Glycans and glycan-binding proteins in immune regulation: A concise introduction to glycobiology for the allergist

Cells are endowed with a rich surface coat of glycans that are carried as glycoproteins and glycolipids on the outer leaflets of their plasma membranes and constitute a major molecular interface between cells and their environment. Each cell's glycome, the sum of its diverse glycan structures, comprises a distinct cellular signature defined by expression levels of the enzymes responsible for glycan biosynthesis. This signature can be read by complementary glycan-binding proteins (GBPs) that translate glycan recognition into function. Nowhere is this more evident than in the immune system, where glycans and GBPs are integral to pathogen recognition and control of inflammatory responses. Glycobiology, the study of glycan structures and their functions, increasingly provides insight into immunoregulatory mechanisms and thereby provides opportunities for therapeutic intervention. This review briefly examines the makeup of the human glycome and the GBPs that translate glycan recognition into function and provides examples of glycan recognition events that are responsible for immune system regulation to promote wider appreciation of this rapidly expanding area of research. Cells are endowed with a rich surface coat of glycans that are carried as glycoproteins and glycolipids on the outer leaflets of their plasma membranes and constitute a major molecular interface between cells and their environment. Each cell's glycome, the sum of its diverse glycan structures, comprises a distinct cellular signature defined by expression levels of the enzymes responsible for glycan biosynthesis. This signature can be read by complementary glycan-binding proteins (GBPs) that translate glycan recognition into function. Nowhere is this more evident than in the immune system, where glycans and GBPs are integral to pathogen recognition and control of inflammatory responses. Glycobiology, the study of glycan structures and their functions, increasingly provides insight into immunoregulatory mechanisms and thereby provides opportunities for therapeutic intervention. This review briefly examines the makeup of the human glycome and the GBPs that translate glycan recognition into function and provides examples of glycan recognition events that are responsible for immune system regulation to promote wider appreciation of this rapidly expanding area of research. Cells are composed of 4 major classes of molecules: nucleic acids, proteins, glycans, and lipids. Among these, glycans inhabit a special niche at cell surfaces, where they are involved in intermolecular and cell-cell recognition events that define and control cell interactions and functions.1Paulson J.C. Blixt O. Collins B.E. Sweet spots in functional glycomics.Nat Chem Biol. 2006; 2: 238-248Crossref PubMed Scopus (339) Google Scholar, 2Varki A. Evolutionary forces shaping the Golgi glycosylation machinery: why cell surface glycans are universal to living cells.Cold Spring Harb Perspect Biol. 2011; 3: a005462Crossref Scopus (114) Google Scholar Glycan recognition is often mediated by complementary glycan-binding proteins (GBPs), each of which carries a specific carbohydrate recognition domain that confers glycan-binding specificity.3Taylor M.E. Drickamer K. Convergent and divergent mechanisms of sugar recognition across kingdoms.Curr Opin Struct Biol. 2014; 28C: 14-22Crossref Scopus (52) Google Scholar Other functional domains on GBPs translate glycan binding into appropriate cellular responses. Nowhere is this more evident than in the immune system, where glycans and GBPs on the surfaces of immune cells are involved in an evolutionary chess match of immune activation and regulation within a dynamic pathogen landscape.2Varki A. Evolutionary forces shaping the Golgi glycosylation machinery: why cell surface glycans are universal to living cells.Cold Spring Harb Perspect Biol. 2011; 3: a005462Crossref Scopus (114) Google Scholar, 4Marth J.D. Grewal P.K. Mammalian glycosylation in immunity.Nat Rev Immunol. 2008; 8: 874-887Crossref PubMed Scopus (542) Google Scholar, 5van Kooyk Y. Rabinovich G.A. Protein-glycan interactions in the control of innate and adaptive immune responses.Nat Immunol. 2008; 9: 593-601Crossref PubMed Scopus (586) Google Scholar, 6Rabinovich G.A. van KY, Cobb BA. Glycobiology of immune responses.Ann N Y Acad Sci. 2012; 1253: 1-15Crossref PubMed Scopus (208) Google Scholar Humans have more than 80 different GBPs (also known as lectins) in at least 12 structural families (see http://www.imperial.ac.uk/research/animallectins). Knowledge of the glycans and GBPs that underlie and regulate immune function provides previously unanticipated opportunities for rational design of targeted therapies for immune dysfunction, some of which show promise in the clinic.7Ernst B. Magnani J.L. From carbohydrate leads to glycomimetic drugs.Nat Rev Drug Discov. 2009; 8: 661-677Crossref PubMed Scopus (656) Google Scholar, 8Wun T. Styles L. DeCastro L. Telen M.J. Kuypers F. Cheung A. et al.Phase 1 study of the E-selectin inhibitor GMI 1070 in patients with sickle cell anemia.PLoS One. 2014; 9: e101301Crossref PubMed Scopus (57) Google Scholar This review introduces the key players in human glycobiology: the human glycome (the totality of human glycan structures) and GBP families that decipher the glycan code. Examples of the roles of glycans and GBPs in infectious disease, inflammation, and control of immune responses follow. An accompanying review in this issue of the Journal provides a more focused perspective of the roles of glycans and GBPs in patients with allergic diseases.9Bochner B.S. Zimmermann N. Role of siglecs and related glycan-binding proteins in immune responses and immunoregulation.J Allergy Clin Immunol. 2015; 135: 598-608Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Every cell in nature has a rich and diverse surface glycan coat that constitutes its interface with the environment.2Varki A. Evolutionary forces shaping the Golgi glycosylation machinery: why cell surface glycans are universal to living cells.Cold Spring Harb Perspect Biol. 2011; 3: a005462Crossref Scopus (114) Google Scholar Although the complexity and diversity of glycans throughout nature are truly daunting, human glycans are more circumscribed and amenable to structure-function studies using rapidly improving analytic techniques. A basic understanding of the building blocks, major structural themes, and general biosynthetic machinery of the human glycome provides the context for understanding glycomic regulation in the immune system. The human glycome is built primarily from just 9 monosaccharide building blocks (Fig 1),10Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Marth J.D. et al.Symbol nomenclature for glycan representation.Proteomics. 2009; 9: 5398-5399Crossref PubMed Scopus (143) Google Scholar each of which is a 6-membered ring (5 carbons and oxygen) further defined by the identity and stereochemistry of the molecular constituents on each ring carbon (mostly hydroxyl groups). Monosaccharides are enzymatically linked to form linear and branched oligosaccharides on protein and lipid carriers. Because each monosaccharide can link to any of up to 4 hydroxyls on another monosaccharide in one of 2 configurations (α or β) and in either linear or branched arrays, an impressive diversity of distinct structures can be created from just a few building blocks. Whereas 3 different amino acids can combine to form 6 distinct tripeptides, 3 different monosaccharides can combine to form more than a thousand distinct trisaccharides. The diversity of glycan structures found on cells is not random but is carefully controlled by gene expression. Each cell's glycome is defined by the expression of the genes responsible for glycan biosynthesis.11Nairn A.V. York W.S. Harris K. Hall E.M. Pierce J.M. Moremen K.W. Regulation of glycan structures in animal tissues: transcript profiling of glycan-related genes.J Biol Chem. 2008; 283: 17298-17313Crossref PubMed Scopus (165) Google Scholar These include genes coding glycosyltransferases (approximately 200 in the human genome), glycosidases, glycan precursor biosynthetic enzymes, and transporters that together represent more than 3% of all human genes. Cell-specific expression of a distinct suite of glycan biosynthetic genes generates each cell's glycan “persona,” a face to the world that regulates its intracellular and intercellular molecular interactions. The cell's glycome varies among cell types, during differentiation, and in response to outside stimuli, providing a rich layer of regulation of cellular functions. It is estimated that human glycosyltransferases create approximately 7000 potential terminal glycan-binding determinants, the basic unit of glycan recognition by GBPs (up to 5-6 sugars in a specific grouping).12Cummings R.D. The repertoire of glycan determinants in the human glycome.Mol Biosyst. 2009; 5: 1087-1104Crossref PubMed Scopus (370) Google Scholar Because oligosaccharide chains are hydrophilic and often charged, they spread out in space, and even minor changes, such as linkage position, linkage configuration, or even the stereochemistry of a single hydroxyl group, provide the basis for the specificity of GBP recognition and downstream functional consequences. The interplay between glycan biosynthetic gene expression, glycan structure, GBP recognition, and biological function is the focus of the field of glycobiology. Glycans at cell surfaces are classified as glycolipids or glycoproteins (Fig 1). Glycolipids have their hydrophobic lipid tail firmly embedded in the outer leaflet of the plasma membrane, with their hydrophilic oligosaccharide chain extending out into the extracellular space. In most cells glycolipids comprise a small percentage of total plasma membrane lipids. In contrast, nearly all proteins at the cell surface are glycoproteins, including single- and multiple-pass transmembrane and secreted proteins. Glycans on proteins are classified based on their covalent linkage to the polypeptide: an asparagine (N-linked) or a serine or threonine (O-linked). N-linked protein glycosylation is initiated during protein translation in the endoplasmic reticulum, whereas O-linked glycosylation occurs after translation in the Golgi apparatus. In both cases the glycans are further modified and elaborated in the Golgi apparatus on their way to the cell surface. Whereas O-linked glycans are built stepwise and can be as small as a single sugar, N-linked glycans are prebuilt on a special lipid carrier and transferred as a characteristic 14-sugar block onto the nascent polypeptide, where the block is trimmed and then further elaborated. Most mature O-linked oligosaccharides are small, whereas N-linked glycans are larger. A study of the adult human lung glycome (http://www.functionalglycomics.org/) revealed that the most abundant O-linked glycans have 2 to 6 sugar residues as linear or short single-branched oligosaccharides. In contrast, the major human lung N-linked glycans have 10 to 20 residues, most as highly branched structures (typically 3-5 branches). In addition to N- and O-linked oligosaccharides, glycoproteins can also carry very long (up to approximately 100 sugars) repeating disaccharides linked to serine and threonine residues. These glycoproteins are classified as a separate family, the proteoglycans, to reflect their special structural features. The best known proteoglycans are heparan and chondroitin sulfates. Their long anionic sugar chains, termed glycosaminoglycans, are further substituted with sulfate groups, making them even larger, more charged, and more structurally diverse. One other member of the glycosaminoglycan family, hyaluronic acid, has the same repeating sugar units but is not attached to a protein, remains unsulfated, and reaches molecular weights in the millions of daltons. Proteoglycans and glycosaminoglycans, fascinating molecules deserving of their own review in the control of immune responses, are not addressed further here.13Frey H. Schroeder N. Manon-Jensen T. Iozzo R.V. Schaefer L. Biological interplay between proteoglycans and their innate immune receptors in inflammation.FEBS J. 2013; 280: 2165-2179Crossref PubMed Scopus (194) Google Scholar Glycan recognition by GBPs is most often focused on the outermost grouping of sugars on a glycoprotein or glycolipid oligosaccharide. In man and other mammals oligosaccharides are often terminated with sialic acid, the largest and most structurally complex of the monosaccharides that make up mammalian glycans (Fig 1). Evolution has often used sialic acid's structural complexity in developing specificity for GBP recognition.14Cohen M. Varki A. The sialome—far more than the sum of its parts.OMICS. 2010; 14: 455-464Crossref PubMed Scopus (174) Google Scholar This is true for some pathogens that use sialic acid to gain access to host cells and for the immune system, which uses sialic acid for immune cell recognition, trafficking, and regulation. Examples of both sialic acid–dependent and independent GBPs and their roles in immune system function follow. Because glycans are dominant chemical determinants on the surfaces of every cell of every living organism, the GBPs that translate glycan recognition into function are also found throughout biology, from viral hemagglutinins to bacterial adhesins to plant and animal lectins.3Taylor M.E. Drickamer K. Convergent and divergent mechanisms of sugar recognition across kingdoms.Curr Opin Struct Biol. 2014; 28C: 14-22Crossref Scopus (52) Google Scholar Every GBP is characterized by a unique carbohydrate recognition domain with precisely spaced amino acid functional groups that selectively engage a particular glycan target. The carbohydrate recognition domain is found in association with other protein domains that translate recognition into appropriate biological responses. Understanding GBPs, their targets, and their functions provides insight into molecular processes in immune regulation that are amenable to therapeutic intervention. Among the first GBPs discovered were viral hemagglutinins, with influenza virus remaining the clearest example. When exposed to cultured influenza virus, human red blood cells agglutinated in a manner that spontaneously reversed over time.15Hirst G.K. Adsorption of influenza hemagglutinins and virus by red blood cells.J Exp Med. 1942; 76: 195-209Crossref PubMed Scopus (120) Google Scholar The virus-treated red blood cells remained intact, but after reversal of agglutination, they were resistant to agglutination with fresh virus. The virus receptor on the red blood cell had been removed. Eventually, the factor released from red blood cells after incubation with virus was identified as sialic acid. The viral hemagglutinin was identified as a sialic acid–specific GBP, and the viral enzyme that removed the receptor was identified as a sialidase (also called neuraminidase).16Gottschalk A. The influenza virus neuraminidase.Nature. 1958; 181: 377-378Crossref PubMed Scopus (25) Google Scholar Influenza viruses bind to sialic acid–terminated oligosaccharides on human airway epithelium, enter cells and replicate, and then bud out in large numbers. The slower-acting sialidase then comes into play by removing the sialic acids on the budded virus particles themselves, allowing them to disengage, disseminate, and bind to sialic acids on fresh target cells to continue the replication cycle. The rational design of sialic acid analogs (sialomimetics) able to inhibit sialidase and break this cycle led to the development of 2 first-line anti-influenza drugs, Relenza (GlaxoSmithKline, Research Triangle Park, NC) and Tamiflu (Genentech, South San Francisco, Calif).17von Itzstein M. Wu W.Y. Kok G.B. Pegg M.S. Dyason J.C. Jin B. et al.Rational design of potent sialidase-based inhibitors of influenza virus replication.Nature. 1996; 363: 418-423Crossref Scopus (1759) Google Scholar, 18von Itzstein M. The war against influenza: discovery and development of sialidase inhibitors.Nat Rev Drug Discov. 2007; 6: 967-974Crossref PubMed Scopus (595) Google Scholar In the presence of these drugs, sialic acids on the surfaces of freshly budded virions persist and engage the hemagglutinin on adjacent virions so that they clump together and do not disseminate, halting the ongoing infection. The carbohydrate-binding domain of influenza hemagglutinin is specific for the particular way in which a terminal sialic acid is linked to the rest of the oligosaccharide chain. Avian influenza virus hemagglutinins bind to sialic acids linked to the 3-position hydroxyl of a galactose, whereas human influenza virus hemagglutinins bind to sialic acid linked to the 6-position hydroxyl of galactose.19Stevens J. Blixt O. Paulson J.C. Wilson I.A. Glycan microarray technologies: tools to survey host specificity of influenza viruses.Nat Rev Microbiol. 2006; 4: 857-864Crossref PubMed Scopus (295) Google Scholar Although this seems like a modest structural difference, the shift from binding 3- to 6-linked sialic acid marks its transition from a deadly bird disease to a deadly human disease, and the Centers for Disease Control and Prevention is using that difference to track the potential conversion of avian to human influenza viruses worldwide.20Stevens J. Blixt O. Chen L.M. Donis R.O. Paulson J.C. Wilson I.A. Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity.J Mol Biol. 2008; 381: 1382-1394Crossref PubMed Scopus (179) Google Scholar Many other viral, bacterial, and parasitic pathogens also use glycans as adhesive targets, the study of which might provide insights into their pathophysiology and provide new antimicrobial drugs.21Bernardi A. Jimenez-Barbero J. Casnati A. De C.C. Darbre T. Fieschi F. et al.Multivalent glycoconjugates as anti-pathogenic agents.Chem Soc Rev. 2013; 42: 4709-4727Crossref PubMed Scopus (437) Google Scholar Evolution has retained abundant glycans on all human cells despite the potent pathogens that take advantage of them, demonstrating their fundamental physiologic roles. The first human GBP was discovered serendipitously in the late 1960s. In the intervening years, more than 80 human GBPs representing a dozen different structural families have been discovered (see http://www.imperial.ac.uk/research/animallectins). Although they have been found to play diverse roles, their expression and functions in the immune system continue to garner the most attention.4Marth J.D. Grewal P.K. Mammalian glycosylation in immunity.Nat Rev Immunol. 2008; 8: 874-887Crossref PubMed Scopus (542) Google Scholar, 5van Kooyk Y. Rabinovich G.A. Protein-glycan interactions in the control of innate and adaptive immune responses.Nat Immunol. 2008; 9: 593-601Crossref PubMed Scopus (586) Google Scholar, 22Macauley M.S. Crocker P.R. Paulson J.C. Siglec-mediated regulation of immune cell function in disease.Nat Rev Immunol. 2014; 14: 653-666Crossref PubMed Scopus (681) Google Scholar, 23Johnson J.L. Jones M.B. Ryan S.O. Cobb B.A. The regulatory power of glycans and their binding partners in immunity.Trends Immunol. 2013; 34: 290-298Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Examples from 3 families demonstrating different ways in which glycans control immunity will be presented here: galectins, selectins (a subclass of C-type lectins), and siglecs (Fig 2).24Rabinovich G.A. Toscano M.A. Turning ‘sweet’ on immunity: galectin-glycan interactions in immune tolerance and inflammation.Nat Rev Immunol. 2009; 9: 338-352Crossref PubMed Scopus (714) Google Scholar, 25von Andrian U.H. Mackay C.R. T-cell function and migration. Two sides of the same coin.N Engl J Med. 2000; 343: 1020-1034Crossref PubMed Scopus (1237) Google Scholar, 26Chang Y.C. Nizet V. The interplay between Siglecs and sialylated pathogens.Glycobiology. 2014; 24: 818-825Crossref PubMed Scopus (96) Google Scholar A general description of each of these GBP families is followed by specific examples of the roles of selected family members in immune system function. Galectins are a family of evolutionarily conserved small soluble secreted GBPs (10 in human subjects) with wide tissue distribution and a variety of regulatory effects on innate and adaptive immune responses.27Liu F.T. Rabinovich G.A. Galectins: regulators of acute and chronic inflammation.Ann N Y Acad Sci. 2010; 1183: 158-182Crossref PubMed Scopus (320) Google Scholar As their name implies, they are galactose-binding lectins, and their major glycan targets are galactose-terminated oligosaccharides on a subset of glycoproteins and glycolipids. Galectins spontaneously form dimers and higher oligomers that cross-link target glycans on cell surfaces, altering their residency time, distribution, and downstream signaling. By selectively cross-linking appropriate glycans on immune cell-surface glycoproteins, such as the T-cell receptor (TCR), CD45, and CD43 (among others), galectins direct immune cell maturation, survival, and activation.24Rabinovich G.A. Toscano M.A. Turning ‘sweet’ on immunity: galectin-glycan interactions in immune tolerance and inflammation.Nat Rev Immunol. 2009; 9: 338-352Crossref PubMed Scopus (714) Google Scholar, 25von Andrian U.H. Mackay C.R. T-cell function and migration. Two sides of the same coin.N Engl J Med. 2000; 343: 1020-1034Crossref PubMed Scopus (1237) Google Scholar, 26Chang Y.C. Nizet V. The interplay between Siglecs and sialylated pathogens.Glycobiology. 2014; 24: 818-825Crossref PubMed Scopus (96) Google Scholar, 27Liu F.T. Rabinovich G.A. Galectins: regulators of acute and chronic inflammation.Ann N Y Acad Sci. 2010; 1183: 158-182Crossref PubMed Scopus (320) Google Scholar, 28Garner O.B. Baum L.G. Galectin-glycan lattices regulate cell-surface glycoprotein organization and signalling.Biochem Soc Trans. 2008; 36: 1472-1477Crossref PubMed Scopus (171) Google Scholar The selectins (E-, L-, and P-selectins) are a subset of the larger calcium-dependent (C-type) lectin family.29Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1.Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar They are single-pass transmembrane cell adhesion proteins, each with an externally facing carbohydrate recognition domain that binds to a grouping of sugars that include appropriately spaced sialic acid and fucose residues. E- and P-selectins are expressed on vascular endothelial cells in response to inflammatory signals and then bind to glycans on passing leukocytes to initiate inflammation. L-selectin is expressed on leukocytes and binds to glycans on the lumens of high endothelial venules, initiating migration of leukocytes from blood to lymph. The 3 selectins are examples of subspecialization within the diverse C-type lectin family, which includes other important immunoregulatory lectins, such as mannose-binding lectin, dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin, Dectins, and Mincle (among others).30Hoving J.C. Wilson G.J. Brown G.D. Signalling C-type lectin receptors, microbial recognition and immunity.Cell Microbiol. 2014; 16: 185-194Crossref PubMed Scopus (185) Google Scholar Sialic acid–binding immunoglobulin-like lectins (siglecs) are the most recently discovered family of human GBPs.22Macauley M.S. Crocker P.R. Paulson J.C. Siglec-mediated regulation of immune cell function in disease.Nat Rev Immunol. 2014; 14: 653-666Crossref PubMed Scopus (681) Google Scholar The family is comprised of 14 members in humans, most of which are selectively expressed on subsets of hematopoietic cells. They are single-pass transmembrane proteins with varying numbers of externally facing immunoglobulin-like domains terminated with a carbohydrate recognition immunoglobulin-like domain that binds to sialic acid–terminated glycans. Many members also have immunoregulatory sequences (immunoreceptor tyrosine-based inhibitory motifs or immunoreceptor tyrosine-based activation motifs) on their cytoplasmic tails, indicating their roles in immunoregulatory transmembrane signaling. Although every siglec requires a sialic acid in its glycan-binding target, the family has evolved to take advantage of the many ways in which sialic acids are arranged in larger glycan structures. Like the influenza virus hemagglutinin, some siglecs bind only to sialic acid when it is linked to the 3-hydroxyl of galactose, and others bind only to sialic acid linked to the 6-hydroxyl of galactose. Yet others prefer sialic acid linked to the 8-hydroxyl of another sialic acid, and some require other spaced molecular constituents (eg, sulfates) on their oligosaccharide targets. In a metaphorical sea of cell-surface sialic acids, these glycan-binding specificities provide selective recognition to siglec family members that are key to their functions in immune regulation. GBPs, which are expressed on immune cells of all types, serve diverse regulatory rolls in leukocyte trafficking, pathogen recognition, antigen processing, immune activation, and immunosuppression.4Marth J.D. Grewal P.K. Mammalian glycosylation in immunity.Nat Rev Immunol. 2008; 8: 874-887Crossref PubMed Scopus (542) Google Scholar, 5van Kooyk Y. Rabinovich G.A. Protein-glycan interactions in the control of innate and adaptive immune responses.Nat Immunol. 2008; 9: 593-601Crossref PubMed Scopus (586) Google Scholar, 23Johnson J.L. Jones M.B. Ryan S.O. Cobb B.A. The regulatory power of glycans and their binding partners in immunity.Trends Immunol. 2013; 34: 290-298Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Glycans on immune cells are also recognized by GBPs on nonimmune cells and tissues (like those on the vascular endothelium) that bind to leukocyte glycans. The 3 vignettes from this extensive area of research that follow (Fig 2) demonstrate paradigms for glycan-mediated immune regulation. Additional examples that are especially relevant to allergic diseases are detailed in an accompanying article in this issue of the Journal.9Bochner B.S. Zimmermann N. Role of siglecs and related glycan-binding proteins in immune responses and immunoregulation.J Allergy Clin Immunol. 2015; 135: 598-608Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar The adaptive immune response is carefully tuned to protect against autoimmunity. One mechanism by which this occurs is through galectins binding to N-linked glycans on the TCR.31Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation.Nature. 2001; 409: 733-739Crossref PubMed Scopus (755) Google Scholar, 32Dennis J.W. Lau K.S. Demetriou M. Nabi I.R. Adaptive regulation at the cell surface by N-glycosylation.Traffic. 2009; 10: 1569-1578Crossref PubMed Scopus (170) Google Scholar Because glycoproteins, including the TCR, traverse the Golgi apparatus, their nascent N-linked glycans are subject to modification through a suite of glycosidases and glycosyltransferases that act in concert to generate mature glycans on selected protein targets. The TCR (along with many other glycoproteins) is subject to the action of the glycosyltransferase mannoside N-acetylglucosaminyltransferase 5, the product of the MGAT5 gene. This leads to elongation of one of its N-linked glycan branches with repeated Gal-GlcNAc repeats, which are high-affinity receptors for galectins, such as galectin-3. Once the TCR reaches the T-cell surface, the Gal-GlcNAc repeats on the TCR recruit galectins, which cross-link the TCRs into a cell-surface lattice that restricts the TCR clustering required for T-cell activation. The effect of galectin-TCR lattice formation is that the T cells require higher agonist concentrations to trigger activation. Thus the action of a particular Golgi glycosyltransferase in T cells is responsible for fine-tuning its responsiveness to agonists. In a potential feedback loop, activation of the TCR regulates expression of Golgi glycosidases and glycosyltransferases to shift the balance of N-linked glycans on the TCR toward those with enhanced galectin binding.33Chen H.L. Li C.F. Grigorian A. Tian W. Demetriou M. T cell receptor signaling co-regulates multiple Golgi genes to enhance N-glycan branching.J Biol Chem. 2009; 284: 32454-32461Crossref PubMed Scopus (45) Google Scholar In this way initial TCR activation might be followed by glycan-regulated desensitization. The function of this pathway in immune regulation was demonstrated in Mgat5 knockout mice, which display worse outcomes (enhanced sensitivity) in models of autoimmunity, including autoimmune glomerulonephritis, delayed-type hypersensitivity, and experimental autoimmune encephalomyelitis (an animal model for multiple sclerosis).31Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation.Nature. 2001; 409: 733-739Crossref PubMed Scopus (755) Google Scholar In human subjects genome-wide association studies revealed hypomorphic MGAT5 variants as factors in multiple sclerosis susceptibility.34Li C.F. Zhou R.W. Mkhikian H. Newton B.L. Yu Z. Demetriou M. Hypomorphic MGAT5 polymorphisms promote multiple sclerosis cooperatively with MGAT1 and interleukin-2 and 7 receptor variants.J Neuroimmunol. 2013; 256: 71-76Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Remarkably, oral administration of N-acetylglucosamine to mice increased the galectin-binding N-glycan structures on their T cells, reduced TCR signaling, and reduced the symptoms of experimental autoimmunity.35Grigorian A. Lee S.U. Tian W. Chen I.J. Gao G. Mendelsohn R. et al.Control of T Cell-mediated autoimmunity by metabolite flux to N-glycan biosynthesis.J Biol Chem. 2007; 282: 20027-20035Crossref PubMed