Proximity-Induced Protein Deglycosylation by Endogenous O-GlcNAcase.

O-GlcNAcylation is an important post-translational modification that regulates numerous cellular processes, yet tools enabling selective removal of O-GlcNAc from individual proteins via endogenous O-GlcNAcase (OGA) in living cells remain limited. Here, we report De-O-GlcNAcylation-targeting chimeras (DOGTACs), a chemically induced proximity strategy that selectively reduces O-GlcNAc from target proteins by recruiting endogenous OGA. Initial designs incorporating potent competitive OGA inhibitors efficiently engaged OGA but failed to induce de-O-GlcNAcylation, revealing that catalytic competence is essential for productive proximity-driven editing. By attenuating inhibitor potency while retaining OGA engagement, we developed optimized DOGTACs that promote concentration- and time-dependent, target-specific de-O-GlcNAcylation in living cells without perturbing global O-GlcNAc levels. Furthermore, we successfully applied DOGTAC to additional target proteins including FKBP12F36V fused proteins and endogenous CK2α across different cell lines. Besides, we found DOGTAC (R)-C4 treatment could reduce the stability of FKBP12F36V fused-NUP98-KDM5A in cells. Collectively, this work established attenuated competitive inhibitors as recruitment modules for catalytic enzyme engagement and a novel strategy, DOGTAC, for targeted de-O-GlcNAcylation via endogenous OGA recruitment in living cells.