T Cell Receptor Signaling Co-regulates Multiple Golgi Genes to Enhance N-Glycan Branching
2009; Elsevier BV; Volume: 284; Issue: 47 Linguagem: Inglês
10.1074/jbc.m109.023630
ISSN1083-351X
AutoresHung-Lin Chen, Carey Fei Li, Ani Grigorian, Wenqiang Tian, Michael A. Demetriou,
Tópico(s)Peptidase Inhibition and Analysis
ResumoT cell receptor (TCR) signaling enhances β1,6GlcNAc-branching in N-glycans, a phenotype that promotes growth arrest and inhibits autoimmunity by increasing surface retention of cytotoxic T lymphocyte antigen-4 (CTLA-4) via interactions with galectins. N-Acetylglucosaminyltransferase V (MGAT5) mediates β1,6GlcNAc-branching by transferring N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to N-glycan substrates produced by the sequential action of Golgi α1,2-mannosidase I (MIa,b,c), MGAT1, α1,2-mannosidase II (MII, IIx), and MGAT2. Here we report that TCR signaling enhances mRNA levels of MIa,b,c and MII,IIx in parallel with MGAT5, whereas limiting levels of MGAT1 and MGAT2. Blocking the increase in MI or MII enzyme activity induced by TCR signaling with deoxymannojirimycin or swainsonine, respectively, limits β1,6GlcNAc-branching, suggesting that enhanced MI and MII activity are both required for this phenotype. MGAT1 and MGAT2 have an ∼250- and ∼20-fold higher affinity for UDP-GlcNAc than MGAT5, respectively, and increasing MGAT1 expression paradoxically inhibits β1,6GlcNAc branching by limiting UDP-GlcNAc supply to MGAT5, suggesting that restricted changes in MGAT1 and MGAT2 mRNA levels in TCR-stimulated cells serves to enhance availability of UDP-GlcNAc to MGAT5. Together, these data suggest that TCR signaling differentially regulates multiple N-glycan-processing enzymes at the mRNA level to cooperatively promote β1,6GlcNAc branching, and by extension, CTLA-4 surface expression, T cell growth arrest, and self-tolerance. T cell receptor (TCR) signaling enhances β1,6GlcNAc-branching in N-glycans, a phenotype that promotes growth arrest and inhibits autoimmunity by increasing surface retention of cytotoxic T lymphocyte antigen-4 (CTLA-4) via interactions with galectins. N-Acetylglucosaminyltransferase V (MGAT5) mediates β1,6GlcNAc-branching by transferring N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to N-glycan substrates produced by the sequential action of Golgi α1,2-mannosidase I (MIa,b,c), MGAT1, α1,2-mannosidase II (MII, IIx), and MGAT2. Here we report that TCR signaling enhances mRNA levels of MIa,b,c and MII,IIx in parallel with MGAT5, whereas limiting levels of MGAT1 and MGAT2. Blocking the increase in MI or MII enzyme activity induced by TCR signaling with deoxymannojirimycin or swainsonine, respectively, limits β1,6GlcNAc-branching, suggesting that enhanced MI and MII activity are both required for this phenotype. MGAT1 and MGAT2 have an ∼250- and ∼20-fold higher affinity for UDP-GlcNAc than MGAT5, respectively, and increasing MGAT1 expression paradoxically inhibits β1,6GlcNAc branching by limiting UDP-GlcNAc supply to MGAT5, suggesting that restricted changes in MGAT1 and MGAT2 mRNA levels in TCR-stimulated cells serves to enhance availability of UDP-GlcNAc to MGAT5. Together, these data suggest that TCR signaling differentially regulates multiple N-glycan-processing enzymes at the mRNA level to cooperatively promote β1,6GlcNAc branching, and by extension, CTLA-4 surface expression, T cell growth arrest, and self-tolerance. IntroductionT cell activation and differentiation induce a number of functionally important changes in cell surface N-glycosylation (1.Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (729) Google Scholar, 2.Morgan R. Gao G. Pawling J. Dennis J.W. Demetriou M. Li B. J. Immunol. 2004; 173: 7200-7208Crossref PubMed Scopus (130) Google Scholar, 3.Comelli E.M. Sutton-Smith M. Yan Q. Amado M. Panico M. Gilmartin T. Whisenant T. Lanigan C.M. Head S.R. Goldberg D. Morris H.R. Dell A. Paulson J.C. J. Immunol. 2006; 177: 2431-2440Crossref PubMed Scopus (97) Google Scholar, 4.Toscano M.A. Bianco G.A. Ilarregui J.M. Croci D.O. Correale J. Hernandez J.D. Zwirner N.W. Poirier F. Riley E.M. Baum L.G. Rabinovich G.A. Nat. Immunol. 2007; 8: 825-834Crossref PubMed Scopus (498) Google Scholar). For example, activation signaling leads to replacement of α2,6-linked sialic acid with α1,3-linked galactose on the termini of N-acetyllactosamine (Galβ1,4GlcNAc) units. N-Acetyllactosamine is the ligand for the galectin family of carbohydrate-binding proteins, and the addition of terminal α2,6-linked sialic acid by the ST6Gal1 transferase inhibits galectin binding (4.Toscano M.A. Bianco G.A. Ilarregui J.M. Croci D.O. Correale J. Hernandez J.D. Zwirner N.W. Poirier F. Riley E.M. Baum L.G. Rabinovich G.A. Nat. Immunol. 2007; 8: 825-834Crossref PubMed Scopus (498) Google Scholar, 5.Amano M. Galvan M. He J. Baum L.G. J. Biol. Chem. 2003; 278: 7469-7475Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 6.Stowell S.R. Arthur C.M. Mehta P. Slanina K.A. Blixt O. Leffler H. Smith D.F. Cummings R.D. J. Biol. Chem. 2008; 283: 10109-10123Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 7.Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W.E. Yagi F. Kasai K. Biochim. Biophys. Acta. 2002; 1572: 232-254Crossref PubMed Scopus (813) Google Scholar). T cell differentiation leads to enhanced expression of ST6Gal1 in TH2 relative to TH1/TH17 cells, thereby reducing sensitivity to galectin-1-induced apoptosis(4.Toscano M.A. Bianco G.A. Ilarregui J.M. Croci D.O. Correale J. Hernandez J.D. Zwirner N.W. Poirier F. Riley E.M. Baum L.G. Rabinovich G.A. Nat. Immunol. 2007; 8: 825-834Crossref PubMed Scopus (498) Google Scholar). Galectins bind surface glycoproteins in proportion to N-acetyllactosamine content, forming a molecular lattice that regulates distribution of glycoproteins to membrane microdomains (i.e. lipid rafts) and inhibits their loss to endocytosis(1.Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (729) Google Scholar, 8.Partridge E.A. Le Roy C. Di Guglielmo G.M. Pawling J. Cheung P. Granovsky M. Nabi I.R. Wrana J.L. Dennis J.W. Science. 2004; 306: 120-124Crossref PubMed Scopus (583) Google Scholar, 9.Nieminen J. Kuno A. Hirabayashi J. Sato S. J. Biol. Chem. 2007; 282: 1374-1383Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 10.Brewer C.F. Miceli M.C. Baum L.G. Curr. Opin. Struct. Biol. 2002; 12: 616-623Crossref PubMed Scopus (371) Google Scholar, 11.Ahmad N. Gabius H.J. André S. Kaltner H. Sabesan S. Roy R. Liu B. Macaluso F. Brewer C.F. J. Biol. Chem. 2004; 279: 10841-10847Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 12.Chen I.J. Chen H.L. Demetriou M. J. Biol. Chem. 2007; 282: 35361-35372Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 13.Grigorian A. Torossian S. Demetriou M. Immunol. Rev. 2009; 230: 232-246Crossref PubMed Scopus (102) Google Scholar). Binding avidity of galectins for glycoproteins is regulated by the number of attached N-glycans (i.e. N = occupied N-X-(S/T) sites, X ≠ P), an encoded feature of protein sequences, as well as the degree of Golgi-mediated GlcNAc branching, which together determine N-acetyllactosamine content in glycoproteins(14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). The number of N-X-(S/T) sites varies widely between different glycoproteins, with growth-promoting membrane glycoproteins (e.g. T cell receptor (TCR)) 2The abbreviations used are: TCRT cell receptorGEMGM1-enriched lipid microdomainsEAEexperimental autoimmune encephalomyelitisMIα-mannosidase IMIIα-mannosidase IIL-PHAleukoagglutininFITCfluorescein isothiocyanateFACSfluorescence-activated cell sorterELISAenzyme-linked immunosorbent assayPBMCperipheral blood mononuclear cellMBPmyelin basic proteinILinterleukinIFNinterferonSWswainsonineDMNdeoxymannojirimycinMAPKmitogen-activated protein kinase. generally displaying large numbers of N-X-(S/T) sites (i.e. n > 5), whereas growth inhibitory receptors (e.g. cytotoxic T lymphocyte antigen-4 (CTLA-4)) have few N-glycans (n ≤ 4)(14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). The large difference in galectin avidity for high and low multiplicity receptors allows Golgi-mediated changes in GlcNAc branching to differentially control surface retention (i.e. endocytosis rates) of these receptors and therefore transitions between growth and arrest signaling (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar).In resting T cells, where endocytosis is minimal, galectin-3 binds the TCR and the tyrosine phosphatase CD45 but partitions them to different membrane compartments by opposing F-actin targeting (12.Chen I.J. Chen H.L. Demetriou M. J. Biol. Chem. 2007; 282: 35361-35372Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Galectin binding prevents spontaneous TCR oligomerization in the absence of antigen, thereby blocking recruitment of multiple adaptor proteins and CD4-Lck to TCR, F-actin-mediated transfer to GM1-enriched lipid microdomains (GEMs), and activation signaling by Lck. In parallel, galectin partitions CD45 to GEMs by counteracting F-actin-mediated exclusion, dampening Lck activation. Upon encounter with antigen, GEMs cluster at the immune synapse formed at the contact site between T cells and antigen-presenting cells, with the galectin lattice reducing TCR/CD4-Lck and increasing CD45 concentration to dampen T cell activation and TH1 differentiation (1.Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (729) Google Scholar, 2.Morgan R. Gao G. Pawling J. Dennis J.W. Demetriou M. Li B. J. Immunol. 2004; 173: 7200-7208Crossref PubMed Scopus (130) Google Scholar, 12.Chen I.J. Chen H.L. Demetriou M. J. Biol. Chem. 2007; 282: 35361-35372Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). After activation, membrane turnover increases in T cell blasts, markedly limiting surface levels of the growth inhibitor CTLA-4 via constitutive endocytosis (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). GlcNAc branching increases in blasting T cells, enhancing CTLA-4 affinity for galectins and opposing surface loss to endocytosis (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Thus, GlcNAc branching in N-glycans negatively regulates T cell growth early by raising T cell activation thresholds and later by enhancing CTLA-4-mediated growth arrest.GlcNAc branching is regulated by activities of the medial Golgi N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) (15.Schachter H. Glycobiology. 1991; 1: 453-461Crossref PubMed Scopus (167) Google Scholar), as well as metabolic supply of their shared substrate UDP-GlcNAc (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 16.Grigorian A. Lee S.U. Tian W. Chen I.J. Gao G. Mendelsohn R. Dennis J.W. Demetriou M. J. Biol. Chem. 2007; 282: 20027-20035Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Mgat1, Mgat2, Mgat4, and Mgat5 transfer N-acetylglucosamine from UDP-GlcNAc to N-glycans transiting the medial Golgi, forming mono-, bi-, tri-, and tetra-antennary GlcNAc-branched N-glycans (see Fig. 1A). β1,6GlcNAc-branched N-glycans produced by Mgat5 are preferentially extended by poly-N-acetyllactosamine, generating high affinity ligands for galectins (7.Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W.E. Yagi F. Kasai K. Biochim. Biophys. Acta. 2002; 1572: 232-254Crossref PubMed Scopus (813) Google Scholar, 17.Cummings R.D. Kornfeld S. J. Biol. Chem. 1984; 259: 6253-6260Abstract Full Text PDF PubMed Google Scholar). Mgat5-deficient 129/Sv mice display T cell hyperactivity in vivo, as exemplified by enhanced type IV hypersensitivity, spontaneous kidney autoimmunity, and increased experimental autoimmune encephalomyelitis (EAE) (1.Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (729) Google Scholar), an animal model of multiple sclerosis. Susceptibility of inbred mouse strains to EAE varies widely and correlates with strain-specific deficiencies in GlcNAc branching in T cells (18.Lee S.U. Grigorian A. Pawling J. Chen I.J. Gao G. Mozaffar T. McKerlie C. Demetriou M. J. Biol. Chem. 2007; 282: 33725-33734Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The EAE-susceptible PL/J strain intrinsically possesses reduced basal activities of Mgat1, Mgat2, and Mgat5 and develops a spontaneous multiple sclerosis-like disease that is markedly enhanced by Mgat5+/− and Mgat5−/− backgrounds in a gene dose-dependent manner. Autoimmunity is a complex trait that is influenced by environmental inputs. In this regard, N-glycan GlcNAc branching is regulated by the nutrient environment and metabolism via hexosamine pathway production of UDP-GlcNAc, the sugar nucleotide donor for Mgat1, Mgat2, Mgat4, and Mgat5 (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 16.Grigorian A. Lee S.U. Tian W. Chen I.J. Gao G. Mendelsohn R. Dennis J.W. Demetriou M. J. Biol. Chem. 2007; 282: 20027-20035Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Overexpression of Mgat1, an enzyme that has an ∼250-fold lower Km for UDP-GlcNAc than Mgat5, paradoxically reduces β1,6GlcNAc branching by limiting UDP-GlcNAc supply to Mgat5 (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Metabolic supplements to the hexosamine pathway suppress TCR signaling, T cell activation/proliferation, TH1 differentiation, CTLA-4 endocytosis, and autoimmunity (EAE and autoimmune diabetes). TCR signaling results in sequential increases in glucose flux, UDP-GlcNAc production, β1,6GlcNAc branching, and finally, CTLA-4 surface expression.These data implicate TCR signaling-mediated enhancement of GlcNAc branching as a critical regulator of CTLA-4 surface retention and autoimmunity. In addition to the Mgat enzymes, Golgi α-mannosidase I (MI) and II (MII) are required for GlcNAc branching (15.Schachter H. Glycobiology. 1991; 1: 453-461Crossref PubMed Scopus (167) Google Scholar, 19.Akama T.O. Nakagawa H. Wong N.K. Sutton-Smith M. Dell A. Morris H.R. Nakayama J. Nishimura S. Pai A. Moremen K.W. Marth J.D. Fukuda M.N. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8983-8988Crossref PubMed Scopus (57) Google Scholar, 20.Herscovics A. Biochimie. 2001; 83: 757-762Crossref PubMed Scopus (92) Google Scholar). MI acts upstream and MII acts downstream of Mgat1, steps required for the action of Mgat2 and production of bi-, tri-, and tetra-antennary N-glycans (see Fig. 1A). In mammals, the Mgat1, Mgat2, and Mgat5 enzymes are encoded by single genes, whereas three genes encode MI (MIa = MAN1A1, MIb = MAN1A2, MIc = MAN1C1) and two encode MII (MAN2A1, MAN2A2). In T cells, MAN2A1 and MAN2A2 are both expressed, and loss of the former is reported to have little effect on GlcNAc branching or T cell growth (21.Chui D. Sellakumar G. Green R. Sutton-Smith M. McQuistan T. Marek K. Morris H. Dell A. Marth J. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 1142-1147Crossref PubMed Scopus (179) Google Scholar). Effects of targeted deletion of other Golgi α-mannosidase genes in T cells have not been explored (22.Tremblay L.O. Nagy Kovács E. Daniels E. Wong N.K. Sutton-Smith M. Morris H.R. Dell A. Marcinkiewicz E. Seidah N.G. McKerlie C. Herscovics A. J. Biol. Chem. 2007; 282: 2558-2566Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 23.Akama T.O. Nakagawa H. Sugihara K. Narisawa S. Ohyama C. Nishimura S. O'Brien D.A. Moremen K.W. Millan J.L. Fukuda M.N. Science. 2002; 295: 124-127Crossref PubMed Scopus (124) Google Scholar). Here we report that TCR signaling enhances expression of all five Golgi α-mannosidase genes in parallel with Mgat5, an activity required for optimal production of β1,6GlcNAc-branched N-glycans. In contrast, TCR signaling has little or negative effect on Mgat1 and Mgat2 expression, presumably to increase supply of UDP-GlcNAc to Mgat4 and Mgat5 and promote GlcNAc branching. Our data suggest that TCR signaling differentially regulates multiple Golgi enzymes at the mRNA levels to enhance GlcNAc branching in T cell blasts, and subsequently, growth arrest by CTLA-4.DISCUSSIONGlcNAc branching of N-glycans attached to cell surface glycoproteins such as TCR, CD45, and CTLA-4 titrates binding to galectins, forming a molecular lattice that negatively regulates T cell growth and susceptibility to autoimmunity. TCR signaling up-regulates MGAT5 gene expression and metabolic supply of UDP-GlcNAc to the Golgi to promote GlcNAc branching and CTLA-4 retention at the cell surface. Here we find that TCR signaling differentially regulates mRNA expression of multiple genes upstream of Mgat5 in the Golgi to enhance GlcNAc branching. MGAT5 mRNA expression strongly correlates with the three Golgi MI and the two Golgi MII genes. Limiting TCR signaling-mediated increases in MI or MII enzyme activity confirms that this phenotype is required to enhance GlcNAc branching. In contrast, TCR signaling-mediated changes in mRNA levels in MGAT1 and MGAT2 do not correlate with MGAT5/MI/MII genes, being reduced when MGAT5 expression is maximally increased. MAGT1, MAGT2, and MAGT5 act sequentially, utilize UDP-GlcNAc as substrate, and are absolutely required for biosynthesis of β1,6GlcNAc-branched N-glycans. However, MGAT1 and MGAT2 have 200- and 20-fold lower Km for UDP-GlcNAc than MGAT5, suggesting unequal competition for substrate in the medial Golgi (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Indeed, overexpression of MGAT1 reduces GlcNAc branching by reducing supply of UDP-GlcNAc to downstream GlcNAc transferases (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). We conclude that positive regulation of GlcNAc-branched N-glycans by TCR signaling, and by extension, CTLA-4 surface retention and inhibition of autoimmunity, require coordinated up-regulation of MI, MII, and Mgat5 activities and UDP-GlcNAc biosynthesis, coupled with limited changes/suppression of Mgat1 and Mgat2.MAN2A1-deficient resting T cells from C57BL/6 mice are reported to display little change in GlcNAc-branched N-glycans, suggesting that MAN2A2 supplies sufficient MII activity to compensate for this loss (21.Chui D. Sellakumar G. Green R. Sutton-Smith M. McQuistan T. Marek K. Morris H. Dell A. Marth J. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 1142-1147Crossref PubMed Scopus (179) Google Scholar). Whether this is also true in activated T cells requires further investigation; however, it is consistent with our conclusion that up-regulation of total MII activity, via increases in both MAN2A1 and MAN2A2 mRNA, is the physiologically relevant change. Similarly, experimentally blocking up-regulation of a single MI gene in activated T cells is unlikely to be biologically significant; rather, enhanced MI activity distributed over increases in the three genes is likely the relevant phenotype required to increase GlcNAc branching in activated T cells.Enhanced GlcNAc branching in activated T cells promotes cell surface retention of CTLA-4 (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar), a critical negative regulator of autoimmunity (35.Waterhouse P. Penninger J.M. Timms E. Wakeham A. Shahinian A. Lee K.P. Thompson C.B. Griesser H. Mak T.W. Science. 1995; 270: 985-988Crossref PubMed Scopus (2354) Google Scholar). Coordinated up-regulation of MGAT5, MAN1A1, MAN1A2, MAN1C1, MAN2A1, and MAN2A2 mRNA downstream of TCR signaling suggests a common transcriptional regulator(s) activated by the phosphatidylinositol 3-kinase/ERK pathway. Defining the molecular mechanism for this co-regulation should provide new critical regulators of GlcNAc branching and expand the hexosamine/Golgi gene network regulating CTLA-4 surface retention. Genetic variations in network genes are prime candidates for regulation of human autoimmunity.Increasing the expression of MGAT1 reduces GlcNAc branching by reducing availability of UDP-GlcNAc to Mgat4 and Mgat5, suggesting that limiting Mgat1 activity is necessary for optimal increases in GlcNAc branching. MGAT1 and MGAT2 are simple genes with two and one exons, respectively, whereas MGAT5 has 16 exons with long intervening introns. It is tempting to speculate that these differences evolved to permit continuous and consistent production of glycoproteins with a minimum affinity for galectin (i.e. mono- and biantennary N-glycans by Mgat1 and Mgat2) and limited but tightly regulated production of high affinity galectin ligands by Mgat5. As exemplified in T cells, our data suggest that coordinated expression of multiple N-glycan pathway genes and UDP-GlcNAc production by phosphatidylinositol 3-kinase/ERK signaling is necessary for optimal regulation of GlcNAc branching, and when disturbed, may lead to diseases such as autoimmunity, cancer, and type 2 diabetes (1.Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (729) Google Scholar, 36.Demetriou M. Nabi I.R. Coppolino M. Dedhar S. Dennis J.W. J. Cell Biol. 1995; 130: 383-392Crossref PubMed Scopus (250) Google Scholar, 37.Granovsky M. Fata J. Pawling J. Muller W.J. Khokha R. Dennis J.W. Nat. Med. 2000; 6: 306-312Crossref PubMed Scopus (461) Google Scholar, 38.Ohtsubo K. Takamatsu S. Minowa M.T. Yoshida A. Takeuchi M. Marth J.D. Cell. 2005; 123: 1307-1321Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). IntroductionT cell activation and differentiation induce a number of functionally important changes in cell surface N-glycosylation (1.Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (729) Google Scholar, 2.Morgan R. Gao G. Pawling J. Dennis J.W. Demetriou M. Li B. J. Immunol. 2004; 173: 7200-7208Crossref PubMed Scopus (130) Google Scholar, 3.Comelli E.M. Sutton-Smith M. Yan Q. Amado M. Panico M. Gilmartin T. Whisenant T. Lanigan C.M. Head S.R. Goldberg D. Morris H.R. Dell A. Paulson J.C. J. Immunol. 2006; 177: 2431-2440Crossref PubMed Scopus (97) Google Scholar, 4.Toscano M.A. Bianco G.A. Ilarregui J.M. Croci D.O. Correale J. Hernandez J.D. Zwirner N.W. Poirier F. Riley E.M. Baum L.G. Rabinovich G.A. Nat. Immunol. 2007; 8: 825-834Crossref PubMed Scopus (498) Google Scholar). For example, activation signaling leads to replacement of α2,6-linked sialic acid with α1,3-linked galactose on the termini of N-acetyllactosamine (Galβ1,4GlcNAc) units. N-Acetyllactosamine is the ligand for the galectin family of carbohydrate-binding proteins, and the addition of terminal α2,6-linked sialic acid by the ST6Gal1 transferase inhibits galectin binding (4.Toscano M.A. Bianco G.A. Ilarregui J.M. Croci D.O. Correale J. Hernandez J.D. Zwirner N.W. Poirier F. Riley E.M. Baum L.G. Rabinovich G.A. Nat. Immunol. 2007; 8: 825-834Crossref PubMed Scopus (498) Google Scholar, 5.Amano M. Galvan M. He J. Baum L.G. J. Biol. Chem. 2003; 278: 7469-7475Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 6.Stowell S.R. Arthur C.M. Mehta P. Slanina K.A. Blixt O. Leffler H. Smith D.F. Cummings R.D. J. Biol. Chem. 2008; 283: 10109-10123Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 7.Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W.E. Yagi F. Kasai K. Biochim. Biophys. Acta. 2002; 1572: 232-254Crossref PubMed Scopus (813) Google Scholar). T cell differentiation leads to enhanced expression of ST6Gal1 in TH2 relative to TH1/TH17 cells, thereby reducing sensitivity to galectin-1-induced apoptosis(4.Toscano M.A. Bianco G.A. Ilarregui J.M. Croci D.O. Correale J. Hernandez J.D. Zwirner N.W. Poirier F. Riley E.M. Baum L.G. Rabinovich G.A. Nat. Immunol. 2007; 8: 825-834Crossref PubMed Scopus (498) Google Scholar). Galectins bind surface glycoproteins in proportion to N-acetyllactosamine content, forming a molecular lattice that regulates distribution of glycoproteins to membrane microdomains (i.e. lipid rafts) and inhibits their loss to endocytosis(1.Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (729) Google Scholar, 8.Partridge E.A. Le Roy C. Di Guglielmo G.M. Pawling J. Cheung P. Granovsky M. Nabi I.R. Wrana J.L. Dennis J.W. Science. 2004; 306: 120-124Crossref PubMed Scopus (583) Google Scholar, 9.Nieminen J. Kuno A. Hirabayashi J. Sato S. J. Biol. Chem. 2007; 282: 1374-1383Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 10.Brewer C.F. Miceli M.C. Baum L.G. Curr. Opin. Struct. Biol. 2002; 12: 616-623Crossref PubMed Scopus (371) Google Scholar, 11.Ahmad N. Gabius H.J. André S. Kaltner H. Sabesan S. Roy R. Liu B. Macaluso F. Brewer C.F. J. Biol. Chem. 2004; 279: 10841-10847Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 12.Chen I.J. Chen H.L. Demetriou M. J. Biol. Chem. 2007; 282: 35361-35372Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 13.Grigorian A. Torossian S. Demetriou M. Immunol. Rev. 2009; 230: 232-246Crossref PubMed Scopus (102) Google Scholar). Binding avidity of galectins for glycoproteins is regulated by the number of attached N-glycans (i.e. N = occupied N-X-(S/T) sites, X ≠ P), an encoded feature of protein sequences, as well as the degree of Golgi-mediated GlcNAc branching, which together determine N-acetyllactosamine content in glycoproteins(14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). The number of N-X-(S/T) sites varies widely between different glycoproteins, with growth-promoting membrane glycoproteins (e.g. T cell receptor (TCR)) 2The abbreviations used are: TCRT cell receptorGEMGM1-enriched lipid microdomainsEAEexperimental autoimmune encephalomyelitisMIα-mannosidase IMIIα-mannosidase IIL-PHAleukoagglutininFITCfluorescein isothiocyanateFACSfluorescence-activated cell sorterELISAenzyme-linked immunosorbent assayPBMCperipheral blood mononuclear cellMBPmyelin basic proteinILinterleukinIFNinterferonSWswainsonineDMNdeoxymannojirimycinMAPKmitogen-activated protein kinase. generally displaying large numbers of N-X-(S/T) sites (i.e. n > 5), whereas growth inhibitory receptors (e.g. cytotoxic T lymphocyte antigen-4 (CTLA-4)) have few N-glycans (n ≤ 4)(14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). The large difference in galectin avidity for high and low multiplicity receptors allows Golgi-mediated changes in GlcNAc branching to differentially control surface retention (i.e. endocytosis rates) of these receptors and therefore transitions between growth and arrest signaling (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar).In resting T cells, where endocytosis is minimal, galectin-3 binds the TCR and the tyrosine phosphatase CD45 but partitions them to different membrane compartments by opposing F-actin targeting (12.Chen I.J. Chen H.L. Demetriou M. J. Biol. Chem. 2007; 282: 35361-35372Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Galectin binding prevents spontaneous TCR oligomerization in the absence of antigen, thereby blocking recruitment of multiple adaptor proteins and CD4-Lck to TCR, F-actin-mediated transfer to GM1-enriched lipid microdomains (GEMs), and activation signaling by Lck. In parallel, galectin partitions CD45 to GEMs by counteracting F-actin-mediated exclusion, dampening Lck activation. Upon encounter with antigen, GEMs cluster at the immune synapse formed at the contact site between T cells and antigen-presenting cells, with the galectin lattice reducing TCR/CD4-Lck and increasing CD45 concentration to dampen T cell activation and TH1 differentiation (1.Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (729) Google Scholar, 2.Morgan R. Gao G. Pawling J. Dennis J.W. Demetriou M. Li B. J. Immunol. 2004; 173: 7200-7208Crossref PubMed Scopus (130) Google Scholar, 12.Chen I.J. Chen H.L. Demetriou M. J. Biol. Chem. 2007; 282: 35361-35372Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). After activation, membrane turnover increases in T cell blasts, markedly limiting surface levels of the growth inhibitor CTLA-4 via constitutive endocytosis (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). GlcNAc branching increases in blasting T cells, enhancing CTLA-4 affinity for galectins and opposing surface loss to endocytosis (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Thus, GlcNAc branching in N-glycans negatively regulates T cell growth early by raising T cell activation thresholds and later by enhancing CTLA-4-mediated growth arrest.GlcNAc branching is regulated by activities of the medial Golgi N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) (15.Schachter H. Glycobiology. 1991; 1: 453-461Crossref PubMed Scopus (167) Google Scholar), as well as metabolic supply of their shared substrate UDP-GlcNAc (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 16.Grigorian A. Lee S.U. Tian W. Chen I.J. Gao G. Mendelsohn R. Dennis J.W. Demetriou M. J. Biol. Chem. 2007; 282: 20027-20035Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Mgat1, Mgat2, Mgat4, and Mgat5 transfer N-acetylglucosamine from UDP-GlcNAc to N-glycans transiting the medial Golgi, forming mono-, bi-, tri-, and tetra-antennary GlcNAc-branched N-glycans (see Fig. 1A). β1,6GlcNAc-branched N-glycans produced by Mgat5 are preferentially extended by poly-N-acetyllactosamine, generating high affinity ligands for galectins (7.Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W.E. Yagi F. Kasai K. Biochim. Biophys. Acta. 2002; 1572: 232-254Crossref PubMed Scopus (813) Google Scholar, 17.Cummings R.D. Kornfeld S. J. Biol. Chem. 1984; 259: 6253-6260Abstract Full Text PDF PubMed Google Scholar). Mgat5-deficient 129/Sv mice display T cell hyperactivity in vivo, as exemplified by enhanced type IV hypersensitivity, spontaneous kidney autoimmunity, and increased experimental autoimmune encephalomyelitis (EAE) (1.Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (729) Google Scholar), an animal model of multiple sclerosis. Susceptibility of inbred mouse strains to EAE varies widely and correlates with strain-specific deficiencies in GlcNAc branching in T cells (18.Lee S.U. Grigorian A. Pawling J. Chen I.J. Gao G. Mozaffar T. McKerlie C. Demetriou M. J. Biol. Chem. 2007; 282: 33725-33734Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The EAE-susceptible PL/J strain intrinsically possesses reduced basal activities of Mgat1, Mgat2, and Mgat5 and develops a spontaneous multiple sclerosis-like disease that is markedly enhanced by Mgat5+/− and Mgat5−/− backgrounds in a gene dose-dependent manner. Autoimmunity is a complex trait that is influenced by environmental inputs. In this regard, N-glycan GlcNAc branching is regulated by the nutrient environment and metabolism via hexosamine pathway production of UDP-GlcNAc, the sugar nucleotide donor for Mgat1, Mgat2, Mgat4, and Mgat5 (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 16.Grigorian A. Lee S.U. Tian W. Chen I.J. Gao G. Mendelsohn R. Dennis J.W. Demetriou M. J. Biol. Chem. 2007; 282: 20027-20035Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Overexpression of Mgat1, an enzyme that has an ∼250-fold lower Km for UDP-GlcNAc than Mgat5, paradoxically reduces β1,6GlcNAc branching by limiting UDP-GlcNAc supply to Mgat5 (14.Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Metabolic supplements to the hexosamine pathway suppress TCR signaling, T cell activation/proliferation, TH1 differentiation, CTLA-4 endocytosis, and autoimmunity (EAE and autoimmune diabetes). TCR signaling results in sequential increases in glucose flux, UDP-GlcNAc production, β1,6GlcNAc branching, and finally, CTLA-4 surface expression.These data implicate TCR signaling-mediated enhancement of GlcNAc branching as a critical regulator of CTLA-4 surface retention and autoimmunity. In addition to the Mgat enzymes, Golgi α-mannosidase I (MI) and II (MII) are required for GlcNAc branching (15.Schachter H. Glycobiology. 1991; 1: 453-461Crossref PubMed Scopus (167) Google Scholar, 19.Akama T.O. Nakagawa H. Wong N.K. Sutton-Smith M. Dell A. Morris H.R. Nakayama J. Nishimura S. Pai A. Moremen K.W. Marth J.D. Fukuda M.N. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8983-8988Crossref PubMed Scopus (57) Google Scholar, 20.Herscovics A. Biochimie. 2001; 83: 757-762Crossref PubMed Scopus (92) Google Scholar). MI acts upstream and MII acts downstream of Mgat1, steps required for the action of Mgat2 and production of bi-, tri-, and tetra-antennary N-glycans (see Fig. 1A). In mammals, the Mgat1, Mgat2, and Mgat5 enzymes are encoded by single genes, whereas three genes encode MI (MIa = MAN1A1, MIb = MAN1A2, MIc = MAN1C1) and two encode MII (MAN2A1, MAN2A2). In T cells, MAN2A1 and MAN2A2 are both expressed, and loss of the former is reported to have little effect on GlcNAc branching or T cell growth (21.Chui D. Sellakumar G. Green R. Sutton-Smith M. McQuistan T. Marek K. Morris H. Dell A. Marth J. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 1142-1147Crossref PubMed Scopus (179) Google Scholar). Effects of targeted deletion of other Golgi α-mannosidase genes in T cells have not been explored (22.Tremblay L.O. Nagy Kovács E. Daniels E. Wong N.K. Sutton-Smith M. Morris H.R. Dell A. Marcinkiewicz E. Seidah N.G. McKerlie C. Herscovics A. J. Biol. Chem. 2007; 282: 2558-2566Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 23.Akama T.O. Nakagawa H. Sugihara K. Narisawa S. Ohyama C. Nishimura S. O'Brien D.A. Moremen K.W. Millan J.L. Fukuda M.N. Science. 2002; 295: 124-127Crossref PubMed Scopus (124) Google Scholar). Here we report that TCR signaling enhances expression of all five Golgi α-mannosidase genes in parallel with Mgat5, an activity required for optimal production of β1,6GlcNAc-branched N-glycans. In contrast, TCR signaling has little or negative effect on Mgat1 and Mgat2 expression, presumably to increase supply of UDP-GlcNAc to Mgat4 and Mgat5 and promote GlcNAc branching. Our data suggest that TCR signaling differentially regulates multiple Golgi enzymes at the mRNA levels to enhance GlcNAc branching in T cell blasts, and subsequently, growth arrest by CTLA-4.
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