PROJECT SUMMARY X-linked intellectual disability (XLID) affects approximately 1 in 500 males. Recently, we, and others, have discovered multiple unique missense mutations in the X-linked gene encoding O-GlcNAc transferase (OGT) that are causal for XLID. These mutations generate variants with amino acid substitutions in either the TPR domain or catalytic domain of OGT. The modification of Ser/Thr residues of thousands of nuclear and cytosolic proteins by the addition of a single glycan (O-linked N-acetylglucosamine, O-GlcNAc) by OGT can impact the stability, localization, activity, and protein-protein interactions of the modified proteins. Similar to phosphorylation, the O- GlcNAc modification is inducible and dynamic. However, unlike phosphorylation, which is mediated by hundreds of kinases and a smaller set of phosphatases, O-GlcNAc modification results from the activity of a single transferase (OGT) that can be removed by a single hydrolase (O-GlcNAc hydrolase, OGA). We have generated considerable published and preliminary data including biochemical characterization of several OGT-TPR variants, engineering of an isotope-based approach to quantitatively compare site-specific O-GlcNAc between samples and establish turnover rates, generation of Cas9-engineered RUES-1 stem cells harboring the XLID missense mutations, and defining the OGT TPR interactome including defining interactors, such as the epigenetic regulator TET2 and the synapse scaffolding protein PCLO, that show reduced interactions with XLID TPR variants. Based on our preliminary data, we will test the hypothesis that OGT-TPR variants have an altered interactome and define the impact of loss of interaction (Aim 1A). In Aim 1B, we will characterize novel XLID variants in the catalytic domain of OGT that, based on modeling and in cellulo expression studies, we hypothesize will be Km variants for the sugar nucleotide donor and perform genome wide screens to identify regulators of OGT expression that might be able to ameliorate the deficits of a catalytic variant. Finally, given the nearly identical and overlapping patient phenotypes for both TPR and catalytic domain XLID OGT variants, we will test the hypothesis that all variants share common downstream alterations, likely in gene regulation that may be modulated by TET2 and associated proteins (Aim 2) and/or synaptic activity modulated by PCLO and associated proteins (Aim 3) based on preliminary and published data. The successful completion of these aims, geared towards elucidating a mechanistic understanding of the impact of XLID OGT variants, will advance both the intellectual disability and O-GlcNAc fields.

PROJECT NARRATIVE This proposal is focused on elucidating the regulation and structure/function relationships of a single enzyme, OGT, involved in post-translational modification of thousands of protein substrates. This work builds on our recent discovery that mutations in the OGT gene are causal for X-linked intellectual disability. The proposed efforts will uncover a mechanistic understanding of the impact of the disease variants and lay the groundwork for being able to ameliorate consequences of the disability.