Identification of O-GalNAc-modified proteins interacting with GALNT3 using proximity labeling method

Protein glycosylation is a widely occurring post-translational modification catalyzed by glycosyltransferases and is involved in various biological functions. O– N-acetylgalactosamine (O-GalNAc) modification is catalyzed by the N-acetylgalactosaminyltransferase (GALNT) family and represents a truncated form of O-glycosylation, which is closely associated with the development of various diseases. The O-GalNAc modification has long been considered to occur exclusively within the secretory pathway, targeting only membrane and secreted proteins. However, recent study has reported the nuclear localization of GALNT3, suggesting that O-GalNAc modification can also occur in the nucleus. Currently, there is no comprehensive glycoproteomic characterization of O-GalNAc modifications in the nucleus. Here, we employed an efficient proximity labeling approach based on a mutant biotin ligase, TurboID. By generating a fusion of GALNT3 with TurboID, biotin labeling was performed in living cells, enabling the biotinylation of proteins that interact with GALNT3. Using this strategy, delineated 25 high-confidence and 10 potential O-GalNAc-modified sites across 26 characterized proteins and 4 uncharacterized proteins. Additionally, 52 putative O-GalNAc-modified peptides originating from 33 distinct proteins and 19 uncharacterized proteins were identified. The majority of which were nuclear-localized and reported to be O-GalNAc-modified for the first time.