=coreh AAV7,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV8&maxresults=20 AAV8,] [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=AAV9&maxresults=20 AAV9.]) Glycan array analyses have been used to identify glycans recognized by the capsids of these viruses, which includes terminal sialic acid containing oligosaccharides as well as those which terminate in galatose <ref name="Wu 2006"/>(and unpublished data).

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Mavis Agbandje-McKenna, Thilo Stehle

Reovirus hemagglutinin (sigma 1)

2011-03-21T19:56:39Z

Carole Weaver: /* Knockout mouse lines */

'''Mammalian orthoreoviruses (reoviruses)''' are useful models for studies of viral receptor recognition and the pathogenesis of viral disease. Reovirus also efficiently lyses tumor cells in experimental animals<ref>Duncan, M.R., Stanish, S.M., and Cox, D.C. Differential sensitivity of normal and transformed human cells to reovirus infection. J. Virol. 28:444-449, 1978.</ref><ref> Coffey, M.C., Strong, J.E., Forsyth, P.A., and Lee, P.W. Reovirus therapy of tumors with activated Ras pathway. Science 282:1332-1334, 1998.</ref> and has shown efficacy in clinical trials for aggressive and refractory human tumors<ref>Stoeckel, J., and Hay, J.G. Drug evaluation: Reolysin--wild-type reovirus as a cancer therapeutic. Curr. Opin. Mol. Ther. 8:249-260, 2006.</ref><ref>Twigger, K., Vidal, L., White, C.L., De Bono, J.S., Bhide, S., Coffey, M., Thompson, B., Vile, R.G., Heinemann, L., Pandha, H.S., et al. Enhanced in vitro and in vivo cytotoxicity of combined reovirus and radiotherapy. Clin. Cancer Res. 14:912-923, 2008. </ref>. Reovirus forms double-shelled particles<ref>Dryden, K.A., Wang, G., Yeager, M., Nibert, M.L., Coombs, K.M., Furlong, D.B., Fields, B.N., and Baker, T.S. Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction. J. Cell Biol. 122:1023-1041, 1993.</ref> that contain a segmented dsRNA genome. The reovirus sigma 1 protein is a long, fiber-like molecule that extends from the virion surface<ref>Furlong, D.B., Nibert, M.L., and Fields, B.N. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles. J. Virol. 62:246-256, 1988.</ref> and mediates viral attachment<ref>Weiner, H.L., Ault, K.A., and Fields, B.N. Interaction of reovirus with cell surface receptors. I. Murine and human lymphocytes have a receptor for the hemagglutinin of reovirus type 3. J. Immunol. 124:2143-2148, 1980.</ref><ref>Lee, P.W.K., Hayes, E.C., and Joklik, W.K. Protein σ1 is the reovirus cell attachment protein. Virology 108:156-163, 1981.</ref>. The three human serotypes (T1, T2, and T3) differ in cell tropism, which correlates directly with receptor-binding properties of sigma 1. Sialic acid serves as an essential receptor for T3 reovirus on murine erythroleukemia (MEL) cells<ref name="Rubin1992">Rubin, D.H., Wetzel, J.D., Williams, W.V., Cohen, J.A., Dworkin, C., and Dermody, T.S. Binding of type 3 reovirus by a domain of the σ1 protein important for hemagglutination leads to infection of murine erythroleukemia cells. J. Clin. Invest. 90:2536-2542, 1992.</ref>, and it functions as a coreceptor on murine L929 (L) cells<ref name="Gentsch1985">Gentsch, J.R., and Pacitti, A.F. Effect of neuraminidase treatment of cells and effect of soluble glycoproteins on type 3 reovirus attachment to murine L cells. J. Virol. 56:356-364, 1985.</ref><ref name="Pacitti1987">Pacitti, A., and Gentsch, J.R. Inhibition of reovirus type 3 binding to host cells by sialylated glycoproteins is mediated through the viral attachment protein. J. Virol. 61:1407-1415, 1987. </ref><ref name="name4">.</ref><ref name="Rubin1992"></ref><ref name="Nibert1995">Nibert, M.L., Chappell, J.D., and Dermody, T.S. Infectious subvirion particles of reovirus type 3 Dearing exhibit a loss in infectivity and contain a cleaved σ1 protein. J. Virol. 69:5057-5067, 1995.</ref>. Residues involved in sialic acid-binding map to the center of the long fiber, close to the midpoint of the molecule<ref name="Chappell2002">Chappell, J.D., Prota, A., Dermody, T.S., and Stehle, T. Crystal structure of reovirus attachment protein σ1 reveals evolutionary relationship to adenovirus fiber. EMBO J. 21:1-11, 2002.</ref>, in a repetitive structural region known as the triple β-spiral. The T1 sigma 1 protein binds to cell-surface glycans of unknown structure.

The triple β-spiral of '''sigma 1''' functions as a trimerization domain and defines a novel carbohydrate-recognition motif. Other carbohydrate-recognition domains, such as those of the C-type lectin superfamily<ref>Weis, W.I., Taylor, M.E., and Drickamer, K. The C-type lectin superfamily in the immune system. Immunol. Rev. 163:19-34, 1998.</ref> or the sialic acid-binding domains in the Siglec family of adhesion proteins<ref>Crocker, P.R., and Varki, A. Siglecs in the immune system. Immunology 103:137-145, 2001.</ref> (see [http://glycobank.mit.edu/glycoWiki/Main_Page Siglec paradigms]), have been described, but none are formed by a repetitive, fiber-like structure such as the one present in sigma 1. In fact, the domain in sigma 1 that binds sialic acid constitutes a carbohydrate-binding “cassette” that could be endowed with altered ligand-binding properties or grafted onto other trimeric structures and used to create avidity for carbohydrates. For example, the adenovirus fiber shaft could be licensed with sialic acid-binding capacity using this approach. These properties render the sigma 1 protein unique among the structurally known glycan-binding moieties.

== CFG Participating Investigators contributing to the understanding of this paradigm ==
CFG Participating Investigators (PIs) contributing to the understanding of sigma 1 include: Terence Dermody, Thilo Stehle

== Progress toward understanding this GBP paradigm ==

=== Carbohydrate ligands ===
Reovirus strains of all three serotypes are capable of binding to carbohydrates, which is a property mediated by viral attachment protein sigma 1<ref name="Chappell2000">Chappell, J.D., Duong, J.L., Wright, B.W., and Dermody, T.S. Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses. J. Virol. 74:8472-8479, 2000.</ref>. Substantial evidence indicates that serotype 3 reoviruses bind to sialic acid, whereas the identity of carbohydrates bound by serotype 1 and 2 reoviruses is less clear. Serotype 3 reoviruses bind to alpha-linked sialic acid in either alpha2,3 or alpha2,6 linkages on a variety of cell types<ref name="Chappell2000">.</ref><ref name="Gentsch1985">.</ref><ref name="name4">Paul, R.W., Choi, A.H., and Lee, P.W.K. The a-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus. Virology 172:382-385, 1989.</ref><ref>Dermody, T.S., Nibert, M.L., Bassel-Duby, R., and Fields, B.N. Sequence diversity in S1 genes and S1 translation products of 11 serotype 3 reovirus strains. J. Virol. 64:4842-4850, 1990.</ref><ref name="Chappell1997">Chappell, J.D., Gunn, V.L., Wetzel, J.D., Baer, G.S., and Dermody, T.S. Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein s1. J. Virol. 71:1834-1841, 1997.</ref>. Hemagglutination by serotype 3 reoviruses is mediated by interactions with alpha-linked sialic acid on several glycosylated erythrocyte proteins such as glycophorin A<ref>Gentsch, J.R., and Pacitti, A.F. Differential interaction of reovirus type 3 with sialylated receptor components on animal cells. Virology 161:245-248, 1987.</ref><ref>Paul, R.W., and Lee, P.W.K. Glycophorin is the reovirus receptor on human erythrocytes. Virology 159:94-101, 1987.</ref>. Reovirus strain T3D binds to sialoglycophorin, but not to asialoglycophorin, with an avidity of ~ 5 x 10e-9 M<ref name="Barton2001a">Barton, E.S., Connolly, J.L., Forrest, J.C., Chappell, J.D., and Dermody, T.S. Utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening. J. Biol. Chem. 276:2200-2211, 2001.</ref>.

Serotype 1 reoviruses also appear to bind to sialic acid in some contexts. Reovirus strain T1L, but not strain T3D, binds to the apical surface of microfold (M) cells, but not to enterocytes, in tissue sections of rabbit Peyer’s patches<ref name="name10">Helander, A., Silvey, K.J., Mantis, N.J., Hutchings, A.B., Chandran, K., Lucas, W.T., Nibert, M.L., and Neutra, M.R. The viral s1 protein and glycoconjugates containing a2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces. J. Virol. 77:7964-7977, 2003.</ref>. Binding is inhibited by pre-incubation of the tissue sections with neuraminidase or with lectins that specifically recognize alpha2-3-linked sialic acid.

=== Cellular expression of GBP and ligands ===
Sialic acid serves as an essential receptor for type 3 reoviruses on murine erythroleukemia (MEL) cells<ref name="Chappell1997">.</ref><ref name="Rubin1992"></ref>. Sialic acid also functions as a coreceptor on murine L929 cells<ref name="Gentsch1985">.</ref><ref name="name4">.</ref><ref name="Rubin1992">.</ref><ref name="Pacitti1987">.</ref><ref name="Nibert1995">.</ref> and human HeLa cells<ref name="Barton2001a">.</ref>. Although not all serotype 3 strains are capable of binding to sialic acid, the majority bind to this carbohydrate.

Serotype 1 reoviruses are incapable of infecting MEL cells, which support infection only by sialic-acid-binding strains<ref name="Rubin1992">.</ref>. Serotype 1 reoviruses also are insensitive to the growth-inhibitory effects of neuraminidase treatment of L929 cells<ref name="Nibert1995">.</ref>. However, binding of serotype 1 reoviruses to intestinal M cells is diminished by neuraminidase treatment<ref name="name10">.</ref>. The explanation for this discrepancy is not known.

Reovirus employs a multi-step mechanism of viral attachment in which a low-affinity interaction with sialic acid serves to tether the virion to target cells and precedes a high-affinity interaction with JAM-A<ref name="Barton2001a">.</ref>, an immunoglobulin superfamily protein engaged by reovirus<ref name="Barton2001b">Barton, E.S., Forrest, J.C., Connolly, J.L., Chappell, J.D., Liu, Y., Schnell, F., Nusrat, A., Parkos, C.A., and Dermody, T.S. Junction adhesion molecule is a receptor for reovirus. Cell 104:441-451, 2001.</ref><ref>Prota, A.E., Campbell, J.A., Schelling, P., Forrest, J.C., Peters, T.R., Watson, M.J., Aurrand-Lions, M., Imhof, B., Dermody, T.S., and Stehle, T. Crystal structure of human junctional adhesion molecule 1: implications for reovirus binding. Proc. Natl. Acad. Sci. U. S. A. 100:5366-5371, 2003.</ref><ref>Campbell, J.A., Shelling, P., Wetzel, J.D., Johnson, E.M., Wilson, G.A.R., Forrest, J.C., Aurrand-Lions, M., Imhof, B., Stehle, T., and Dermody, T.S. Junctional adhesion molecule-A serves as a receptor for prototype and field-isolate strains of mammalian reovirus. J. Virol. 79:7967-7978, 2005.</ref>. By virtue of its rapid association rate, virus binding to sialic acid adheres the virion to the cell surface, thereby enabling it to diffuse laterally until it encounters JAM-A. Such lateral diffusion has been reported for influenza virus<ref>Sagik, B., Puck, T., and Levine, S. Quantitative aspects of the spontaneous elution of influenza virus from red cells. J. Exp. Med. 99:251-260, 1954.</ref> and phage T4<ref>Wilson, J.H., Luftig, R.B., and Wood, W.B. Interaction of bacteriophage T4 tail fiber components with a lipopolysaccharide fraction from Escherichia coli. J. Mol. Biol. 51:423-434, 1970.</ref>. After attachment, reovirus is internalized by receptor-mediated endocytosis<ref>Borsa, J., Morash, B.D., Sargent, M.D., Copps, T.P., Lievaart, P.A., and Szekely, J.G. Two modes of entry of reovirus particles into L cells. J. Gen. Virol. 45:161-170, 1979.</ref><ref>Borsa, J., Sargent, M.D., Lievaart, P.A., and Copps, T.P. Reovirus: evidence for a second step in the intracellular uncoating and transcriptase activation process. Virology 111:191-200, 1981.</ref><ref>Ehrlich, M., Boll, W., Van Oijen, A., Hariharan, R., Chandran, K., Nibert, M.L., and Kirchhausen, T. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118:591-605, 2004.</ref><ref name="Maginnis2006">Maginnis, M.S., Forrest, J.C., Kopecky-Bromberg, S.A., Dickeson, S.K., Santoro, S.A., Zutter, M.M., Nemerow, G.R., Bergelson, J.M., and Dermody, T.S. b1 integrin mediates internalization of mammalian reovirus. J. Virol. 80:2760-2770, 2006.</ref><ref>Sturzenbecker, L.J., Nibert, M.L., Furlong, D.B., and Fields, B.N. Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle. J. Virol. 61:2351-2361, 1987.</ref><ref name="Maginnis2008">Maginnis, M.S., Mainou, B.A., Derdowski, A.M., Johnson, E.M., Zent, R., and Dermody, T.S. NPXY motifs in the b1 integrin cytoplasmic tail are required for functional reovirus entry. J. Virol. 82:3181-3191, 2008.</ref> using a mechanism dependent on beta 1 integrin<ref name="Maginnis2006">.</ref><ref name="Maginnis2008">.</ref>.
=== Biosynthesis of ligands ===
 
=== Structure ===
Structural analysis of the C-terminal half of reovirus attachment protein sigma 1 from strain T3D (residues 246-455) reveals a trimeric structure, in which each monomer is composed of a slender tail and a compact head<ref name="Chappell2002">.</ref>. The C-terminal residues that form the head domain (310-455) consist of two Greek-key motifs that fold into a beta-barrel. The sigma 1 head domain binds to JAM-A<ref name="Barton2001b">.</ref><ref>Kirchner, E., Guglielmi, K.M., Strauss, H.M., Dermody, T.S., and Stehle, T. Structure of reovirus s1 in complex with its receptor junctional adhesion molecule-A. PLoS Path. 4:e1000235, 2008.</ref>. N-terminal residues in the crystallized fragment form a portion of the tail, residues 246-309, which consists of three beta-spiral repeats. Each repeat is composed of two short beta-strands connected by a four-residue beta-turn that has either a proline or a glycine residue at its third position<ref name="Chappell2002">.</ref>. A surface-exposed, variable loop links successive repeats, and trimerization generates an unusual triple beta-spiral motif that also is observed in the adenovirus fiber<ref>van Raaij, M.J., Mitraki, A., Lavigne, G., and Cusack, S. A triple b-spiral in the adenovirus fibre shaft reveals a new structural motif for a fibrous protein. Nature 401:935-938, 1999.</ref>, bacteriophage PRD1 P5 protein<ref>Merckel, M.C., Huiskonen, J.T., Bamford, D.H., Goldman, A., and Tuma, R. The structure of the bacteriophage PRD1 spike sheds light on the evolution of viral capsid architecture. Mol. Cell 18:161-170, 2005.</ref>, and avian reovirus attachment protein sigma C<ref>Guardado, C.P., Fox, G.C., Hermo Parrado, X.L., Llamas-Saiz, A.L., Costas, C., Martinez-Costas, J., Benavente, J., and van Raaij, M.J. Structure of the carboxy-terminal receptor-binding domain of avian reovirus fibre sigmaC. J. Mol. Biol. 354:137-149, 2005.</ref>.

In addition to the three beta-spiral repeats observed in the crystallized sigma 1 fragment, sequence analysis suggests that residues 167-249 in the T3D sigma 1 tail form an additional five N-terminal beta-spiral repeats<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">Guglielmi, K.M., Johnson, E.M., Stehle, T., and Dermody, T.S. Attachment and cell entry of mammalian orthoreovirus. Curr. Top. Microbiol. Immunol. 309:1-38, 2006.</ref>. Alternatively, these residues may form a combination of beta-spiral repeats and alpha-helical coiled-coil, as suggested by sequence analysis<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">.</ref><ref name="Nibert1990">Nibert, M.L., Dermody, T.S., and Fields, B.N. Structure of the reovirus cell-attachment protein: a model for the domain organization of s1. J. Virol. 64:2976-2989, 1990.</ref> and an observed narrowing in this region in a composite negative-stain electron micrograph<ref>Fraser, R.D.B., Furlong, D.B., Trus, B.L., Nibert, M.L., Fields, B.N., and Steven, A.C. Molecular structure of the cell-attachment protein of reovirus: correlation of computer-processed electron micrographs with sequence-based predictions. J. Virol. 64:2990-3000, 1990.</ref>. The structure of N-terminal residues 1-160 of sigma 1 is unknown. However, a repeating heptad sequence motif is predictive of an amphipathic alpha-helix, which likely assembles into an alpha-helical coiled-coil in the trimer<ref name="Chappell2002">.</ref><ref name="Guglielmi2006">.</ref><ref name="Nibert1990">.</ref>.

Although the structure of sigma 1 in complex with sialic acid is not yet available, studies using expressed proteins indicate that the region of T3D sigma 1 required for sialic binding resides near the midpoint of the tail, whereas a region just N-terminal to the head domain of T1L sigma 1 binds to carbohydrate<ref name="Chappell2000">.</ref>. In both T1L and T3D sigma 1, interactions with carbohydrate are mediated by a region of predicted beta-spiral<ref name="Chappell2002">.</ref>. Adaptation of non-sialic-acid-binding reoviruses to growth in MEL cells results in amino acid substitutions at residues 198, 202, and 204 of sigma 1 that confer sialic-acid-binding capacity on the resultant viruses<ref name="Chappell1997">.</ref>. Molecular modeling of the sigma 1 tail, based on available structure and sequence data, suggests that these residues are surface-exposed and proximal to one another in the predicted beta-spiral region<ref name="Chappell2002">.</ref>. Thus, residues 198, 202, and 204 are likely to contribute to a sialic-acid-binding site in T3D sigma 1.

=== Biological roles of GBP-ligand interaction ===
Sialic acid binding serves an important role in reovirus tropism and pathogenesis in vivo<ref>Barton, E.S., Youree, B.E., Ebert, D.H., Forrest, J.C., Connolly, J.L., Valyi-Nagy, T., Washington, K., Wetzel, J.D., and Dermody, T.S. Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease. J. Clin. Invest. 111:1823-1833, 2003.</ref>. Strain T3SA+, which binds to sialic acid, and strain T3SA-, which does not bind to this carbohydrate, produce equivalent titers in the intestine of newborn mice following peroral inoculation. However, T3SA+ spreads more rapidly from the intestine to sites of secondary replication and produces higher titers in the spleen, liver, and brain. Mice infected with T3SA+, but not T3SA-, develop clinical evidence of bile duct obstruction including jaundice and steatorrhea. Liver tissue from mice infected with T3SA+ show intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in human infants, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ binds 100-fold more efficiently than T3SA- to human cholangiocarcinoma cells. Thus, binding to sialic acid targets reovirus to bile duct epithelium and produces a disease reminiscent of infantile biliary atresia.

== CFG resources used in investigations ==
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=reovirus&maxresults=20 CFG database search results for "reovirus"].

=== Glycan profiling ===
Not performed.

=== Glycogene microarray ===
Not performed.

=== Knockout mouse lines ===
Not applicable.

=== Glycan array ===
Experiments in progress. No definitive results have been obtained thus far.

== Related GBPs ==
The attachment protein of adenovirus, fiber, is a structural homolog of sigma 1. At least one adenovirus serotype (Ad37; [http://www.functionalglycomics.org/glycomics/search/jsp/landing.jsp?query=ad37&maxresults=20 CFG data]) is known to bind glycan receptors via residues in the fiber protein<ref>Burmeister WP, Guilligay D, Cusack S, Wadell G, Arnberg N: Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites. Journal of Virology 78:7727-7736, 2004.</ref>. The actual binding site is not homologous. However, information about reovirus glycan binding could also be used to engineer adenovirus fiber proteins (or other trimeric fiber-like proteins) that possess novel glycan-binding properties.

== References ==
<references/>

== Acknowledgements ==
The CFG is grateful to the following PIs for their contributions to this wiki page: Terence Dermody, Mavis McKenna, Thilo Stehle.