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

Sung-Uk Lee,Carey F Li,Michael Demetriou,Judy Pawling,James W Dennis,Ani Grigorian,Christie-Lynn Mortales

doi:10.1371/journal.pone.0214253

Abstract

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.

Highlights

Cell surface and secreted proteins are co- and post-translationally modified on Asn (N) by the addition of carbohydrates (N-glycans) in the endoplasmic reticulum
To increase hydrophobicity and membrane permeability, we examined GlcNAc analogs modified using one or more acetyl substituents at various positions (Fig 1B) and tested their ability to raise N-glycan branching in human CD4+ T cells
Cells were activated with PMA/ionomycin or plate-bound anti-CD3ε plus soluble anti-CD28 and assessed by flow cytometry with Phaseolus vulgaris leukoagglutinin (L-PHA), a plant lectin that binds β1,6GlcNAc-branched N-glycans produced by the Mgat5 enzyme (Fig 1A) and serves as an overall marker of N-glycan branching [2,3,9,10,31]

Summary

Introduction

Cell surface and secreted proteins are co- and post-translationally modified on Asn (N) by the addition of carbohydrates (N-glycans) in the endoplasmic reticulum. Nglycan branching drives Treg differentiation by promoting IL-2Rα (CD25) surface expression and signaling in T cell blasts, with loss of branching virtually eliminating induction of Tregs [10]. Consistent with these phenotypes, reductions in N-glycan branching promote proinflammatory T cell responses and autoimmunity in both mice and humans [3,12,13,14,15,16,17]

Methods

Results

Conclusion