Novel Tools to Characterize OGT-XLID - Project Summary Thousands of nuclear, cytosolic, and mitochondrial proteins are dynamically modified and regulated by monosaccharides of O-linked b-N-acetylglucosamine (O-GlcNAc). The cycling of O-GlcNAc is regulated by the concerted actions of enzymes encoded by just two genes: the O-GlcNAc transferase (OGT) and the O- GlcNAcase that add and remove O-GlcNAc, respectively. OGT has an additional critical activity; cleavage and activation of the epigenetic regulator Host Cell Factor 1 (HCF1). Underscoring its importance, OGT plays key roles in reproductive health (for example, gestational diabetes and placental function), transformation and tumorigenesis, cardiovascular disease, neurodegeneration, and mammalian development. Despite its essential roles in development and disease, our ability to interrogate OGT function remains severely limited. This gap in technology is particularly urgent given that over 25 pathogenic OGT mutations have been identified in individuals with X-linked intellectual disability (OGT-XLID; OMIM #300255), a neurodevelopmental disorder characterized by cognitive impairment, developmental delay, and behavioral abnormalities. Existing assays for measuring OGT enzymatic activity are low-throughput, technically complex, and often rely on specialized or commercially unavailable probes. These limitations have restricted their use in both basic and translational research, creating a significant barrier to progress. As a result, our understanding of OGT function, regulation, and substrate targeting remains limited. To address this critical need, we propose to develop and validate AlphaLISA-based assays for high-throughput quantification of OGT glycosyltransferase and protease activities. This platform will reduce assay time and cost, eliminate the use of radioisotopes, and support automation. A central challenge in the development of a glycosyltransferase assay is the identification of O- GlcNAc-readers that bind glycopeptides/glycoproteins at high affinity, the product of the OGT glycosyltransferase reaction. These studies will leverage biolayer inferometry to identify ideal OGT substrate/O-GlcNAc reader pairs. In parallel, we will adapt existing reporter to generate an AlphaLISA protease assay. The catalytic parameters and sensitivity of OGT activity measured in Alpha-format will be benchmarked against gold-standard methods and validated in cell and tissue lysates. While these tools are expected to have broad applicability, our ultimate goal is to characterize OGT mutations associated with X-linked intellectual disability (XLID). We anticipate that these assays will enable the regular interrogation of OGT enzymatic function, facilitating routine characterization of XLID models and detection of aberrant OGT activity in patient-derived samples. Ultimately, this work will accelerate the diagnosis and molecular characterization of XLID, a devastating neurodevelopmental disorder.
