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.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Antigens, Neoplasm / chemistry
-
Antigens, Neoplasm / genetics
-
Antigens, Neoplasm / metabolism*
-
Biological Assay
-
Catalytic Domain
-
Drug Delivery Systems / methods
-
Gene Expression
-
Glycosylation
-
HEK293 Cells
-
Histone Acetyltransferases / chemistry
-
Histone Acetyltransferases / genetics
-
Histone Acetyltransferases / metabolism*
-
Humans
-
Hyaluronoglucosaminidase / chemistry
-
Hyaluronoglucosaminidase / genetics
-
Hyaluronoglucosaminidase / metabolism*
-
Hydrolysis
-
JNK Mitogen-Activated Protein Kinases / genetics
-
JNK Mitogen-Activated Protein Kinases / metabolism*
-
Membrane Glycoproteins / genetics
-
Membrane Glycoproteins / metabolism*
-
Nuclear Pore Complex Proteins / genetics
-
Nuclear Pore Complex Proteins / metabolism*
-
Plasmids / chemistry
-
Plasmids / metabolism
-
Protein Binding
-
Recombinant Fusion Proteins / chemistry
-
Recombinant Fusion Proteins / genetics
-
Recombinant Fusion Proteins / metabolism*
-
Single-Domain Antibodies / chemistry*
-
Single-Domain Antibodies / metabolism
-
Sp1 Transcription Factor / genetics
-
Sp1 Transcription Factor / metabolism*
-
Transcription Factors / genetics
-
Transcription Factors / metabolism*
-
Transfection / methods
Substances
-
Antigens, Neoplasm
-
JunB protein, human
-
Membrane Glycoproteins
-
Nuclear Pore Complex Proteins
-
Recombinant Fusion Proteins
-
Single-Domain Antibodies
-
Sp1 Transcription Factor
-
SP1 protein, human
-
Transcription Factors
-
nuclear pore protein p62
-
Histone Acetyltransferases
-
JNK Mitogen-Activated Protein Kinases
-
OGA protein, human
-
Hyaluronoglucosaminidase