Project Summary/Abstract
O-GlcNAc is a single N-acetylglucosamine coupled to serine and threonine residues of nuclear and cytoplasmic
proteins. Analogous to phosphorylation, O-GlcNAc signaling is dynamic, rapidly added and removed from
proteins in a site-specific manner in response to cellular perturbations and extracellular cues. Because both
modifications occur on the same residues it is hypothesized that there is a functional crosstalk between O-
GlcNAc and phosphorylation, where one may affect deposition or removal the other. Unlike phosphorylation,
however, which is catalyzed by over 500 kinases and roughly 300 phosphatases, the mammalian genome only
encodes a single O-GlcNAc transferase (OGT) and a single hydrolase (OGA). While many kinases recognize
specific amino acid sequences in their substrates, the determinants guiding OGT are unclear and likely manifold.
This intracellular glycosylation is implicated in nearly every cellular process from gene expression and signal
transduction to cell division and differentiation. Despite the ubiquitous nature of this post-translational
modification in health and disease, the specific functions of OGT and the basic principles of O-GlcNAc signaling
remain almost entirely elusive. This gap in our knowledge has been largely due to the major lack of tools and
technologies available to study O-GlcNAc signaling or perturb the essential OGT.
Here, we aim to uncover the basic principles of OGT and O-GlcNAc signaling, and their role in transcriptional
regulation of cellular differentiation. We have recently developed a highly sensitive and specific enrichment
reagent to analyze O-GlcNAc-modified peptides from cells and tissues by mass spectrometry. Using these new
anti-O-GlcNAc antibodies we will elucidate the global, site-specific temporal dynamics of O-GlcNAc signaling
during the transition from totipotency to naïve and primed pluripotency. Combined with phosphoprotemic profiling
of the same samples, we will monitor for crosstalk between these two post-translational modifications. To gain
insight into how OGT targets its diverse array of substrates, we will deconvolute the extensive OGT interactome
employing chemical crosslinking and biochemical fractionation, followed by mass spectrometric analysis. To
explore how OGT uses adaptor proteins to targets substrates, we will use an innovative approach, degrading
specific OGT interacting proteins and assessing changes in downstream O-GlcNAc signaling using our new
quantitative glycoproteomic approach. Integrating these two research programs, we will create a holistic, high-
resolution understanding of the principles of O-GlcNAc signaling. The MIRA mechanism will not only enable the
investigation of basic O-GlcNAc biology, but will provide the flexibility to conduct data-driven follow-up, functional
analyses of dynamic O-GlcNAc/crosstalk sites and distinct OGT complexes, and their role in transcriptional
regulation of some of the earliest development decisions.
