Boyce, MichaelCox, Nathan James2018-05-312019-05-012018https://hdl.handle.net/10161/16884<p>O-linked β-N-acetylglucosamine (O-GlcNAc) exerts myriad effects on protein localization, activation, inhibition, stability, conformational changes, or degradation. However, the biochemical effects of O-GlcNAc on the vast majority of substrates is unknown. Recently, we and others have shown that several coat protein complex II (COPII) components including SEC23A, SEC24C, and SEC31A are O-GlcNAcylated. The COPII coat complex consists of protein coated carriers that mediate secretory trafficking from the endoplasmic reticulum. To determine the effects of O-GlcNAc on COPII we used a combination of chemical, biochemical, cellular and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. We demonstrate that individual O-GlcNAcylation sites of SEC23A are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of cranio-lenticulo-sutural dysplasia (CLSD). </p><p> Next, we developed a proteomic workflow to address the challenges of identifying and quantifying novel changes in substrate O-GlcNAcylation in response to a stimulus. Current methods of O-GlcNAcome enrichment suffer from issues with specificity, reproducibility, time-resolution, or require specialized hardware. We developed a novel, unbiased glycoproteomics workflow to survey global changes in O-GlcNAc in response to stimuli. Our approach utilizes both stable isotope labeling with amino acids in cell culture (SILAC) for quantitation and metabolic labeling of O-GlcNAc for enrichment. Using our glycoproteomics workflow we examined the effects of brefeldin A (BFA), a fungal metabolite that disrupts vesicle trafficking, and cytokine deprivation on a pro-B cell line. We identified changes in the O-GlcNAcylation of Coatomer subunit gamma-1 (COPG) a coat protein complex I (COPI) component in response to BFA. Interestingly, COPI mediates traffic from the Golgi to the ER, as well as within the Golgi, and is the specific target of BFA. O-GlcNAcylation of COPI components may have effects similar to O-GlcNAc on COPII, possibly altering membrane binding or the trafficking of specific cargo. </p><p> Finally, we identified a candidate O-GlcNAc-mediated binding part of SEC23A using a combination chemical biology tools and mass spectrometry (MS). We identified ankycorbin, a vertebrate specific protein with no known function, as the candidate SEC23A O-GlcNAc-mediated binding partner. However, our attempts to validate this interaction were inconclusive. </p><p>Overall, this work examines the role of O-GlcNAc in the vertebrate secretory pathway. We demonstrate the effects of O-GlcNAc on SEC23A in the COPII pathway and identify a potentially novel method of COPI protein trafficking regulation via the O-GlcNAcylation of COPG.</p>PharmacologyCellular biologyBiochemistryChemical biologyCOPICOPIIO-GlcNAcDissertation