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.

天冬酰胺（Asn）连接的N-连接糖基化的N-乙酰葡糖胺（GlcNAc）分支可抑制促炎性T细胞应答，并对多发性硬化症（Multiple Sclerosis, MS）等自身免疫疾病模型具有干预作用。代谢通过调控己糖胺通路（hexosamine pathway）从头合成UDP-GlcNAc——其为高尔基体分支酶的供体底物——来控制T细胞的N-糖链分支。活化的T细胞会将代谢模式从氧化磷酸化（oxidative phosphorylation）转换为有氧糖酵解（aerobic glycolysis）与谷氨酰胺分解（glutaminolysis），这会减少葡萄糖与谷氨酰胺流入己糖胺通路的通量，进而抑制UDP-GlcNAc的从头合成与N-糖链分支。将GlcNAc通过补救合成途径整合入己糖胺通路，可克服这种代谢抑制，恢复UDP-GlcNAc合成与N-糖链分支，从而促进抗炎性调节性T细胞（Treg）的分化，而非促炎性辅助性T细胞17（TH17）与辅助性T细胞1（TH1）的分化，以此抑制自身免疫。然而，由于缺乏细胞表面转运体（cell surface transporter），GlcNAc的生物活性受到限制，且需要高剂量才能通过巨胞饮作用（macropinocytosis）进入细胞。本研究报道，GlcNAc-6-乙酸酯是一种更优异的GlcNAc前体药物（pro-drug）形式。氨基糖的乙酰化可提升细胞膜通透性，随后经细胞质酯酶（cytoplasmic esterases）脱乙酰化，使氨基糖能够通过补救途径整合入己糖胺通路。对GlcNAc进行全乙酰化与双乙酰化会导致T细胞产生毒性，而仅在6位（而非3位）进行单乙酰化，可在不引发显著毒性的前提下，较普通GlcNAc更显著地提升N-糖链分支水平。GlcNAc-6-乙酸酯可在实验性自身免疫性脑脊髓炎（Experimental Autoimmune Encephalomyelitis, EAE，一种MS小鼠模型）中抑制T细胞活化/增殖、TH1/TH17应答，并延缓疾病进展。综上，GlcNAc-6-乙酸酯可提供一种更优化的治疗策略，用于提升N-糖链分支水平、抑制促炎性T细胞应答，并治疗多发性硬化症等自身免疫疾病。