N‐glycosylation is critical for the biogenesis of small extracellular vesicles

Small extracellular vesicles (sEVs) play an important role in communication between cells by mediating the unconventional secretion of nucleotides and proteins, which contributes to a wide range of biological and pathological phenomena such as cell differentiation, immune response, neurodegenerative diseases, and tumor progression. The biogenesis of sEVs is regulated by multiple molecular machineries, generating considerably heterogeneous vesicle populations, i.e. , 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 discovered that 2‐deoxyglucose (2‐DG), a well‐known glycolysis inhibitor, suppressed the secretion of E xosome‐like, A sparagine‐linked GL ycosylation‐dependent, and E ndosomal and S ecretory pathways associated vesicles (EAGLES) by impairing asparagine‐linked glycosylation (N‐glycosylation) in mouse melanoma cells. Mechanistically, 2‐DG was metabolically incorporated into N‐glycan precursors, causing the precursor degradation and partial hypoglycosylation. N‐glycosylation blockade by Stt3a silencing was sufficient to inhibit EAGLES secretion. In contrast, N‐glycosylation blockade barely influenced exosomal secretion of tetraspanin proteins. Functionally, N‐glycosylation at the specific sites of the hepatocyte growth factor receptor, a cargo protein of EAGLES, facilitated its sorting into the vesicles. These results uncover a link between N‐glycosylation and unconventional vesicle secretion, and suggest that N‐glycosylation regulates the biogenesis of certain types of sEVs through cargo protein sorting.