Glycometabolic Regulation of the Biogenesis of Small Extracellular Vesicles

The biogenesis of small extracellular vesicles (sEVs) is regulated by multiple molecular machineries generating considerably heterogeneous vesicle populations, including exosomes and non-exosomal vesicles, with distinct cargo molecules. However, the role of carbohydrate metabolism in generating such vesicle heterogeneity remains largely elusive. Here, we discover that 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, suppresses the secretion of non-exosomal vesicles by impairing asparagine-linked glycosylation (N-glycosylation) in mouse melanoma cells. Mechanistically, 2-DG is metabolically incorporated into N-glycan precursors, causing precursor degradation and partial hypoglycosylation. N-glycosylation blockade by Stt3a silencing is sufficient to inhibit non-exosomal vesicle secretion. In contrast, N-glycosylation blockade barely influences exosomal secretion of tetraspanin proteins. Functionally, N-glycosylation at specific sites of the hepatocyte growth factor receptor, a cargo protein of non-exosomal vesicles, facilitates its sorting into vesicles. These results uncover a link between N-glycosylation and unconventional vesicle secretion and suggest that N-glycosylation facilitates sEV biogenesis through cargo protein sorting.