Site-specific O-GlcNAc MAbs: New Tools for Glycoproteomics

DESCRIPTION (provided by applicant): O-glycosylation of serine and threonine of nuclear and cytoplasmic proteins by a single beta-N- acetyl-D-glucosamine moiety (O-GlcNAc) is a ubiquitous post-translational modification that is highly dynamic and fluctuates in response to cellular stimuli. O-GlcNAc often competes with protein phosphorylation, and these two modifications have extensive crosstalk in the regulation of signaling, transcription, and the functions of oncogenes and tumor suppressors. Diabetes, thebiological driver for this application, is a prime example of a disease where increased levels of insulin resistance, inhibition of the anti-apoptotic action of insulin, alteration of circulating O-GlcNAc is known to disrupt normal signaling, and has beenassociated with the induction of adipocytokine levels, deregulation of gluconeogenesis, and modulation of insulin gene transcription. We will utilize a new immunogen strategy to develop site-specific O-GlcNac antibodies to four sites of O-GlcNAc modification on three proteins that play a role in signaling suppression in diabetes. Consequently, if we are successful, the mAbs generated in this initial study will have an immediate impact on diabetes research in addition to providing a test system for this strategy. We predict that at the end of phase 1, we will have demonstrated a strategy that will allow GlycoScientific to produce a wide range of O-GlcNAc site-specific MAbs, at a known and contained cost. We feel that these will have far reaching implications in disease research. PUBLIC HEALTH RELEVANCE: O-glycosylation of serine and threonine of nuclear and cytoplasmic proteins by a single beta-N- acetyl-D-glucosamine moiety (O-GlcNAc) is a ubiquitous post-translational modification that is highlydynamic and fluctuates in response to cellular stimuli. O-GlcNAc often competes with protein phosphorylation, and these two modifications have extensive crosstalk in the regulation of signaling, transcription, and the functions of oncogenes and tumor suppressors. Diabetes, the biological driver for this application, is a prime example of a disease where increased levels of insulin resistance, inhibition of the anti-apoptotic action of insulin, alteration of circulating O-GlcNAc is known to disrupt normal signaling, and has been associated with the induction of adipocytokine levels, deregulation of gluconeogenesis, and modulation of insulin gene transcription. We will utilize a new immunogen strategy to develop site-specific O-GlcNac antibodies to four sitesof O-GlcNAc modification on three proteins that play a role in signaling suppression in diabetes. Consequently, if we are successful, the mAbs generated in this initial study will have an immediate impact on diabetes research in addition to providing a test system for this strategy.