Versatile <i>O</i>‑GlcNAc Transferase Assay for High-Throughput Identification of Enzyme Variants, Substrates, and Inhibitors

The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single <i>N</i>-acetylglucosamine (<i>O</i>-GlcNAcylation) is critical for many important cellular processes. Cellular <i>O</i>-GlcNAc levels are highly regulated by two enzymes: <i>O</i>-GlcNAc transferase (OGT) is responsible for GlcNAc addition and <i>O</i>-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