N-acetylglucosamine inhibits inflammation and neurodegeneration markers in multiple sclerosis: a mechanistic trial.

BACKGROUND: In the demyelinating disease multiple sclerosis (MS), chronic-active brain inflammation, remyelination failure and neurodegeneration remain major issues despite immunotherapy. While B cell depletion and blockade/sequestration of T and B cells potently reduces episodic relapses, they act peripherally to allow persistence of chronic-active brain inflammation and progressive neurological dysfunction. N-acetyglucosamine (GlcNAc) is a triple modulator of inflammation, myelination and neurodegeneration. GlcNAc promotes biosynthesis of Asn (N)-linked-glycans, which interact with galectins to co-regulate the clustering/signaling/endocytosis of multiple glycoproteins simultaneously. In mice, GlcNAc crosses the blood brain barrier to raise N-glycan branching, suppress inflammatory demyelination by T and B cells and trigger stem/progenitor cell mediated myelin repair. MS clinical severity, demyelination lesion size and neurodegeneration inversely associate with a marker of endogenous GlcNAc, while in healthy humans, age-associated increases in endogenous GlcNAc promote T cell senescence. OBJECTIVES AND METHODS: An open label dose-escalation mechanistic trial of oral GlcNAc at 6 g (n = 18) and 12 g (n = 16) for 4 weeks was performed in MS patients on glatiramer acetate and not in relapse from March 2016 to December 2019 to assess changes in serum GlcNAc, lymphocyte N-glycosylation and inflammatory markers. Post-hoc analysis examined changes in serum neurofilament light chain (sNfL) as well as neurological disability via the Expanded Disability Status Scale (EDSS). RESULTS: Prior to GlcNAc therapy, high serum levels of the inflammatory cytokines IFNγ, IL-17 and IL-6 associated with reduced baseline levels of a marker of endogenous serum GlcNAc. Oral GlcNAc therapy was safe, raised serum levels and modulated N-glycan branching in lymphocytes. Glatiramer acetate reduces TH1, TH17 and B cell activity as well as sNfL, yet the addition of oral GlcNAc dose-dependently lowered serum IFNγ, IL-17, IL-6 and NfL. Oral GlcANc also dose-dependently reduced serum levels of the anti-inflammatory cytokine IL-10, which is increased in the brain of MS patients. 30% of treated patients displayed confirmed improvement in neurological disability, with an average EDSS score decrease of 0.52 points. CONCLUSIONS: Oral GlcNAc inhibits inflammation and neurodegeneration markers in MS patients despite concurrent immunomodulation by glatiramer acetate. Blinded studies are required to investigate GlcNAc's potential to control residual brain inflammation, myelin repair and neurodegeneration in MS.

The Cancer Moonshot Immuno-Oncology Translational Network at 5: accelerating cancer immunotherapies.

The Immuno-Oncology Translational Network (IOTN) was established in 2018 as part of the Cancer Moonshot. In 2022, President Joe Biden set new goals to reduce the cancer death rate by half within 25 years and improve the lives of people with cancer and cancer survivors. The IOTN is focused on accelerating translation of cancer immunology research, from bench to bedside, and improving immunotherapy outcomes across a wide array of cancers in the adult population. The unique structure and team science approach of the IOTN is designed to accelerate discovery and evaluation of novel immune-based therapeutic and prevention strategies. In this article, we describe IOTN progress to date, including new initiatives and the development of a robust set of resources to advance cancer immunology research. We summarize new insights by IOTN researchers, some of which are ripe for translation for several types of cancers. Looking to the future, we identify barriers to the translation of immuno-oncology concepts into clinical trials and key areas for action and improvements that are suitable for high-yield investments. Based on these experiences, we recommend novel National Institutes of Health funding mechanisms and development of new resources to address these barriers.

Age-associated impairment of T cell immunity is linked to sex-dimorphic elevation of N-glycan branching.

Impaired T cell immunity with aging increases mortality from infectious disease. The branching of Asparagine-linked glycans is a critical negative regulator of T cell immunity. Here we show that branching increases with age in females more than males, in naïve more than memory T cells, and in CD4+ more than CD8+ T cells. Female sex hormones and thymic output of naïve T cells (TN) decrease with age, however neither thymectomy nor ovariectomy altered branching. Interleukin-7 (IL-7) signaling was increased in old female more than male mouse TN cells, and triggered increased branching. N-acetylglucosamine, a rate-limiting metabolite for branching, increased with age in humans and synergized with IL-7 to raise branching. Reversing elevated branching rejuvenated T cell function and reduced severity of Salmonella infection in old female mice. These data suggest sex-dimorphic antagonistic pleiotropy, where IL-7 initially benefits immunity through TN maintenance but inhibits TN function by raising branching synergistically with age-dependent increases in N-acetylglucosamine.

The Crossroads of Glycoscience, Infection, and Immunology.

Advances in experimental capabilities in the glycosciences offer expanding opportunities for discovery in the broad areas of immunology and microbiology. These two disciplines overlap when microbial infection stimulates host immune responses and glycan structures are central in the processes that occur during all such encounters. Microbial glycans mediate host-pathogen interactions by acting as surface receptors or ligands, functioning as virulence factors, impeding host immune responses, or playing other roles in the struggle between host and microbe. In the context of the host, glycosylation drives cell-cell interactions that initiate and regulate the host response and modulates the effects of antibodies and soluble immune mediators. This perspective reports on a workshop organized jointly by the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research in May 2020. The conference addressed the use of emerging glycoscience tools and resources to advance investigation of glycans and their roles in microbe-host interactions, immune-mediated diseases, and immune cell recognition and function. Future discoveries in these areas will increase fundamental scientific understanding and have the potential to improve diagnosis and treatment of infections and immune dysregulation.

Association of a Marker of N-Acetylglucosamine With Progressive Multiple Sclerosis and Neurodegeneration.

Importance: N-glycan branching modulates cell surface receptor availability, and its deficiency in mice promotes inflammatory demyelination, reduced myelination, and neurodegeneration. N-acetylglucosamine (GlcNAc) is a rate-limiting substrate for N-glycan branching, but, to our knowledge, endogenous serum levels in patients with multiple sclerosis (MS) are unknown. Objective: To investigate a marker of endogenous serum GlcNAc levels in patients with MS. Design, Setting, and Participants: A cross-sectional discovery study and cross-sectional confirmatory study were conducted at 2 academic MS centers in the US and Germany. The discovery study recruited 54 patients with MS from an outpatient clinic as well as 66 healthy controls between April 20, 2010, and June 21, 2013. The confirmatory study recruited 180 patients with MS from screening visits at an academic MS study center between April 9, 2007, and February 29, 2016. Serum samples were analyzed from December 2, 2013, to March 2, 2015. Statistical analysis was performed from February 23, 2020, to March 18, 2021. Main Outcomes and Measures: Serum levels of GlcNAc plus its stereoisomers, termed N-acetylhexosamine (HexNAc), were assessed using targeted tandem mass spectroscopy. Secondary outcomes (confirmatory study) comprised imaging and clinical disease markers. Results: The discovery cohort included 66 healthy controls (38 women; mean [SD] age, 42 [20] years), 33 patients with relapsing-remitting MS (RRMS; 25 women; mean [SD] age, 50 [11] years), and 21 patients with progressive MS (PMS; 14 women; mean [SD] age, 55 [7] years). The confirmatory cohort included 125 patients with RRMS (83 women; mean [SD] age, 40 [9] years) and 55 patients with PMS (22 women; mean [SD] age, 49 [80] years). In the discovery cohort, the mean (SD) serum level of GlcNAc plus its stereoisomers (HexNAc) was 710 (174) nM in healthy controls and marginally reduced in patients with RRMS (mean [SD] level, 682 [173] nM; P = .04), whereas patients with PMS displayed markedly reduced levels compared with healthy controls (mean [SD] level, 548 [101] nM; P = 9.55 × 10-9) and patients with RRMS (P = 1.83 × 10-4). The difference between patients with RRMS (mean [SD] level, 709 [193] nM) and those with PMS (mean [SD] level, 405 [161] nM; P = 7.6 × 10-18) was confirmed in the independent confirmatory cohort. Lower HexNAc serum levels correlated with worse expanded disability status scale scores (ρ = -0.485; P = 4.73 × 10-12), lower thalamic volume (t = 1.7; P = .04), and thinner retinal nerve fiber layer (B = 0.012 [SE = 7.5 × 10-11]; P = .008). Low baseline serum HexNAc levels correlated with a greater percentage of brain volume loss at 18 months (t = 1.8; P = .04). Conclusions and Relevance: This study suggests that deficiency of GlcNAc plus its stereoisomers (HexNAc) may be a biomarker for PMS. Previous preclinical, human genetic, and ex vivo human mechanistic studies revealed that N-glycan branching and/or GlcNAc may reduce proinflammatory responses, promote myelin repair, and decrease neurodegeneration. Combined, the data suggest that GlcNAc deficiency may be associated with progressive disease and neurodegeneration in patients with MS.

N-Glycan Branching Decouples B Cell Innate and Adaptive Immunity to Control Inflammatory Demyelination.

B cell depletion potently reduces episodes of inflammatory demyelination in multiple sclerosis (MS), predominantly through loss of innate rather than adaptive immunity. However, molecular mechanisms controlling innate versus adaptive B cell function are poorly understood. N-glycan branching, via interactions with galectins, controls endocytosis and signaling of cell surface receptors to control cell function. Here we report that N-glycan branching in B cells dose dependently reduces pro-inflammatory innate responses by titrating decreases in Toll-like receptor-4 (TLR4) and TLR2 surface expression via endocytosis. In contrast, a minimal level of N-glycan branching maximizes surface retention of the B cell receptor (BCR) and the CD19 co-receptor to promote adaptive immunity. Branched N-glycans inhibit antigen presentation by B cells to reduce T helper cell-17 (TH17)/TH1 differentiation and inflammatory demyelination in mice. Thus, N-glycan branching negatively regulates B cell innate function while promoting/maintaining adaptive immunity via BCR, providing an attractive therapeutic target for MS.

Effect of vitamin D supplementation on N-glycan branching and cellular immunophenotypes in MS.

OBJECTIVE: To investigate the effect of cholecalciferol (vitamin D3) supplementation on peripheral immune cell frequency and N-glycan branching in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: Exploratory analysis of high-dose (20 400 IU) and low-dose (400 IU) vitamin D3 supplementation taken every other day of an 18-month randomized controlled clinical trial including 38 RRMS patients on stable immunomodulatory therapy (NCT01440062). We investigated cholecalciferol treatment effects on N-glycan branching using L-PHA stain (phaseolus vulgaris leukoagglutinin) at 6 months and frequencies of T-, B-, and NK-cell subpopulations at 12 months with flow cytometry. RESULTS: High-dose supplementation did not change CD3+ T cell subsets, CD19+ B cells subsets, and NK cells frequencies, except for CD8+ T regulatory cells, which were reduced in the low-dose arm compared to the high-dose arm at 12 months. High-dose supplementation decreased N-glycan branching on T and NK cells, measured as L-PHA mean fluorescence intensity (MFI). A reduction of N-glycan branching in B cells was not significant. In contrast, low-dose supplementation did not affect N-glycan branching. Changes in N-glycan branching did not correlate with cell frequencies. INTERPRETATION: Immunomodulatory effect of vitamin D may involve regulation of N-glycan branching in vivo. Vitamin D3 supplementation did at large not affect the frequencies of peripheral immune cells.

N-Glycan Branching Is Required for Development of Mature B Cells.

Galectins have been implicated in inhibiting BCR signaling in mature B cells but promoting pre-BCR signaling during early development. Galectins bind to branched N-glycans attached to cell surface glycoproteins to control the distribution, clustering, endocytosis, and signaling of surface glycoproteins. During T cell development, N-glycan branching is required for positive selection of thymocytes, inhibiting both death by neglect and negative selection via enhanced surface retention of the CD4/CD8 coreceptors and limiting TCR clustering/signaling, respectively. The role of N-glycan branching in B cell development is unknown. In this study, we report that N-glycan branching is absolutely required for development of mature B cells in mice. Elimination of branched N-glycans in developing B cells via targeted deletion of N-acetylglucosaminyl transferase I (Mgat1) markedly reduced cellularity in the bone marrow and/or spleen and inhibited maturation of pre-, immature, and transitional stage 2 B cells. Branching deficiency markedly reduced surface expression of the pre-BCR/BCR coreceptor CD19 and promoted spontaneous death of pre-B cells and immature B cells in vitro. Death was rescued by low-dose pre-BCR/BCR stimulation but exacerbated by high-dose pre-BCR/BCR stimulation as well as antiapoptotic BclxL overexpression in pre-B cells. Branching deficiency also enhanced Nur77 induction, a marker of negative selection. Together, these data suggest that, as in T cells, N-glycan branching promotes positive selection of B cells by augmenting pre-BCR/BCR signaling via CD19 surface retention, whereas limiting negative selection from excessive BCR engagement.

Cancer Moonshot Immuno-Oncology Translational Network (IOTN): accelerating the clinical translation of basic discoveries for improving immunotherapy and immunoprevention of cancer.

Despite regulatory approval of several immune-based treatments for cancer in the past decade, a number of barriers remain to be addressed in order to fully harness the therapeutic potential of the immune system and provide benefits for patients with cancer. As part of the Cancer Moonshot initiative, the Immuno-Oncology Translational Network (IOTN) was established to accelerate the translation of basic discoveries to improve immunotherapy outcomes across the spectrum of adult cancers and to develop immune-based approaches that prevent cancers before they occur. The IOTN currently consists of 32 academic institutions in the USA. By leveraging cutting-edge preclinical research in immunotherapy and immunoprevention, open data and resource sharing, and fostering highly collaborative team science across the immuno-oncology ecosystem, the IOTN is designed to accelerate the generation of novel mechanism-driven immune-based cancer prevention and therapies, and the development of safe and effective personalized immuno-oncology approaches.

N-acetylglucosamine drives myelination by triggering oligodendrocyte precursor cell differentiation.

Myelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis. Supplying oral GlcNAc to lactating mice drives primary myelination in newborn pups via secretion in breast milk, whereas genetically blocking N-glycan branching markedly inhibits primary myelination. In adult mice with toxin (cuprizone)-induced demyelination, oral GlcNAc prevents neuro-axonal damage by driving myelin repair. In MS patients, endogenous serum GlcNAc levels inversely correlated with imaging measures of demyelination and microstructural damage. Our data identify N-glycan branching and GlcNAc as critical regulators of primary myelination and myelin repair and suggest that oral GlcNAc may be neuroprotective in demyelinating diseases like MS.

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.

Cell Surface N-Glycans Influence Electrophysiological Properties and Fate Potential of Neural Stem Cells.

Understanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage.

Long-term plasma exchange as maintenance therapy for cerebellar-type Hashimoto's encephalopathy, a case report.

Hashimoto's encephalopathy (HE) is a presumed autoimmune disorder associated with anti-thyroid autoantibodies and signs and symptoms of encephalopathy. A sub-type of HE is associated with cerebellar dysfunction and ataxia. Immunosuppressive therapy, particularly corticosteroid treatment, is utilized in the majority of cases. Short-term apheresis has been reported with variable patient responses. Here we report the case of a 72 year-old female with an ∼15 year history of cerebellar type HE that had profound improvement in symptoms after long-term apheresis treatment over an ∼2 year period. Following an induction phase, twice-weekly maintenance apheresis of 1 plasma volume reversed long-standing severe gait ataxia that had required a walker, as well as mild cognitive symptoms. This paralleled reductions in anti-thyroid antibody levels. Holidays from apheresis lasting several weeks and/or reductions in maintenance apheresis frequency to once per-week resulted in re-expression of ataxia and cognitive impairments along with a rise in anti-thyroid antibody levels. An apheresis dose-effect was observed whereby parallel rise and fall in both symptomatology and antibody levels would mirror duration between apheresis intervals. To our knowledge, this is the first report of profound therapeutic benefit and a dose-response relationship to long-term apheresis in cerebellar-type HE. This case suggests that maintenance apheresis be considered in responsive patients, particularly in those with contraindications to medical immunosuppression.

Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation.

Rapidly proliferating cells switch from oxidative phosphorylation to aerobic glycolysis plus glutaminolysis, markedly increasing glucose and glutamine catabolism. Although Otto Warburg first described aerobic glycolysis in cancer cells >90 years ago, the primary purpose of this metabolic switch remains controversial. The hexosamine biosynthetic pathway requires glucose and glutamine for de novo synthesis of UDP-GlcNAc, a sugar-nucleotide that inhibits receptor endocytosis and signaling by promoting N-acetylglucosamine branching of Asn (N)-linked glycans. Here, we report that aerobic glycolysis and glutaminolysis co-operatively reduce UDP-GlcNAc biosynthesis and N-glycan branching in mouse T cell blasts by starving the hexosamine pathway of glucose and glutamine. This drives growth and pro-inflammatory TH17 over anti-inflammatory-induced T regulatory (iTreg) differentiation, the latter by promoting endocytic loss of IL-2 receptor-α (CD25). Thus, a primary function of aerobic glycolysis and glutaminolysis is to co-operatively limit metabolite supply to N-glycan biosynthesis, an activity with widespread implications for autoimmunity and cancer.

Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis.

Essential biological systems employ self-correcting mechanisms to maintain cellular homeostasis. Mammalian cell function is dynamically regulated by the interaction of cell surface galectins with branched N-glycans. Here we report that N-glycan branching deficiency triggers the Golgi to generate bioequivalent N-glycans that preserve galectin-glycoprotein interactions and cellular homeostasis. Galectins bind N-acetyllactosamine (LacNAc) units within N-glycans initiated from UDP-GlcNAc by the medial-Golgi branching enzymes as well as the trans-Golgi poly-LacNAc extension enzyme ß1,3-N-acetylglucosaminyltransferase (B3GNT). Marginally reducing LacNAc content by limiting N-glycans to three branches results in T-cell hyperactivity and autoimmunity; yet further restricting branching does not produce a more hyperactive state. Rather, new poly-LacNAc extension by B3GNT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by redistribution of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules. These data demonstrate the functional equivalency of structurally dissimilar N-glycans and suggest a self-correcting feature of the Golgi that sustains cellular homeostasis.

Genome-Wide Analysis of Gene-Gene and Gene-Environment Interactions Using Closed-Form Wald Tests.

Despite the successful discovery of hundreds of variants for complex human traits using genome-wide association studies, the degree to which genes and environmental risk factors jointly affect disease risk is largely unknown. One obstacle toward this goal is that the computational effort required for testing gene-gene and gene-environment interactions is enormous. As a result, numerous computationally efficient tests were recently proposed. However, the validity of these methods often relies on unrealistic assumptions such as additive main effects, main effects at only one variable, no linkage disequilibrium between the two single-nucleotide polymorphisms (SNPs) in a pair or gene-environment independence. Here, we derive closed-form and consistent estimates for interaction parameters and propose to use Wald tests for testing interactions. The Wald tests are asymptotically equivalent to the likelihood ratio tests (LRTs), largely considered to be the gold standard tests but generally too computationally demanding for genome-wide interaction analysis. Simulation studies show that the proposed Wald tests have very similar performances with the LRTs but are much more computationally efficient. Applying the proposed tests to a genome-wide study of multiple sclerosis, we identify interactions within the major histocompatibility complex region. In this application, we find that (1) focusing on pairs where both SNPs are marginally significant leads to more significant interactions when compared to focusing on pairs where at least one SNP is marginally significant; and (2) parsimonious parameterization of interaction effects might decrease, rather than increase, statistical power.

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.

Encoding asymmetry of the N-glycosylation motif facilitates glycoprotein evolution.

Protein N-glycosylation is found in all domains of life and has a conserved role in glycoprotein folding and stability. In animals, glycoproteins transit through the Golgi where the N-glycans are trimmed and rebuilt with sequences that bind lectins, an innovation that greatly increases structural diversity and redundancy of glycoprotein-lectin interaction at the cell surface. Here we ask whether the natural tension between increasing diversity (glycan-protein interactions) and site multiplicity (backup and status quo) might be revealed by a phylogenic examination of glycoproteins and NXS/T(X ≠ P) N-glycosylation sites. Site loss is more likely by mutation at Asn encoded by two adenosine (A)-rich codons, while site gain is more probable by generating Ser or Thr downstream of an existing Asn. Thus mutations produce sites at novel positions more frequently than the reversal of recently lost sites, and therefore more paths though sequence space are made available to natural selection. An intra-species comparison of secretory and cytosolic proteins revealed a departure from equilibrium in sequences one-mutation-away from NXS/T and in (A) content, indicating strong selective pressures and exploration of N-glycosylation positions during vertebrate evolution. Furthermore, secretory proteins have evolved at rates proportional to N-glycosylation site number, indicating adaptive interactions between the N-glycans and underlying protein. Given the topology of the genetic code, mutation of (A) is more often nonsynonomous, and Lys, another target of many PTMs, is also encoded by two (A)-rich codons. An examination of acetyl-Lys sites in proteins indicated similar evolutionary dynamics, consistent with asymmetry of the target and recognition portions of modified sites. Our results suggest that encoding asymmetry is an ancient mechanism of evolvability that increases diversity and experimentation with PTM site positions. Strong selective pressures on PTMs may have contributed to the A+T → G+C shift in genome-wide nucleotide composition during metazoan radiation.

Hypomorphic MGAT5 polymorphisms promote multiple sclerosis cooperatively with MGAT1 and interleukin-2 and 7 receptor variants.

Deficiency of the Golgi N-glycan branching enzyme Mgat5 in mice promotes T cell hyperactivity, endocytosis of CTLA-4 and autoimmunity, including a spontaneous multiple sclerosis (MS)-like disease. Multiple genetic and environmental MS risk factors lower N-glycan branching in T cells. These include variants in interleukin-2 receptor-α (IL2RA), interleukin-7 receptor-α (IL7RA), and MGAT1, a Golgi branching enzyme upstream of MGAT5, as well as vitamin D3 deficiency and Golgi substrate metabolism. Here we describe linked intronic variants of MGAT5 that are associated with reduced N-glycan branching, CTLA-4 surface expression and MS (p=5.79×10(-9), n=7,741), the latter additive with the MGAT1, IL2RA and IL7RA MS risk variants (p=1.76×10(-9), OR=0.67-1.83, n=3,518).