Increasing cell permeability of N-acetylglucosamine via 6-acetylation enhances capacity to suppress T-helper 1 (TH1)/TH17 responses and autoimmunity.

N-acetylglucosamine (GlcNAc) branching of Asn (N)-linked glycans inhibits pro-inflammatory T cell responses and models of autoimmune diseases such as Multiple Sclerosis (MS). Metabolism controls N-glycan branching in T cells by regulating de novo hexosamine pathway biosynthesis of UDP-GlcNAc, the donor substrate for the Golgi branching enzymes. Activated T cells switch metabolism from oxidative phosphorylation to aerobic glycolysis and glutaminolysis. This reduces flux of glucose and glutamine into the hexosamine pathway, thereby inhibiting de novo UDP-GlcNAc synthesis and N-glycan branching. Salvage of GlcNAc into the hexosamine pathway overcomes this metabolic suppression to restore UDP-GlcNAc synthesis and N-glycan branching, thereby promoting anti-inflammatory T regulatory (Treg) over pro-inflammatory T helper (TH) 17 and TH1 differentiation to suppress autoimmunity. However, GlcNAc activity is limited by the lack of a cell surface transporter and requires high doses to enter cells via macropinocytosis. Here we report that GlcNAc-6-acetate is a superior pro-drug form of GlcNAc. Acetylation of amino-sugars improves cell membrane permeability, with subsequent de-acetylation by cytoplasmic esterases allowing salvage into the hexosamine pathway. Per- and bi-acetylation of GlcNAc led to toxicity in T cells, whereas mono-acetylation at only the 6 > 3 position raised N-glycan branching greater than GlcNAc without inducing significant toxicity. GlcNAc-6-acetate inhibited T cell activation/proliferation, TH1/TH17 responses and disease progression in Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of MS. Thus, GlcNAc-6-Acetate may provide an improved therapeutic approach to raise N-glycan branching, inhibit pro-inflammatory T cell responses and treat autoimmune diseases such as MS.

N-glycosylation bidirectionally extends the boundaries of thymocyte positive selection by decoupling Lck from Ca²âº signaling.

Positive selection of diverse yet self-tolerant thymocytes is vital to immunity and requires a limited degree of T cell antigen receptor (TCR) signaling in response to self peptide-major histocompatibility complexes (self peptide-MHCs). Affinity of newly generated TCR for peptide-MHC primarily sets the boundaries for positive selection. We report that N-glycan branching of TCR and the CD4 and CD8 coreceptors separately altered the upper and lower affinity boundaries from which interactions between peptide-MHC and TCR positively select T cells. During thymocyte development, N-glycan branching varied approximately 15-fold. N-glycan branching was required for positive selection and decoupled Lck signaling from TCR-driven Ca(2+) flux to simultaneously promote low-affinity peptide-MHC responses while inhibiting high-affinity ones. Therefore, N-glycan branching imposes a sliding scale on interactions between peptide-MHC and TCR that bidirectionally expands the affinity range for positive selection.

Heteromorphic variants of chromosome 9.

BACKGROUND: Heterochromatic variants of pericentromere of chromosome 9 are reported and discussed since decades concerning their detailed structure and clinical meaning. However, detailed studies are scarce. Thus, here we provide the largest ever done molecular cytogenetic research based on >300 chromosome 9 heteromorphism carriers. RESULTS: In this study, 334 carriers of heterochromatic variants of chromosome 9 were included, being 192 patients from Western Europe and the remainder from Easter-European origin. A 3-color-fluorescence in situ hybridization (FISH) probe-set directed against for 9p12 to 9q13~21.1 (9het-mix) and 8 different locus-specific probes were applied for their characterization. The 9het-mix enables the characterization of 21 of the yet known 24 chromosome 9 heteromorphic patterns. In this study, 17 different variants were detected including five yet unreported; the most frequent were pericentric inversions (49.4%) followed by 9qh-variants (23.9%), variants of 9ph (11.4%), cenh (8.2%), and dicentric- (3.8%) and duplication-variants (3.3%). For reasons of simplicity, a new short nomenclature for the yet reported 24 heteromorphic patterns of chromosome 9 is suggested. Six breakpoints involved in four of the 24 variants could be narrowed down using locus-specific probes. CONCLUSIONS: Based on this largest study ever done in carriers of chromosome 9 heteromorphisms, three of the 24 detailed variants were more frequently observed in Western than in Eastern Europe. Besides, there is no clear evidence that infertility is linked to any of the 24 chromosome 9 heteromorphic variants.

A prospective randomized study comparing fibrin sealant to manual compression for the treatment of anastomotic suture-hole bleeding in expanded polytetrafluoroethylene grafts.

OBJECTIVE: The ideal hemostatic agent for treatment of suture-line bleeding at vascular anastomoses has not yet been established. This study evaluated whether the use of a fibrin sealant containing 500 IU/mL thrombin and synthetic aprotinin (FS; marketed in the United States under the name TISSEEL) is beneficial for treatment of challenging suture-line bleeding at vascular anastomoses of expanded polytetrafluoroethylene (ePTFE) grafts, including those further complicated by concomitant antiplatelet therapies. METHODS: Over a 1-year period ending in 2010, ePTFE graft prostheses, including arterio-arterial bypasses and arteriovenous shunts, were placed in 140 patients who experienced suture-line bleeding that required treatment after completion of anastomotic suturing. Across 24 US study sites, 70 patients were randomized and treated with FS and 70 with manual compression (control). The primary end point was the proportion of patients who achieved hemostasis at the study suture line at 4 minutes after start of application of FS or positioning of surgical gauze pads onto the study suture line. RESULTS: There was a statistically significant difference in the comparison of hemostasis rates at the study suture line at 4 minutes between FS (62.9%) and control (31.4%) patients (P < .0001), which was the primary end point. Similarly, hemostasis rates in the subgroup of patients on antiplatelet therapies were 64.7% (FS group) and 28.2% (control group). When analyzed by bleeding severity, the hemostatic advantage of FS over control at 4 minutes was similar (27.8% absolute improvement for moderate bleeding vs 32.8% for severe bleeding). Logistic regression analysis (accounting for gender, age, intervention type, bleeding severity, blood pressure, heparin coating of ePTFE graft, and antiplatelet therapies) found a statistically significant treatment effect in the odds ratio (OR) of meeting the primary end point between treatment groups (OR, 6.73; P < .0001), as well as statistically significant effects for intervention type (OR, 0.25; P = .0055) and bleeding severity (OR, 2.59; P = .0209). The safety profile of FS was excellent as indicated by the lack of any related serious adverse events. CONCLUSIONS: The findings from this phase 3 study confirmed that FS is safe and its efficacy is superior to manual compression for hemostasis in patients with peripheral vascular ePTFE grafts. The data also suggest that FS promotes hemostasis independently of the patient's own coagulation system, as shown in a representative population of patients with vascular disease under single- or dual-antiplatelet therapies.

Pathogenesis of multiple sclerosis via environmental and genetic dysregulation of N-glycosylation.

Autoimmune diseases such as multiple sclerosis (MS) result from complex and poorly understood interactions of genetic and environmental factors. A central role for T cells in MS is supported by mouse models, association of the major histocompatibility complex region, and association of critical T cell growth regulator genes such as interleukin-2 receptor (IL-2RA) and interleukin-7 receptor (IL-7RA). Multiple environmental factors (vitamin D(3) deficiency and metabolism) converge with multiple genetic variants (IL-7RA, IL-2RA, MGAT1, and CTLA-4) to dysregulate Golgi N-glycosylation in MS, resulting in T cell hyperactivity, loss of self-tolerance and in mice, a spontaneous MS-like disease with neurodegeneration. Here, we review the genetic and biological interactions that regulate MS pathogenesis through dysregulation of N-glycosylation and how this may enable individualized therapeutic approaches.

Interleukin-2, Interleukin-7, T cell-mediated autoimmunity, and N-glycosylation.

T cell activation and self-tolerance are tightly regulated to provide effective host defense against foreign pathogens while deflecting inappropriate autoimmune responses. Golgi Asn (N)-linked protein glycosylation coregulates homeostatic set points for T cell growth, differentiation, and self-tolerance to influence risk of autoimmune disorders such as multiple sclerosis (MS). Human autoimmunity is a complex trait that develops from intricate and poorly understood interactions between an individual's genetics and their environmental exposures. Recent evidence from our group suggests that in MS, additive and/or epistatic interactions between multiple genetic and environmental risk factors combine to dysregulate a common biochemical pathway, namely Golgi N-glycosylation. Here, we review the multiple regulatory mechanisms controlling N-glycan branching in T cells and autoimmunity, focusing on recent data implicating a critical role for interleukin-2 (IL-2) and IL-7 signaling.

N-acetylglucosamine inhibits T-helper 1 (Th1)/T-helper 17 (Th17) cell responses and treats experimental autoimmune encephalomyelitis.

Current treatments and emerging oral therapies for multiple sclerosis (MS) are limited by effectiveness, cost, and/or toxicity. Genetic and environmental factors that alter the branching of Asn (N)-linked glycans result in T cell hyperactivity, promote spontaneous inflammatory demyelination and neurodegeneration in mice, and converge to regulate the risk of MS. The sugar N-acetylglucosamine (GlcNAc) enhances N-glycan branching and inhibits T cell activity and adoptive transfer experimental autoimmune encephalomyelitis (EAE). Here, we report that oral GlcNAc inhibits T-helper 1 (Th1) and T-helper 17 (Th17) responses and attenuates the clinical severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE when administered after disease onset. Oral GlcNAc increased expression of branched N-glycans in T cells in vivo as shown by high pH anion exchange chromatography, MALDI-TOF mass spectroscopy and FACS analysis with the plant lectin l-phytohemagglutinin. Initiating oral GlcNAc treatment on the second day of clinical disease inhibited MOG-induced EAE as well as secretion of interferon-γ, tumor necrosis factor-α, interleukin-17, and interleukin-22. In the more severe 2D2 T cell receptor transgenic EAE model, oral GlcNAc initiated after disease onset also inhibits clinical disease, except for those with rapid lethal progression. These data suggest that oral GlcNAc may provide an inexpensive and nontoxic oral therapeutic agent for MS that directly targets an underlying molecular mechanism causal of disease.

Genetics and the environment converge to dysregulate N-glycosylation in multiple sclerosis.

How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice, N-glycan branching by the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity, cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis, spontaneous inflammatory demyelination and neurodegeneration, the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3), including genetic variants in interleukin-7 receptor-α (IL7RA*C), interleukin-2 receptor-α (IL2RA*T), MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3), optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway, namely N-glycosylation.

Use of fibrin sealant as a hemostatic agent in expanded polytetrafluoroethylene graft placement surgery.

BACKGROUND: The low thrombogenicity, porosity, and limited elasticity of expanded polytetrafluoroethylene (ePTFE) vascular grafts, although beneficial, may exacerbate the problem of suture-line bleeding at vascular anastomoses and consequently lead to increased operating times. The overall objective of this prospective, randomized, controlled, subject-blinded, multicenter phase 2 study was to evaluate the efficacy and safety of a fibrin sealant containing 500 IU/mL thrombin and synthetic aprotinin (FS; marketed in the United States under the name TISSEEL) for hemostasis in subjects undergoing vascular surgery and receiving prosthetic ePTFE vascular grafts. METHODS: FS was compared with manual compression with surgical gauze pads, a standard of care for hemostasis in vascular surgery. Two FS polymerization/setting times (60 and 120 seconds) were investigated to evaluate influence on the efficacy results. Patients undergoing ePTFE graft placement surgery (N = 73) who experienced bleeding that required treatment after surgical hemostasis were randomized to be treated with FS with clamps opened at 60 seconds (FS-60; N = 26), with FS with clamps opened at 120 seconds (FS-120; N = 24), or with manual compression with surgical gauze pads (control; N = 23). The proportion of subjects achieving hemostasis at 4 minutes (primary endpoint) as well as at 6 and 10 minutes (secondary endpoints) in the three treatment groups was analyzed using logistic regression analysis, taking into account gender, age, type of intervention, severity of bleeding, systolic blood pressure, diastolic blood pressure, heparin coating of the ePTFE graft, and platelet inhibitors. RESULTS: There were substantial differences in the proportion of subjects who achieved hemostasis at the study suture line at 4 minutes from treatment application between FS-120 (62.5%) and control (34.8%) groups (a 79.6% relative improvement). Logistic regression analyses found a statistically significant treatment effect at the 10% level in the odds ratio (OR) of achieving hemostasis at 4 minutes between the FS-120 and control groups (OR = 3.98, p = 0.0991). Furthermore, it has been shown that the perioperative administration of platelet inhibitors significantly influences (OR = 3.89, p = 0.0607) hemostasis rates at the primary endpoint. No statistically significant treatment effects were found for the other factors. Logistic regression analyses performed on the secondary endpoints demonstrated a significant treatment effect of achieving hemostasis at 6 minutes (OR = 9.92, p = 0.0225) and at 10 minutes (OR = 6.70, p = 0.0708) between the FS-120 and control groups. Statistically significant effects in the logistic regression analyses were found at the 10% level in the OR of achieving hemostasis at 6 and 10 minutes, respectively, for the following factors: FS-120 versus control group (OR = 9.92; p = 0.0225 and OR = 6.70; p = 0.0708, respectively), type of intervention (OR = 0.3; p = 0.0775 and OR = 0.25; p = 0.0402, respectively), and heparin coating of the ePTFE prosthesis (OR = 4.83; p = 0.0413 and OR = 3.65; p = 0.0911, respectively). FS was safe and well-tolerated, as indicated by the lack of any related serious adverse events. CONCLUSION: The findings from this phase 2 study support the strong safety profile of FS and suggest that it is an efficacious hemostatic agent in ePTFE graft placement surgery, as well as a useful tool in peripheral vascular surgery applications.

Mgat5 deficiency in T cells and experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease initiated by autoreactive T cells. Mgat5, a gene in the Asn (N-) linked protein glycosylation pathway, associates with MS severity and negatively regulates experimental autoimmune encephalomyelitis (EAE) and spontaneous inflammatory demyelination in mice. N-glycan branching by Mgat5 regulates interaction of surface glycoproteins with galectins, forming a molecular lattice that differentially controls the concentration of surface glycoproteins. T-cell receptor signaling, T-cell proliferation, T(H)1 differentiation, and CTLA-4 endocytosis are inhibited by Mgat5 branching. Non-T cells also contribute to MS pathogenesis and express abundant Mgat5 branched N-glycans. Here we explore whether Mgat5 deficiency in myelin-reactive T cells is sufficient to promote demyelinating disease. Adoptive transfer of myelin-reactive Mgat5(-/-) T cells into Mgat5(+/+) versus Mgat5(-/-) recipients revealed more severe EAE in the latter, suggesting that Mgat5 branching deficiency in recipient naive T cells and/or non-T cells contribute to disease pathogenesis.

Manipulating cell surface glycoproteins by targeting N-glycan-galectin interactions.

The interaction of cell surface receptors and transporters with cognate ligands depends on their concentration, distribution, and organization at the cellular surface. The majority of cell surface receptors and transporters are co- and/or post-translationally modified with asparagine (N)-linked oligosaccharides (glycans). N-Glycan number and structure combine to control the concentration of glycoproteins at the cell surface through interactions with endogenous lectins such as galectins. ER/Golgi enzyme activity and hexosamine pathway supply of Golgi metabolites co-dependently regulate N-glycan biosynthesis and combine to provide adaptive control over cell growth and differentiation. Studies in mice and humans have revealed metabolic and genetic dysregulation of N-glycosylation in T-cell-mediated autoimmunity. In this chapter, we describe methods used to analyze N-glycan-galectin interactions in controlling the distribution and organization of cell surface receptors and transporters.

T cell receptor signaling co-regulates multiple Golgi genes to enhance N-glycan branching.

T cell receptor (TCR) signaling enhances beta1,6GlcNAc-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 beta1,6GlcNAc-branching by transferring N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to N-glycan substrates produced by the sequential action of Golgi alpha1,2-mannosidase I (MIa,b,c), MGAT1, alpha1,2-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 beta1,6GlcNAc-branching, suggesting that enhanced MI and MII activity are both required for this phenotype. MGAT1 and MGAT2 have an approximately 250- and approximately 20-fold higher affinity for UDP-GlcNAc than MGAT5, respectively, and increasing MGAT1 expression paradoxically inhibits beta1,6GlcNAc 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 beta1,6GlcNAc branching, and by extension, CTLA-4 surface expression, T cell growth arrest, and self-tolerance.

T-cell growth, cell surface organization, and the galectin-glycoprotein lattice.

Basal, activation, and arrest signaling in T cells determines survival, coordinates responses to pathogens, and, when dysregulated, leads to loss of self-tolerance and autoimmunity. At the T-cell surface, transmembrane glycoproteins interact with galectins via their N-glycans, forming a molecular lattice that regulates membrane localization, clustering, and endocytosis of surface receptors. Galectin-T-cell receptor (TCR) binding prevents ligand-independent TCR signaling via Lck by blocking spontaneous clustering and CD4-Lck recruitment to TCR, and in turn F-actin transfer of TCR/CD4-Lck complexes to membrane microdomains. Peptide-major histocompatibility complexes overcome galectin-TCR binding to promote TCR clustering and signaling by Lck at the immune synapse. Galectin also localizes the tyrosine phosphatase CD45 to microdomains and the immune synapse, suppressing basal and activation signaling by Lck. Following activation, membrane turnover increases and galectin binding to cytotoxic T-lymphocyte antigen-4 (CTLA-4) enhances surface expression by inhibiting endocytosis, thereby promoting growth arrest. Galectins bind surface glycoproteins in proportion to the branching and number of N-glycans per protein, the latter an encoded feature of protein sequence. N-glycan branching is conditional to the activity of Golgi N-acetylglucosaminyl transferases I, II, IV and V (Mgat1, 2, 4, and 5) and metabolic supply of their donor substrate UDP-GlcNAc. Genetic and metabolic control of N-glycan branching co-regulate homeostatic set-points for basal, activation, and arrest signaling in T cells and, when disturbed, result in T-cell hyperactivity and autoimmunity.

N-glycan processing deficiency promotes spontaneous inflammatory demyelination and neurodegeneration.

Multiple sclerosis (MS) is characterized by inflammatory demyelination of axons and neurodegeneration, the latter inadequately modeled in experimental autoimmune encephalomyelitis (EAE). Susceptibility of inbred mouse strains to EAE is in part determined by major histocompatibility complex haplotype; however, other molecular mechanisms remain elusive. Galectins bind GlcNAc-branched N-glycans attached to surface glycoproteins, forming a molecular lattice that restricts lateral movement and endocytosis of glycoproteins. GlcNAc branching negatively regulates T cell activity and autoimmunity, and when absent in neurons, induces apoptosis in vivo in young adult mice. We find that EAE susceptible mouse strains PL/J, SJL, and NOD have reduced GlcNAc branching. PL/J mice display the lowest levels, partial deficiencies in N-acetylglucosaminyltransferase I, II, and V (i.e. Mgat1, -2, and -5), T cell hyperactivity and spontaneous late onset inflammatory demyelination and neurodegeneration; phenotypes markedly enhanced by Mgat5(+/-) and Mgat5(-/-) backgrounds in a gene dose-dependent manner. Spontaneous disease is transferable and characterized by progressive paralysis, tremor, dystonia, neuronophagia, and axonal damage in both demyelinated lesions and normal white matter, phenocopying progressive MS. Our data identify hypomorphic Golgi processing as an inherited trait that determines susceptibility to EAE, provides a unique spontaneous model of MS, and suggests GlcNAc-branching deficiency may promote T cell-mediated demyelination and neurodegeneration in MS.

Control of T Cell-mediated autoimmunity by metabolite flux to N-glycan biosynthesis.

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, T(H)1 differentiation, and autoimmunity are negatively regulated by beta1,6GlcNAc-branched N-glycans attached to cell surface glycoproteins. Beta1,6GlcNAc-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, beta1,6GlcNAc-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 beta1,6GlcNAc-branched N-glycans in naïve mouse T cells and suppresses T cell receptor signaling, T cell proliferation, CTLA-4 endocytosis, T(H)1 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 beta1,6GlcNAc-branched N-glycans. This suggests metabolic therapy as a potential treatment for autoimmune disease.

Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation.

The number of N-glycans (n) is a distinct feature of each glycoprotein sequence and cooperates with the physical properties of the Golgi N-glycan-branching pathway to regulate surface glycoprotein levels. The Golgi pathway is ultrasensitive to hexosamine flux for the production of tri- and tetra-antennary N-glycans, which bind to galectins and form a molecular lattice that opposes glycoprotein endocytosis. Glycoproteins with few N-glycans (e.g., TbetaR, CTLA-4, and GLUT4) exhibit enhanced cell-surface expression with switch-like responses to increasing hexosamine concentration, whereas glycoproteins with high numbers of N-glycans (e.g., EGFR, IGFR, FGFR, and PDGFR) exhibit hyperbolic responses. Computational and experimental data reveal that these features allow nutrient flux stimulated by growth-promoting high-n receptors to drive arrest/differentiation programs by increasing surface levels of low-n glycoproteins. We have identified a mechanism for metabolic regulation of cellular transition between growth and arrest in mammals arising from apparent coevolution of N-glycan number and branching.