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.
This proposal is focused on understanding the role of the O-GlcNAc transferase (OGT) protein in X-linked intellectual disability (XLID). This work builds on our recent findings that there are mutations in the gene coding for OGT that are causal for XLID. This work lays the groundwork for being able to perform target- specific, hypothesis-driven research into the causes of OGT-mediated XLID by developing and/or applying technologies to uncover the impact of OGT variants on O-GlcNAc cycling and interacting proteins.
