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Discovering Biological Roles of Glycosaminoglycans and Protein O-GlcNAcylation Using Chemical Tools

Citation

Griffin, Matthew Everett (2017) Discovering Biological Roles of Glycosaminoglycans and Protein O-GlcNAcylation Using Chemical Tools. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9610XCH. http://resolver.caltech.edu/CaltechTHESIS:06042017-183300784

Abstract

Carbohydrates surround nearly every cell in the human body. Glycosaminoglycans like chondroitin sulfate and heparan sulfate on the cell surface regulate protein ligand engagement and receptor activation to control a variety of biological processes including development, angiogenesis, and neuronal growth. These polysaccharides exert activity through protein binding to their diverse chemical structures. Therefore, the development of methods to tailor glycosaminoglycan populations at the cell surface with defined structures could provide novel approaches to control biological activity. Herein, two new methods to engineer the cell surface glycocalyx with known glycosaminoglycans are reported. Together, these methods provide complementary short- and long-term approaches to change carbohydrate structures at the cell surface and guide neuronal growth and stem cell differentiation. It is also critical to identify unknown protein-carbohydrate interactions that underlie biological phenomena. Studies delineating novel GAG interactions with an orphan receptor and related soluble ligands are reported herein as well as work towards understanding the biological functions of these newly discovered interactions. These results showcase the utility of chemical biology and biochemical tools to discover and modulate various GAG-protein interactions in diverse biological systems.

Within the cell, thousands of proteins are modified by O-GlcNAc glycosylation, a process that is uniquely catalyzed by a single transferase and hydrolase pair unlike many other post-translational modifications. O-GlcNAcylation functions in many biological contexts including transcription, translation, proteostasis, and metabolism. Key to understanding its effects on these physiological phenomena is the discovery of O-GlcNAc modification sites. However, due to a number of technical challenges, O-GlcNAc proteomics has not progressed nearly as quickly as phosphoproteomics. Thus, developing new methods to enrich O-GlcNAcylated substrates and map modification sites is critical to unravel the myriad functions of O-GlcNAc. Herein, a labeling approach using a chemically cleavable tag is reported as an improved method to capture and release O-GlcNAcylated substrates. Unlike other methods, the cleavable Dde tag is quantitatively removed under mild, neutral conditions and leaves a minimal residual tag on the O-GlcNAcylated peptide to be analyzed. Moreover, the Dde linker outcompetes a previously used UV-cleavable tag both at the protein and peptide enrichment levels. Together, these results highlight the potential usefulness of this method to illuminate novel roles of O-GlcNAcylation in diverse systems.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:chemical biology, glycosaminoglycans, chondroitin sulfate, heparan sulfate, glycan engineering, receptor deorphanization, O-GlcNAc, O-GlcNAcylation, site mapping, proteomics
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Hsieh-Wilson, Linda C.
Thesis Committee:
  • Dougherty, Dennis A. (chair)
  • Ismagilov, Rustem F.
  • Sternberg, Paul W.
  • Hsieh-Wilson, Linda C.
Defense Date:1 June 2017
Funders:
Funding AgencyGrant Number
National Science Foundation Graduate Research FellowshipDGE-1144469
National Institutes of Health (NIH)R01-GM093627
National Institutes of Health (NIH)R01-GM084724
Record Number:CaltechTHESIS:06042017-183300784
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:06042017-183300784
DOI:10.7907/Z9610XCH
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.chembiol.2015.12.007DOIArticle adapted for Chapter 1
http://dx.doi.org/10.1021/ja5005174DOIArticle adapted for Chapter 2
http://dx.doi.org/10.1002/anie.201409258DOIArticle adapted for Chapter 2
http://dx.doi.org/10.1039/C6MB00138FDOIArticle adapted for Chapter 5
ORCID:
AuthorORCID
Griffin, Matthew Everett0000-0001-9549-4418
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10267
Collection:CaltechTHESIS
Deposited By: Matthew Griffin
Deposited On:07 Jun 2017 00:00
Last Modified:23 Jan 2019 18:36

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