Screening Point Mutations of the O‐GlcNAc Hydrolase Enzyme, OGA, to Investigate Potential Regulation

The O‐GlcNAcase enzyme (OGA) hydrolyzes the glycosidic linkage between a GlcNAc and the hydroxyl group on serine and threonine residues on target proteins. OGA is the sole enzyme responsible for removing this cytosolic, post‐translational modification which makes the enzyme itself a likely target for post‐translation modification. Several groups using proteomic analysis have identified phosphorylation sites on OGA, but the functional consequences of these changes have not been fully investigated. In an effort to characterize potential sites of regulation, a plasmid containing 6xHis‐OGA was altered by site‐directed mutagenesis to produce phosphomimetics by substituting an aspartic acid or glutamic acid for a serine or threonine. These mutant OGA enzymes were expressed in E.coli and purified using a two‐step purification of Ni 2+ ‐metal affinity followed by size exclusion. Enzyme activities were investigated using a paranitrophenol‐GlcNAc substrate and were compared to the wild‐type enzyme. The goal of this research is to better understand the function and regulation of OGA, and the role that this important enzyme plays in signaling cascades. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .