Journal
NATURE CHEMICAL BIOLOGY
Volume 17, Issue 5, Pages 593-600Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41589-021-00757-y
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Funding
- National Institutes of Health [U01CA242098-01]
- Burroughs Wellcome Fund, a Career Award at the Scientific Interface
- Sloan Foundation
- Merck Fellowship Fund
- Harvard University
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In this study, a nanobody-fused split O-GlcNAcase (OGA) was developed as an O-GlcNAc eraser for selectively deglycosylating target proteins in cells. Through systematic cellular optimization, a split OGA with reduced inherent deglycosidase activity was identified for selective removal of O-GlcNAc from target proteins guided by nanobody. The system demonstrated generality by using four nanobodies against five target proteins, allowing the study of O-GlcNAc impact on transcription factors c-Jun and c-Fos.
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.
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