Proximity‐Induced Protein Deglycosylation by Endogenous O‐GlcNAcase

ABSTRACT 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 FKBP12 F36V fused proteins and endogenous CK2α across different cell lines. Besides, we found DOGTAC (R)‐C4 treatment could reduce the stability of FKBP12 F36V 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.