1. Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase.
- Author
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Ge Y, Ramirez DH, Yang B, D'Souza AK, Aonbangkhen C, Wong S, and Woo CM
- Subjects
- Antigens, Neoplasm chemistry, Antigens, Neoplasm genetics, Biological Assay, Catalytic Domain, Drug Delivery Systems methods, Gene Expression, Glycosylation, HEK293 Cells, Histone Acetyltransferases chemistry, Histone Acetyltransferases genetics, Humans, Hyaluronoglucosaminidase chemistry, Hyaluronoglucosaminidase genetics, Hydrolysis, JNK Mitogen-Activated Protein Kinases genetics, Membrane Glycoproteins genetics, Nuclear Pore Complex Proteins genetics, Plasmids chemistry, Plasmids metabolism, Protein Binding, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Single-Domain Antibodies metabolism, Sp1 Transcription Factor genetics, Transcription Factors genetics, Transfection methods, Antigens, Neoplasm metabolism, Histone Acetyltransferases metabolism, Hyaluronoglucosaminidase metabolism, JNK Mitogen-Activated Protein Kinases metabolism, Membrane Glycoproteins metabolism, Nuclear Pore Complex Proteins metabolism, Recombinant Fusion Proteins metabolism, Single-Domain Antibodies chemistry, Sp1 Transcription Factor metabolism, Transcription Factors metabolism
- Abstract
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.
- Published
- 2021
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