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• W1975248663 abstract "Autoimmunity is a complex trait disease where the environment influences susceptibility to disease by unclear mechanisms. T cell receptor clustering and signaling at the immune synapse, T cell proliferation, CTLA-4 endocytosis, TH1 differentiation, and autoimmunity are negatively regulated by β1,6GlcNAc-branched N-glycans attached to cell surface glycoproteins. β1,6GlcNAc-branched N-glycan expression in T cells is dependent on metabolite supply to UDP-GlcNAc biosynthesis (hexosamine pathway) and in turn to Golgi N-acetylglucosaminyltransferases Mgat1, -2, -4, and -5. In Jurkat T cells, β1,6GlcNAc-branching in N-glycans is stimulated by metabolites supplying the hexosamine pathway including glucose, GlcNAc, acetoacetate, glutamine, ammonia, or uridine but not by control metabolites mannosamine, galactose, mannose, succinate, or pyruvate. Hexosamine supplementation in vitro and in vivo also increases β1,6GlcNAc-branched N-glycans in naïve mouse T cells and suppresses T cell receptor signaling, T cell proliferation, CTLA-4 endocytosis, TH1 differentiation, experimental autoimmune encephalomyelitis, and autoimmune diabetes in non-obese diabetic mice. Our results indicate that metabolite flux through the hexosamine and N-glycan pathways conditionally regulates autoimmunity by modulating multiple T cell functionalities downstream of β1,6GlcNAc-branched N-glycans. This suggests metabolic therapy as a potential treatment for autoimmune disease. Autoimmunity is a complex trait disease where the environment influences susceptibility to disease by unclear mechanisms. T cell receptor clustering and signaling at the immune synapse, T cell proliferation, CTLA-4 endocytosis, TH1 differentiation, and autoimmunity are negatively regulated by β1,6GlcNAc-branched N-glycans attached to cell surface glycoproteins. β1,6GlcNAc-branched N-glycan expression in T cells is dependent on metabolite supply to UDP-GlcNAc biosynthesis (hexosamine pathway) and in turn to Golgi N-acetylglucosaminyltransferases Mgat1, -2, -4, and -5. In Jurkat T cells, β1,6GlcNAc-branching in N-glycans is stimulated by metabolites supplying the hexosamine pathway including glucose, GlcNAc, acetoacetate, glutamine, ammonia, or uridine but not by control metabolites mannosamine, galactose, mannose, succinate, or pyruvate. Hexosamine supplementation in vitro and in vivo also increases β1,6GlcNAc-branched N-glycans in naïve mouse T cells and suppresses T cell receptor signaling, T cell proliferation, CTLA-4 endocytosis, TH1 differentiation, experimental autoimmune encephalomyelitis, and autoimmune diabetes in non-obese diabetic mice. Our results indicate that metabolite flux through the hexosamine and N-glycan pathways conditionally regulates autoimmunity by modulating multiple T cell functionalities downstream of β1,6GlcNAc-branched N-glycans. This suggests metabolic therapy as a potential treatment for autoimmune disease. Complex trait diseases such as autoimmunity are determined by poorly understood genetic and environmental interactions. The T cell-mediated autoimmune diseases multiple sclerosis (MS) 3The abbreviations used are: MS, multiple sclerosis; MS/MS, tandem mass spectroscopy; NOD, non-obese diabetic; TCR, T cell receptor; EAE, experimental autoimmune encephalomyelitis; l-PHA, P. vulgaris leukoagglutinin; SW, swainsonine; TCR, T cell receptor; FACS, fluorescence-activated cell sorter; CFSE, carboxyfluorescein diacetate succinimidyl ester; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ELISA, enzyme-linked immunosorbent assay; MBP, myelin basic protein; IFN, interferon; IL, interleukin; FITC, fluorescein isothiocyanate. and type 1 diabetes exemplify this problem, where identical twins of Northern European descent are discordant ∼60-70% of the time despite displaying an ∼150-300 times higher risk than the general population prevalence of ∼0.1 and ∼0.4%, respectively (1Ebers G.C. Bulman D.E. Sadovnick A.D. Paty D.W. Warren S. Hader W. Murray T.J. Seland T.P. Duquette P. Grey T. et al.N. Engl. J. Med. 1986; 315: 1638-1642Crossref PubMed Scopus (505) Google Scholar, 2Redondo M.J. Yu L. Hawa M. Mackenzie T. Pyke D.A. Eisenbarth G.S. Leslie R.D. Diabetologia. 2001; 44: 354-362Crossref PubMed Scopus (237) Google Scholar). Genetic-environmental interactions have been established between disease-associated major histocompatibility complex haplotypes and specific pathogen peptides that mimic disease self antigens (3Oldstone M.B. Cell. 1987; 50: 819-820Abstract Full Text PDF PubMed Scopus (799) Google Scholar, 4Wucherpfennig K.W. Strominger J.L. Cell. 1995; 80: 695-705Abstract Full Text PDF PubMed Scopus (1285) Google Scholar). The prevalence of MS and type 1 diabetes changes along north-south gradients, implicating ultraviolet light exposure and production of vitamin D3 in the skin (5Munger K.L. Zhang S.M. O'Reilly E. Hernan M.A. Olek M.J. Willett W.C. Ascherio A. Neurology. 2004; 62: 60-65Crossref PubMed Scopus (850) Google Scholar, 6Green A. Gale E.A. Patterson C.C. 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Immunol. 2005; 23: 447-485Crossref PubMed Scopus (848) Google Scholar). However, molecular mechanisms for genetic-environmental interactions are poorly understood. Salvage of glucosamine by the hexosamine pathway to UDP-GlcNAc is reported to suppress T cell function and EAE in mice by an unknown mechanism (12Ma L. Rudert W.A. Harnaha J. Wright M. Machen J. Lakomy R. Qian S. Lu L. Robbins P.D. Trucco M. Giannoukakis N. J. Biol. Chem. 2002; 277: 39343-39349Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 13Zhang G.X. Yu S. Gran B. Rostami A. J. Immunol. 2005; 175: 7202-7208Crossref PubMed Scopus (57) Google Scholar). De novo biosynthesis of UDP-GlcNAc by the hexosamine pathway utilizes glucose, acetyl-CoA, glutamine, and UTP (see Fig. 1), key allosteric regulators of basic metabolism, suggesting regulation of UDP-GlcNAc supply is integrated with down-stream pathways requiring this sugar-nucleotide. In this regard, the Golgi pathway to β1,6GlcNAc-branched N-glycans is sensitive to cellular UDP-GlcNAc levels (14Sasai K. Ikeda Y. Fujii T. Tsuda T. Taniguchi N. Glycobiology. 2002; 12: 119-127Crossref PubMed Scopus (89) Google Scholar) and plays a role in T cell function (15Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (742) Google Scholar). Mice deficient in Golgi UDP-GlcNAc:β1,6N-acetylglucosaminyltransferase V (Mgat5) display enhanced delayed-type hypersensitivity, spontaneous kidney autoimmunity after 1 year of age, and increased susceptibility to EAE (15Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (742) Google Scholar). Most transmembrane receptors on mammalian cells are modified by N-glycosylation en route to the cell surface. The N-acetylglucosaminyltransferases I, II, IV, and V, encoded by the genes Mgat1, -2, -4a/b, and -5, act sequentially to transfer N-acetyl-d-glucosamine (GlcNAc) from UDP-GlcNAc to N-glycan intermediates in the medial Golgi, producing mono, bi-, tri-, and tetra-antennary N-glycans, respectively (16Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3776) Google Scholar, 17Schachter H. Glycobiology. 1991; 1: 453-461Crossref PubMed Scopus (169) Google Scholar) (see Fig. 1). Branching and particularly the β1,6GlcNAc-branched tetra-antennary N-glycans are sub-saturating, as Mgat1, -2, -4, and -5 display decreasing affinities for UDP-Glc-NAc, and enzyme concentrations also decline across the pathway (see Fig. 1) (17Schachter H. Glycobiology. 1991; 1: 453-461Crossref PubMed Scopus (169) Google Scholar). Galectins, a family of N-acetyllactosamine binding animal lectins, bind cell surface glycoproteins and form a molecular lattice that negatively regulates lateral movement and endocytic loss of surface receptors and transporters (15Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (742) Google Scholar, 18Brewer C.F. Miceli M.C. Baum L.G. Curr. Opin. Struct. Biol. 2002; 12: 616-623Crossref PubMed Scopus (376) Google Scholar, 19Partridge 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 (593) Google Scholar, 20Ohtsubo 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 (343) Google Scholar, 21Nieminen J. Kuno A. Hirabayashi J. Sato S. J. Biol. Chem. 2007; 282: 1374-1383Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Galectins bind to N-glycans on surface glycoproteins with affinities proportional to GlcNAc-branching (Fig. 1) (15Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (742) Google Scholar, 18Brewer C.F. Miceli M.C. Baum L.G. Curr. Opin. Struct. Biol. 2002; 12: 616-623Crossref PubMed Scopus (376) Google Scholar, 19Partridge 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 (593) Google Scholar). β1,6GlcNAc-branched N-glycans are preferentially extended by poly-N-acetyllactosamine (i.e. (Galβ1,4GlcNAcβ1,3-)n), further increasing avidity for galectins. However, increasing the proportion of tri-antennary structures in Mgat5-deficient cells is sufficient to rescue defects in galectin binding and surface retention of receptors (22Lau 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 (682) Google Scholar). In addition, the number of N-glycans, an encoded feature of protein sequence (NX(S/T)), contributes to galectin avidity and regulates surface residency of receptors in a selective manner (22Lau 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 (682) Google Scholar). With increasing hexosamine flux of UDP-GlcNAc through the Golgi to N-glycan Glc-NAc branching, surface galectin binding and retention of glycoproteins with high N-glycan multiplicity (epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, and basic fibroblast growth factor receptors) are enhanced before those with lower multiplicity (transforming growth factor-β receptor, CTLA-4, and GLUT-4) (22Lau 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 (682) Google Scholar). This provides a mechanism for metabolic regulation of cellular transitions between growth (i.e. epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, and basic fibroblast growth factor receptors) and arrest signaling (i.e. transforming growth factor-β receptor, CTLA-4, and GLUT-4) (22Lau 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 (682) Google Scholar). β1,6GlcNAc-branched N-glycans attached to the T cell receptor enhance binding to galectin-3, limiting TCR clustering at the immune synapse and increasing agonist thresholds for TCR signaling (15Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (742) Google Scholar, 22Lau 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 (682) Google Scholar, 23Morgan R. Gao G. Pawling J. Dennis J.W. Demetriou M. Li B. J. Immunol. 2004; 173: 7200-7208Crossref PubMed Scopus (131) Google Scholar). After TCR activation, endocytosis rates are increased and Src-family kinases and phosphatidylinositol 3-kinase/Erk stimulate hexosamine flux and Golgi processing to β1,6GlcNAc-branched N-glycans, thereby enhancing surface retention of the growth suppressor CTLA-4 (22Lau 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 (682) Google Scholar). Therefore, β1,6GlcNAc-branched N-glycans regulate different receptors and at two distinct phases of the T cell response, both serving to suppress T cell activation and autoimmune mechanisms. Here we demonstrate that surface β1,6GlcNAc-branched N-glycans on T cells are regulated by the nutrient environment and metabolite supply to the hexosamine pathway. Hexosamine pathway supplements suppress TCR sensitivity, TH1 differentiation, and autoimmunity by increasing N-glycan GlcNAc-branching in T cells. Our results indicate that genetic and environmental contributions to metabolic homeostasis and Golgi processing regulate N-glycan branching, T cell function, and autoimmunity and suggest new avenues for prevention and treatment of autoimmune disease (Fig. 7). FACS Analysis and in Vitro Proliferation Assays—PL/J mice were congenic at backcross six from our original 129/Sv Mgat5-/- mice (24Granovsky M. Fata J. Pawling J. Muller W.J. Khokha R. Dennis J.W. Nat. Med. 2000; 6: 306-312Crossref PubMed Scopus (469) Google Scholar). NOD mice were obtained from Jackson laboratories at 5 weeks of age. Procedures and protocols with mice were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. Mouse cells were stained with anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CTLA-4 (UC10-4B9), anti-CD28 (37.51), and anti-CD69 (H1.2F3) from eBioscience, Phaseolus vulgaris leukoagglutinating lectin (l-PHA, 4 μg/ml), Lycopersicon esculentum agglutinating lectin (20 μg/ml), and 7-aminoactinomycin D (1 μg/ml) from Sigma. Purified CD3+ T cells (R&D Systems) were labeled with 5 μm 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) in phosphate-buffered saline for 8 min at room temperature and stimulated with plate-bound anti-CD3∊ (2C11, eBioscience) in the presence or absence of GlcNAc and/or swainsonine (SW) (Sigma). Jurkat T cells were cultured in either glutamine-free RPMI 1640, 10% fetal bovine serum, 10/20 mm glucose, or glucose/glutamine-free Dulbecco's modified Eagle's base medium supplemented with 10% fetal bovine serum, 1.5 mm glucose. The indicated monosaccharides and/or metabolites were added daily except glucose, which was added only at time 0 and were titrated until a plateau was reached in l-PHA staining or toxicity was observed. The plateau or highest non-toxic dose is shown. TCR Signaling—1 × 106 purified splenic CD3+ T cells from Mgat5+/+, Mgat5+/-, and Mgat5-/- mice or from purified T cell cultures incubated in the presence or absence of GlcNAc (80 mm) and/or swainsonine (0.25 μm) for 72 h were mixed with 5 × 106 polystyrene beads (6 μm, Polysciences) coated at 4 °C overnight with 0.5 μg/ml anti-CD3∊ antibody (2C11, eBioscience) and pelleted at 5000 rpm for 15 s, incubated at 37 °C for the indicated times, and then solubilized with ice-cold 50 mm Tris, pH 7.2, 300 mm NaCl, 1.0% Triton X-100, protease inhibitor mixture (Roche Applied Science), and 2 mm orthovanadate for 20 min. Cell lysates were separated on Nupage 10% Bis-Tris gels (Invitrogen) under reducing conditions, transferred to polyvinylidene difluoride membranes, and immunoblotted with rabbit anti-phospho-Zap70 antibody (Ab; Cell Signaling Technology), rabbit anti-phospho-LAT Ab (Upstate), and/or anti-actin Ab (Santa Cruz). MS/MS Mass Spectroscopy—For MS/MS mass spectroscopy to determine sugar nucleotide levels, Jurkat T cell pellets (20 × 106 cells) were resuspended in 300 ml of cold methanol:water (1:1) solution containing maltose as an internal standard, vortexed for 10 s, and then pipetted into tubes containing 600 ml of chloroform:methanol (3:2). Maltose (100 pmol) was added to each sample as an internal standard. Samples were vortexed for 1 min and then centrifuged at 14,000 rpm for 5 min at 4 °C. Supernatants were collected, and an equal volume of chloroform: methanol (1:1) was added followed by a second extraction. The pooled aqueous fraction containing the hydrophilic metabolite was dried with a SpeedVac, passed over C18 SepPak in water, dried, and stored at -80 °C. Before injection, the samples were dissolved in 100 ml of methanol:water (1:1). The samples were injected at 150 ml/h and analyzed on a 4000QTRAP (Mass Spectrometer (SCIEX). The metabolites were identified by their transitions in MS/MS and quantified using the Analyst Software (Applied Biosystems-SCIEX), which measured the area under the curve for major fragment ion (M2) corresponding to each parent ion (M1). Collision energy, declustering potential, and ion spray voltage were optimized for each metabolite. UDP-GlcNAc (M1 = 605.9 and M2 = 385.0 with setting declustering potential -40, collision energy -38, and ion spray voltage -3500) and UDP-Gal (M1 = 564.9 and M2 = 323.0 with setting declustering potential +80, collision energy +23, ion spray voltage +5500) were measured in negative and positive, respectively. Maltose was measured in negative mode (M1 = 341 and M2 = 161 with setting declustering potential -40, collision energy -13, and ion spray voltage -4000). Standards for maltose, UDP-GlcNAc, and UDP-Gal showed sensitivity to 15 pmol and linearity to 10 nmol. Data are reported as UDP-HexNAc and UDP-Hex due to potential interconversion of UDP-GlcNAc to UDP-GalNAc and UDP-Gal to UDP-Glc by 4′-epimerase activity, sugar nucleotides not distinguished in M1. In Vivo GlcNAc Treatment—GlcNAc was administered orally in age- and sex-matched PL/J littermate mice by adding GlcNAc to their drinking water at various concentrations. Fresh GlcNAc was given daily for 5 days, with the amount consumed approximated by determining the volume remaining after 24 h. After 5 days of treatment, harvested splenocytes were stained and analyzed by FACS. Cytokine ELISA—Supernatant from splenocyte and/or T cell cultures stimulated with myelin basic protein (MBP) and/or anti-CD3∊ (2C11, eBioscience) in the presence or absence of GlcNAc and/or SW were tested for IFN-γ, IL-6, and/or IL-4 levels by ELISA. Microtiter plates were coated with 50 μl of anti-IFN-γ (1 μg/ml, clone AN-18; eBioscience), anti-IL-6 (1.5 μg/ml, clone MP5-20F3; eBioscience), or anti-IL-4 (2 μg/ml, clone 11B11; eBioscience) overnight at 4 °C. Supernatants were applied at 50 μl/well and incubated for 2 h at room temperature. Captured cytokines were detected using biotinylated anti-IFN-γ (1 μg/ml, clone R4-6A2; eBioscience), anti-IL-6 (1 μg/ml, clone MP5-32C11; eBioscience), or anti-IL-4 (1 μg/ml, clone BVD6-24G2; eBioscience) and detected using avidin horseradish peroxidase (eBioscience) at 1:500× dilution and o-phenylenediamine dihydrochloride tablets (Sigma) according to the manufacturer's protocols. Recombinant IFN-γ, IL-6, or IL-4 (eBioscience) was used as a standard. Adoptive Transfer EAE and NOD Diabetes—Adoptive transfer EAE was induced by subcutaneous immunization of wild-type PL/J mice with 100 μg of bovine MBP (Sigma) emulsified in Complete Freund's Adjuvant containing 4 mg/ml heat-inactivated Mycobacterium tuberculosis (H37 RA; Difco) distributed over three spots on the hind flank. Splenocytes were harvested after 11 days and stimulated in vitro with 50 μg/ml MBP in the presence or absence of 40 mm Glc-NAc (Sigma) added daily. After 96 h of incubation, CD3+ T cells were purified by negative selection (R&D Systems) and injected intraperitoneally into naïve PL/J Mgat5+/- recipient mice. Trypan blue exclusion determined <5% dead cells under both culture conditions. Mice were scored daily for clinical signs of EAE over the next 30 days with the observer blinded to treatment conditions. Mice were examined daily for clinical signs of EAE and scored in a blinded fashion as follows: 0, no disease; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis; 4, forelimb weakness or paralysis and hindlimb paralysis; 5, moribund or dead. GlcNAc was administered orally to female NOD mice by adding fresh GlcNAc at a concentration of 250 μg/ml to the drinking water every 3 days. Spontaneous diabetes in wild-type NOD mice was diagnosed after two consecutive positive tests for glycosuria of ≥2000 mg/dl by Chemstrip (Accu-chek, Roche Applied Science) 1 week apart. T Cell Thresholds and Fractional Changes in β1,6GlcNAc Branching—Conditional or metabolic regulation of T cell activation thresholds could be expected to be sensitive to small incremental changes in N-glycan branching. To test this hypothesis we compared the agonist sensitivity of primary T cells from Mgat5+/+, Mgat5+/-, and Mgat5-/- mice. Mgat5+/- T cells show an ∼20-25% reduction in β1,6GlcNAc-branched N-glycans relative to Mgat5+/+ cells as indicated by l-PHA binding, a plant lectin specific for these structures (Fig. 2A) (15Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (742) Google Scholar, 23Morgan R. Gao G. Pawling J. Dennis J.W. Demetriou M. Li B. J. Immunol. 2004; 173: 7200-7208Crossref PubMed Scopus (131) Google Scholar). Stimulation of purified CD3+ T cells with anti-CD3-coated microbeads demonstrates that Mgat5+/- cells are intermediate compared with Mgat5+/+ and Mgat5-/- cells for TCR signaling as shown by enhanced phosphorylation of Zap-70 and LAT (Fig. 2B). Mgat5+/- T cells are similarly intermediate for TCR-mediated proliferation, as shown by tracking cell division with CFSE (Fig. 2C). To confirm that small reductions in β1,6GlcNAc-branched N-glycans enhance TCR sensitivity, we treated wild type cells with minimal concentrations of SW. SW is a specific inhibitor of mannosidase II that blocks GlcNAc branching in N-glycans and is known to enhance T cell proliferation and TH1 differentiation (23Morgan R. Gao G. Pawling J. Dennis J.W. Demetriou M. Li B. J. Immunol. 2004; 173: 7200-7208Crossref PubMed Scopus (131) Google Scholar, 25Wall K.A. Pierce J.D. Elbein A.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5644-5648Crossref PubMed Scopus (44) Google Scholar). At low levels of anti-CD3 stimulation, partial reductions in β1,6GlcNAc-branched N-glycans by low doses of swainsonine significantly enhance wild type T cell proliferation (Fig. 2D) (25Wall K.A. Pierce J.D. Elbein A.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5644-5648Crossref PubMed Scopus (44) Google Scholar, 26Tulsiani D.R. Harris T.M. Touster O. J. Biol. Chem. 1982; 257: 7936-7939Abstract Full Text PDF PubMed Google Scholar). Thus, changes in T cell activation thresholds can be readily detected with as little as ∼20-25% variations in β1,6GlcNAc-branched N-glycans. Metabolic Flux through the Hexosamine Pathway Regulates β1,6GlcNAc-branched N-Glycans in T Cells—The expression of tri (i.e. β1,4)- and tetra (i.e. β1,6)-antennary GlcNAc-branched N-glycans in cultured cells is sensitive to changes in the intracellular concentration of UDP-GlcNAc (14Sasai K. Ikeda Y. Fujii T. Tsuda T. Taniguchi N. Glycobiology. 2002; 12: 119-127Crossref PubMed Scopus (89) Google Scholar, 22Lau 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 (682) Google Scholar), which enters the Golgi via UDP-GlcNAc transporters (27Hirschberg C.B. Robbins P.W. Abeijon C. Annu. Rev. Biochem. 1998; 67: 49-69Crossref PubMed Scopus (305) Google Scholar). The addition of GlcNAc to cultured cells supplements UDP-GlcNAc pools after uptake by bulk endocytosis, 6-phosphorylation, and conversion to UDP-GlcNAc (Fig. 1) (14Sasai K. Ikeda Y. Fujii T. Tsuda T. Taniguchi N. Glycobiology. 2002; 12: 119-127Crossref PubMed Scopus (89) Google Scholar, 22Lau 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 (682) Google Scholar). Glucose, glutamine, acetyl-CoA, and UTP are metabolites required by the hexosamine pathway for de novo UDP-GlcNAc biosynthesis (Fig. 1). TCR activation of mouse T cells increases Mgat5 gene expression (15Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (742) Google Scholar), glucose uptake (28Frauwirth K.A. Thompson C.B. J. Immunol. 2004; 172: 4661-4665Crossref PubMed Scopus (280) Google Scholar), UDP-HexNAc levels (22Lau 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 (682) Google Scholar), and β1,6GlcNAc-branched N-glycans (23Morgan R. Gao G. Pawling J. Dennis J.W. Demetriou M. Li B. J. Immunol. 2004; 173: 7200-7208Crossref PubMed Scopus (131) Google Scholar). GlcNAc supplementation of activated T cells further enhances UDP-HexNAc levels, increasing the UDP-HexNAc:UDP-Hex ratio up to ∼6-fold (22Lau 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 (682) Google Scholar). To determine whether hexosamine metabolite supplementation enhances expression of cell surface galectin ligands (i.e. N-glycan GlcNAc branching and poly-N-acetyllactosamine) in resting T cells, cells were titrated with various metabolites until a plateau was reached in l-PHA staining or toxicity was observed. Surface levels of β1,6GlcNAc-branched N-glycans (i.e. l-PHA staining) and poly-N-acetyllactosamine (indicated by staining with the plant lectin L. esculentum agglutinin) on non-TCR activated human Jurkat T cells were increased by supplementing the hexosamine pathway with high glucose, GlcNAc, acetoacetate, glutamine, ammonia, or uridine but not with control metabolites mannosamine, galactose, mannose, succinate, or pyruvate (Fig. 3, A and B; supplemental Fig. 1, A and B). Supplementing resting mouse ex vivo splenocytes with GlcNAc, glucose, or uridine in vitro significantly raised β1,6GlcNAc-branched N-glycans in CD4+ T cells (Fig. 4A). In non-T cells, GlcNAc supplementation also increases tri-antennary structures (22Lau 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 (682) Google Scholar), and these are likely also enhanced in supplemented T cells. MS/MS mass spectroscopy confirmed that Glc-NAc, glucose, and uridine supplements raised intracellular UDP-HexNAc levels in non-TCR-activated Jurkat T cells (Fig. 3C). On a per cell basis, UDP-HexNAc levels in Jurkat T cells appear lower than that reported for Chinese hamster ovary cells (29Tomiya N. Ailor E. Lawrence S.M. Betenbaugh M.J. Lee Y.C. Anal. Biochem. 2001; 293: 129-137Crossref PubMed Scopus (173) Google Scholar); however, T cells are smaller and have a markedly higher nuclear to cytoplasmic ratio, making direct comparisons difficult. Co-supplementation of GlcNAc and uridine in resting Jurkat and wild type mouse T cells were additive and more effective than doubling the GlcNAc concentration (Figs. 3D and 4B). Supplementing the drinking water of Mgat5+/- or Mgat5+/+ mice with GlcNAc increased β1,6GlcNAc-branched N-glycans up to ∼40% on CD4+ T cells in vivo (Fig. 4C; supplemental Fig. 2A). Taken together, these data indicate that production of high affinity galectin ligands (e.g. β1,6GlcNAc-branched N-glycans and poly-N-acetyllactosamine) are limited in T cells by the availability of key intermediates of glucose, lipid, nitrogen, and nucleotide metabolism.FIGURE 4Metabolite supply to the hexosamine pathway regulates β1,6GlcNAc-branched N-glycan expression in resting primary mouse T cells. A and B, the indicated monosaccharides and metabolites were cultured with unstimulated mouse splenocytes for 3 days, stained with l-PHA-FITC in triplicate, and analyzed by FACS. The results are representative of at least three independent experiments. Error bars are present in all panels and represent the means ± S.E. of triplicate samples. C, splenocytes from Mgat5+/- mice orally supplemented with GlcNAc for 5 days via their drinking water at the indicated concentrations were stained with l-PHA-FITC in triplicate and analyzed by FACS. On average, mice drank ∼4.6 ± 0.25 ml/day, estimated by determining the amount of GlcNAc solution remaining daily. For a 28.5-g mouse, the approximate mg/kg doses received is 10, 20, 40, 80, and 160. Data shown are an average from two independent experiments, each with triplicate staining. n = number of mice. Error bars are the means ± S.E. p values in B and C are by analysis of variance and the Newman-Keuls multiple comparison test with non-supplemented cells/mice or as indicated. *, p < 0.05; **, p < 0.01; ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image" @default.
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