Essential biological systems are tightly regulated and their dysregulation is often associated with disease states. Recently, plasma membrane dynamics that globally control cell growth and differentiation have been shown to be regulated by the interaction of galectins with branched N-glycans at the cell surface. Galectins bind N-acetyllactosamine units (LacNAc: Gal beta;1,4GlcNAc) in branched N-glycans attached to surface glycoproteins, forming a molecular lattice that controls receptor clustering/surface-retention/signaling. LacNAc density and galectin avidity for N-glycans increase proportional to the number of LacNAc branches initiated via the sequential action of the medial Golgi branching enzymes Mgat1, 2, 4, and 5. Mgat5 deficiency marginally reduces LacNAc content by limiting N-glycans to three branches, yet results in T-cell hyperactivity and autoimmunity in mice. Despite its importance, little is known about endogenous mechanisms that regulate N-glycosylation and the galectin-glycoprotein lattice.
Here we show that multiple MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and Vitamin D3, including genetic variants in interleukin-7 receptor-alpha (IL7RA*C), interleukin-2 receptor-alpha (IL2RA*T), MGAT1 (IVAVT-T) and CTLA-4 (Thr17Ala). Down-regulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IVAVT-T) and Vitamin D3, 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.
We also report a startling homeostatic mechanism that maintains cell surface LacNAc content when branching is severely disrupted. Based on the model of galectin-glycoprotein lattice, restricting N-glycans to a single branch via Mgat2 deficiency or mannosidase II inhibition is expected to result in a dramaticly hyperactive state, when compared to Mgat5 deficiency. However, we find that a marked increase in poly-LacNAc extension maintains LacNAc content and galectin binding to N-glycans, thereby preventing further increases in T cell activity and autoimmunity; phenotypes reversed by targeted deficiency of the poly-LacNAc extension enzyme B3GnT2 or complete blockade of branching. Homeostatic maintenance of LacNAc content in N-glycans appears to be a general mechanism, being present in epithelial, mesenchymal and stem cells. These data define Golgi proofreading and LacNAc content, rather than unique N-glycan structures, as critical regulators of the galectin lattice, cell homeostasis and autoimmunity.