Structure and function of GLUT4’s N-glycan: a novel therapeutic target for type 2 diabetes?

Type 2 diabetes (T2D) is a major healthcare challenge, characterised as dysregulated glucose homeostasis. The glucose transporter GLUT4 is key for maintaining healthy blood glucose levels. Insulin stimulates GLUT4’s transport to the cell surface for glucose uptake into fat and muscle cells, a process which is defective in T2D. While GLUT4 has been intensely studied, little has been researched about the role of its single N-glycan. N-glycosylation is the post-translational addition of a carbohydrate chain to a specific consensus site on a protein, a modification which has a wide variety of functions. A potential function of GLUT4’s N-glycan was identified in the regulation of GLUT4 trafficking. This project aimed to further characterise the function of GLUT4’s N-glycan and elucidate its structure. This was done through a range of methods, including altering the N-glycan’s structure with glycosylation inhibitors and blocking the binding of potential interactors. A HA-GLUT4-GFP HeLa cell line was used, which demonstrates insulin-responsive trafficking of GLUT4 comparable to adipocytes. Using the HA and GFP tags to measure surface GLUT4 as a proportion of whole GLUT4 revealed two roles for GLUT4’s N-glycan in regulating GLUT4 trafficking – one in the intracellular sequestration of GLUT4, and the other in cell surface retention. In addition, analysis of steady state GLUT4 suggested a third role in the regulation of GLUT4 degradation. The structure of GLUT4’s N-glycan was shown to be crucial to each of these functions. Monosaccharide composition analysis revealed the presence of branched polylactosamine in addition to an unidentified monosaccharide not previously discovered in mammalian cells.