O-GlcNAc dynamics and the OGT interactome in variants causal for X-linked intellectual disability

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PROJECT SUMMARY X-linked intellectual disability (XLID) affects approximately 1 in 1,000 males. Recently, we have discovered mutations in the gene encoding O-GlcNAc transferase (OGT) that are causal for XLID. These mutations generate variants with amino acid substitutions in the TPR domains of OGT that are thought to be involved in protein-protein interactions. The modification of Ser/Thr residues of nuclear and cytosolic proteins by the addition of a single glycan (O-linked N-acetylglucosamine, O-GlcNAc) by OGT impacts the stability, localization, activity, and protein-protein interactions of many nuclear and cytosolic proteins. Similar to phosphorylation, thousands of nuclear and cytosolic proteins in mammals are modified by O-GlcNAc. Unlike phosphorylation, which is mediated by a plethora of kinases and a smaller set of phosphatases, O- GlcNAcylation results from the activity of a single transferase (OGT) and can be removed by a single hydrolase (O-GlcNAc hydrolase, OGA). It has been suggested that the O-GlcNAc modification is a regulatory modification in that it has been demonstrated to be globally inducible and dynamic on a small subset of proteins examined. We hypothesize that the TPR variants observed in XLID are altering O-GlcNAc dynamics and/or the OGT interactome. The specific aims leverage our expertise in O-GlcNAc biology and the enzymology of OGT along with our innovative labeling, enrichment, and mass spectrometry-based approaches for site-mapping and interactome identification applied to neural lineages derived from normal or Cas9- engineered human embryonic stem cells. In Aim 1, we develop a novel method for examining the dynamics of both site-specific O-GlcNAc modification and the modified protein and couple this with enrichment strategies and tandem mass spectrometry approaches to define O-GlcNAc cycling rates in a cell type and XLID genotype dependent manner. In Aim 2, we define the OGT interactome using classical co-immunoprecipitation as well as proximity labeling approaches in a cell type and XLID genotype dependent manner. The successful completion of these aims will not only benefit the O-GlcNAc biology community but more importantly will identify specific OGT targets and binding partners impacted by XLID for future detailed hypothesis-driven studies.