This dissertation research explores the molecular aspects of interactions between pro-inflammatory chemokines and the cell surface glycosaminoglycan heparan sulfate. Our goal was to identify the specific attributes of both chemokines and heparan sulfate required for their interactions.
In chapter two, we identified the molecular compositions of heparan sulfate oligosaccharides which bind the CCR2 chemokines MCP-1, -2, and -3. All three chemokines bound to octasaccharides, but not shorter length saccharides. Octasaccharides bearing between 5 and 10 sulfate groups and 0 to 2 acetyl groups were observed to interact with the different chemokines. As indicated by previous studies, increasing sulfation augmented the interactions. Interestingly, our work also identified acetylation as a second factor facilitating binding. The stoichiometry of each formed noncovalent complex was also defined.
In chapter three, we investigated MCP-1: GAG complexes via ion mobility mass spectrometry. Here we uncovered evidence suggesting that in the 2:1 MCP-1: Arixtra complex, the glycosaminoglycan Arixtra interacts with both monomeric subunits of the MCP-1 dimer. We also identified fundamental differences in response to collisional activation between the MCP-1 homodimer, and the MCP-1 dimer: Arixtra complex.
The research described in chapter four focuses on the development of a sequence analysis method for heparan sulfate. The developed method is applied to purified isomers, and in the future will be applicable to the analysis of chemokine-binding heparan sulfate. We showed that the combination of mass spectrometry and nuclear magnetic resonance can be a powerful and sensitive combination in heparan sulfate sequence analysis. Perhaps most significantly, we also showed that ion mobility-mass spectrometry is capable of differentiating heparan sulfate isomers without prior knowledge of structural differences. This last observation may allow the development of extremely sensitive and rapid screening of heparan sulfate libraries by ion mobility-mass spectrometry.
A synopsis and future directions are presented in chapter five. Finally, two unique mass spectrometry-based methods for probing large macromolecular noncovalent complexes (as applied to eIF3) are presented as appendices.
|Advisor:||Leary, Julie A.|
|Commitee:||Lagarias, J. Clark, Stahlberg, Henning|
|School:||University of California, Davis|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- California|
|Source:||DAI-B 71/03, Dissertation Abstracts International|
|Subjects:||Analytical chemistry, Biochemistry|
|Keywords:||Chemokines, Heparan sulfate, Mass spectrometry, Protein-carbohydrate, Protein-protein|
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