Versatile O-GlcNAc Transferase Assay for High-Throughput Identification of Enzyme Variants, Substrates, and InhibitorsClick to copy article linkArticle link copied!
- Eun J. Kim
- Lara K. Abramowitz
- Michelle R. Bond
- Dona C. Love
- Dong W. Kang
- Hans F. Leucke
- Dae W. Kang
- Jong-Seog Ahn
- John A. Hanover
Abstract
![Abstract Image](/cms/10.1021/bc5001774/asset/images/medium/bc-2014-001774_0007.gif)
The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.
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