Abstract
Glycosylation plays a critical role in various biological processes, yet identifying specific glycosyltransferase substrates remains a challenge due to the complexity of glycosylation. Here, we employ proximity labeling with biotin ligases BASU and TurboID to map the proximitome of MGAT3, a glycosyltransferase responsible for the biosynthesis of the bisecting GlcNAc structure, in HEK293T cells. This approach enriched 116 and 189 proteins, respectively, identifying 17 common substrates shared with bisecting GlcNAc-bearing proteome obtained via intact glycopeptide enrichment methods. Gene ontology analysis revealed that the enriched proteins were predominantly localized in the exosome, endoplasmic reticulum, and Golgi apparatus, consistent with subcellular localization of MGAT3 substrates. Notably, four novel substrates, GOLM2, CCDC134, ASPH, and ERO1A, were confirmed to bear bisecting GlcNAc modification, validating the utility of the proximity labeling method. Furthermore, we observed that bisecting GlcNAc modification inhibits breast cancer progression by promoting the degradation of α-galactosidase A (GLA). These findings demonstrate the efficacy of proximity labeling in identifying glycosyltransferase substrates and provide insights into the functional impact of bisecting GlcNAc modification.