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

N-acetylglucosamine (GIcNAc) 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-GIcNAc, 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-GIcNAc synthesis and N-glycan branching. Salvage of GIcNAc into the hexosamine pathway overcomes this metabolic suppression to restore UDP-GIcNAc synthesis and N-glycan branching, thereby promoting anti-inflammatory T regulatory (Treg) over pro-inflammatory T helper (T-H) 17 and T(H)1 differentiation to suppress autoimmunity. However, GIcNAc activity is limited by the lack of a cell surface transporter and requires high doses to enter cells via macropinocytosis. Here we report that GIcNAc-6-acetate is a superior pro-drug form of GIcNAc. 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 GIcNAc led to toxicity in T cells, whereas mono-acetylation at only the 6 > 3 position raised N-glycan branching greater than GIcNAc without inducing significant toxicity. GIcNAc-6-acetate inhibited T cell activation/proliferation, T(H)1/T(H)17 responses and disease progression in Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of MS. Thus, GIcNAc-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.