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).
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.
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.
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.
","abstract_html":"<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>","abstract_has_math":false,"creators":["Cox, Nathan James"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Boyce, Michael"],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018","date_published":"2018","updated_at":"2026-07-24T02:07:21Z","subjects":["Pharmacology","Cellular biology","Biochemistry","Chemical Biology","COPI","COPII","O-GlcNAc"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/10161/16884","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Boyce, Michael"]},{"key":"dc:creator","label":"Author","values":["Cox, Nathan James"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2018-05-31T21:14:12Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2019-05-01T08:17:08Z"]},{"key":"dc:date.issued","label":"Date","values":["2018"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Pharmacology","Cellular biology","Biochemistry","Chemical Biology","COPI","COPII","O-GlcNAc"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10161/16884"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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).
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.
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.
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.
"]},{"key":"dc:title","label":"Title","values":["Role of O-GlcNAc in the Vertebrate Secretory Pathway"]}]}],"canonical_facts":{"dc:contributor.advisor":["Boyce, Michael"],"dc:creator":["Cox, Nathan James"],"dc:date.accessioned":["2018-05-31T21:14:12Z"],"dc:date.available":["2019-05-01T08:17:08Z"],"dc:date.issued":["2018"],"dc:description.abstract":["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).
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.
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.
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.
"],"dc:identifier.uri":["https://hdl.handle.net/10161/16884"],"dc:subject":["Pharmacology","Cellular biology","Biochemistry","Chemical Biology","COPI","COPII","O-GlcNAc"],"dc:title":["Role of O-GlcNAc in the Vertebrate Secretory Pathway"],"dc:type":["Dissertation"]},"updated_at":"2026-07-24T02:07:21Z"}