Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase

O-linked N -acetylglucosamine ( O -GlcNAc) is an essential and dynamic post-translational modification that is 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 the impact of O -GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O -GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O -GlcNAc via the selective removal of O -GlcNAc from individual proteins directly in cells. Fusion of a split form of the protein O -GlcNAcase with nanobodies enables the targeted removal of O -GlcNAc protein modifications, providing a tool for probing the functional roles of specific O -GlcNAc modifications in a cellular context.