Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis

Essential biological systems employ self-correcting mechanisms to maintain cellular homeostasis. Mammalian cell function is dynamically regulated by the interaction of cell surface galectins with branched N-glycans. Here we report that N-glycan branching deficiency triggers the Golgi to generate bioequivalent N-glycans that preserve galectin-glycoprotein interactions and cellular homeostasis. Galectins bind N-acetyllactosamine (LacNAc) units within N-glycans initiated from UDP-GlcNAc by the medial-Golgi branching enzymes as well as the trans-Golgi poly-LacNAc extension enzyme β1,3-N-acetylglucosaminyltransferase (B3GNT). Marginally reducing LacNAc content by limiting N-glycans to three branches results in T-cell hyperactivity and autoimmunity; yet further restricting branching does not produce a more hyperactive state. Rather, new poly-LacNAc extension by B3GNT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by redistribution of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules. These data demonstrate the functional equivalency of structurally dissimilar N-glycans and suggest a self-correcting feature of the Golgi that sustains cellular homeostasis

Cell-extrinsic and intrinsic regulation of N-glycosylation in health and disease

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

T cell senescence by N-glycan branching.

Immune-senescence refers to the aging of the immune system and the resulting deterioration in its ability to fight infections and cancer in older individuals. This phenomenon is reflected in the numerous infectious diseases which show increased severity in the elderly, most recently demonstrated by the COVID-19 pandemic. The trajectory of the COVID-19 pandemic would have differed markedly if the severity of disease in older individuals was similar to healthy young individuals. Therefore, understanding what factors contribute to immune-senescence and how to reverse them is of great importanc

Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation.

Rapidly proliferating cells switch from oxidative phosphorylation to aerobic glycolysis plus glutaminolysis, markedly increasing glucose and glutamine catabolism. Although Otto Warburg first described aerobic glycolysis in cancer cells >90 years ago, the primary purpose of this metabolic switch remains controversial. The hexosamine biosynthetic pathway requires glucose and glutamine for de novo synthesis of UDP-GlcNAc, a sugar-nucleotide that inhibits receptor endocytosis and signaling by promoting N-acetylglucosamine branching of Asn (N)-linked glycans. Here, we report that aerobic glycolysis and glutaminolysis co-operatively reduce UDP-GlcNAc biosynthesis and N-glycan branching in mouse T cell blasts by starving the hexosamine pathway of glucose and glutamine. This drives growth and pro-inflammatory TH17 over anti-inflammatory-induced T regulatory (iTreg) differentiation, the latter by promoting endocytic loss of IL-2 receptor-α (CD25). Thus, a primary function of aerobic glycolysis and glutaminolysis is to co-operatively limit metabolite supply to N-glycan biosynthesis, an activity with widespread implications for autoimmunity and cancer

Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation.

Rapidly proliferating cells switch from oxidative phosphorylation to aerobic glycolysis plus glutaminolysis, markedly increasing glucose and glutamine catabolism. Although Otto Warburg first described aerobic glycolysis in cancer cells >90 years ago, the primary purpose of this metabolic switch remains controversial. The hexosamine biosynthetic pathway requires glucose and glutamine for de novo synthesis of UDP-GlcNAc, a sugar-nucleotide that inhibits receptor endocytosis and signaling by promoting N-acetylglucosamine branching of Asn (N)-linked glycans. Here, we report that aerobic glycolysis and glutaminolysis co-operatively reduce UDP-GlcNAc biosynthesis and N-glycan branching in mouse T cell blasts by starving the hexosamine pathway of glucose and glutamine. This drives growth and pro-inflammatory TH17 over anti-inflammatory-induced T regulatory (iTreg) differentiation, the latter by promoting endocytic loss of IL-2 receptor-α (CD25). Thus, a primary function of aerobic glycolysis and glutaminolysis is to co-operatively limit metabolite supply to N-glycan biosynthesis, an activity with widespread implications for autoimmunity and cancer

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)

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 (MHC) 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 hyper-activity, 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