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Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase
Chia-Wei Hu1, Matthew Worth2, Dacheng Fan1
1Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Nature Chemical Biology
|October 24, 2017
Summary
GlcNAc electrophilic probes (GEPs) help characterize O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) substrate recognition. GEPs enable discovery of OGT residues and genuine substrates, advancing understanding of OGT-substrate binding mechanisms.
Area of Science:
- Biochemistry
- Glycobiology
- Molecular Biology
Background:
- O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is crucial for protein modification.
- The mechanism of OGT's recognition of diverse protein substrates remains largely uncharacterized.
Purpose of the Study:
- To develop and validate GlcNAc electrophilic probes (GEPs) for characterizing OGT-substrate recognition.
- To identify OGT residues critical for protein substrate binding and to discover novel OGT substrates.
Main Methods:
- Mass spectrometry
- X-ray crystallography
- Biochemical assays
- Radiolabeled kinetic assays
- In situ crosslinking using GEPs
Main Results:
- GEPs effectively report the impact of OGT mutations on substrate or sugar binding.
- Identified conserved OGT residues in the N-terminal domain involved in binding different protein substrates.
- Developed an in situ crosslinking method using GEPs to identify weakly or transiently interacting OGT substrates.
Conclusions:
- GEPs offer a powerful tool for dissecting OGT-substrate binding and recognition mechanisms.
- This approach facilitates the discovery of novel OGT substrates and the understanding of OGT's role in cellular processes.
