Project Summary/Abstract
This equipment supplement request is to fund a new, high-throughput, low sample volume sonicator to
efficiently lyse cells and nuclei, while shearing genomic DNA, to propel projects within R35 GM147554.
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 biochemical
fractionation, followed by mass spectrometric analysis. To explore how OGT uses adaptor proteins to
targets substrates, we are 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 requested high-throughput/low volume sonicator will greatly enable the
new and ongoing directions and approaches we are employing to study O-GlcNAc biology.
