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New tools for studying O-GlcNAc glycosylation and chondroitin sulfate proteoglycans and studies on the roles of O-GlcNAc glycosylation on the transcription factor CREB

Citation

Clark, Peter Michael (2011) New tools for studying O-GlcNAc glycosylation and chondroitin sulfate proteoglycans and studies on the roles of O-GlcNAc glycosylation on the transcription factor CREB. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:01132011-095304695

Abstract

The addition and removal of the monosaccharide N-acetyl-D-glucosamine (GlcNAc) to serine and threonine residues of proteins has emerged as a critical regulator of cellular processes. However, studies of O-GlcNAc in such complex systems as the brain have been limited, in part due to the lack of tools. Here we report the development of new tools for studying O-GlcNAc, and the application of these and other tools for studying the roles of O-GlcNAc in the brain. Working from a previously established chemoenzymatic method, we designed an isotopic labeling strategy for probing the dynamics of O-GlcNAc glycosylation using quantitative proteomics. With this tool, we show that O-GlcNAc is dynamically modulated on specific proteins by excitatory stimulation of the brain in vivo. Separately, we improved this chemoenzymatic strategy by integrating [3+2] azide-alkyne cycloaddition chemistry to attach biotin and fluorescent tags to O-GlcNAc residues. These tags allow for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc modified proteins. With this strategy, we identified over 146 novel glycoproteins from the mammalian brain. The transcription factor cAMP-response element binding protein (CREB) is critical for numerous functions in the brain, including neuronal survival, neuronal development, synaptic plasticity, and long-term memory. We show that CREB is highly glycosylated in the brain and discover new glycosylation sites on CREB in neurons. One of these sites is dynamically modulated and is important for regulating CREB. Removal of this glycosylation site alters CREB-mediated functions in vitro and in vivo. These studies are the first demonstration that O-glycosylation at a specific site on a specific protein is critical for neuronal function and behavior. Chondroitin sulfates (CS) are sulfated linear polysaccharides important in neuronal development and viral invasion. Depending on their sulfation patterns, CS molecules differ dramatically in their functions. We developed a computational method to model the structure and function of CS. Using this approach, we show that different CS tetrasaccharides have distinct solution structures. We also modeled the CS binding site on a variety of proteins and discovered that CS may be important in modulating protein-protein interactions.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:glycosylation; post-translational modifications; chemical biology; neurobiology
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Awards:The Herbert Newby McCoy Award, 2011
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Hsieh-Wilson, Linda C.
Thesis Committee:
  • Dougherty, Dennis A. (chair)
  • Dervan, Peter B.
  • Kennedy, Mary B.
  • Hsieh-Wilson, Linda C.
Defense Date:21 December 2010
Funders:
Funding AgencyGrant Number
National Institutes of Health5T32 GM07737
Howard Hughes Medical InstituteUNSPECIFIED
Record Number:CaltechTHESIS:01132011-095304695
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:01132011-095304695
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6223
Collection:CaltechTHESIS
Deposited By: Peter Clark
Deposited On:03 Jun 2014 23:26
Last Modified:22 Aug 2016 21:10

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