Ge, YunRamirez, Daniel H.Yang, BoD’Souza, Alexandria K.Aonbangkhen, ChanatWong, StephanieWoo, Christina M.2023-02-082021-03-08Ge, Yun, Daniel H Ramirez, Bo Yang, Alexandria K D'Souza, Chanat Aonbangkhen, Stephanie Wong, and Christina M Woo. 2021. “Target Protein Deglycosylation in Living Cells by a Nanobody-Fused Split O-GlcNAcase.” Nature Chemical Biology 17 (5): 593–600.1552-44501552-4469https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37374276O-GlcNAc is an essential and dynamic post-translational modification presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins, and use the system to study functions for O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.en-USCell BiologyMolecular BiologyJournal Article2023-02-0810.1038/s41589-021-00757-y