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.

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).

Incorporating parental information into family-based association tests.

Assumptions regarding the true underlying genetic model, or mode of inheritance, are necessary when quantifying genetic associations with disease phenotypes. Here we propose new methods to ascertain the underlying genetic model from parental data in family-based association studies. Specifically, for parental mating-type data, we propose a novel statistic to test whether the underlying genetic model is additive, dominant, or recessive; for parental genotype-phenotype data, we propose three strategies to determine the true mode of inheritance. We illustrate how to incorporate the information gleaned from these strategies into family-based association tests. Because family-based association tests are conducted conditional on parental genotypes, the type I error rate of these procedures is not inflated by the information learned from parental data. This result holds even if such information is weak or when the assumption of Hardy-Weinberg equilibrium is violated. Our simulations demonstrate that incorporating parental data into family-based association tests can improve power under common inheritance models. The application of our proposed methods to a candidate-gene study of type 1 diabetes successfully detects a recessive effect in MGAT5 that would otherwise be missed by conventional family-based association tests.

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.

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.

Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana.

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent--disruption of one class does not affect the other--suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.