Abstract O-GlcNAcylation is an essential post-translational modification, the complete loss of which results in lethality. Despite modifying thousands of nucleocytoplasmic proteins, O-GlcNAc is controlled by just two enzymes: O-GlcNAc transferase (OGT), which adds the modification, and O-GlcNAcase (OGA), which removes it. Disruptions in O-GlcNAc homeostasis, such as an imbalanced OGT/OGA ratio or aberrant O-GlcNAc levels, are implicated in a wide range of human diseases, including Alzheimer’s disease, cancer, intellectual disability, and diabetes. As such, O-GlcNAc and its regulatory enzymes represent valuable therapeutic targets. However, current tools do not permit informative, large-scale drug or genetic screening, hindering the development of O-GlcNAc-targeted therapies. Here, we present a triple fluorescence stem cell sorting approach in which both endogenous OGT and OGA are tagged with spectrally distinct fluorescent proteins and O-GlcNAc levels can be quantified. We demonstrate that this system faithfully reports disruptions in O-GlcNAc homeostasis. Furthermore, we show that the O-GlcNAc feedback regulation is not solely dependent on O-GlcNAc levels, indicating a role for non-catalytic functions of OGT and OGA. Overall, we provide a high-throughput screening platform that enables reliable and quantitative measurement of O-GlcNAc homeostasis, paving the way for identifying compounds and pathways that target protein O-GlcNAcylation.
Yuan et al. (2025) studied this question.