A Genetically Encoded Assay System to Quantify <i>O</i>‐GlcNAc Transferase (OGT) Activity in Live Cells

Abstract O ‐GlcNAc transferase (OGT) catalyzes O ‐GlcNAcylation of many nucleocytoplasmic proteins and plays important roles in regulating diverse cellular functions. Dysregulation of OGT is implicated in various diseases, including cancers and neurodegeneration. Despite its vital roles, little is known about how this enzyme is regulated within cells in part because no current assays directly report on its activity within cells. Here we describe a genetically encoded reporter of cellular OGT glycosyltransferase activity by exploiting the transferase‐dependent proteolytic activity of OGT on host cell factor‐1 (HCF‐1). The reporter comprises sites at which OGT cleaves HCF‐1, which are flanked by two different fluorescent proteins that are linked to either nuclear export or import sequences. OGT‐catalyzed cleavage of this construct leads to separation and independent localization of these two fluorescent proteins. By quantifying their nuclear and cytoplasmic distributions, OGT activity can be measured. We validated this OGT cellular activity reporter (CAR) system using known modulators of the O ‐GlcNAc pathway and assessed the effects of several metabolites on OGT activity. Analyses of the dose‐ and time‐dependent effects of these OGT modulators illustrate the sensitivity and precision of this OGT‐CAR strategy. We envision this OGT‐CAR system will aid in discovering and characterizing modifiers of OGT activity.