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.