The broad goal of this competing renewal focuses on studying carbohydrate:protein, as well as protein:protein non-covalent interactions. In particular, we propose to investigate the binding and interaction of glycosaminoglycans (GAG) with chemokine ligands of the N-terminal G-protein coupled receptors (GPCR), CCR2 and CCR5, as well as exploring possible ternary complexes of all three using Q-TOF/Ion Mobility Mass Spectrometry. Binding of the GAG with chemokine will be explored via incubation of various GAG libraries with the chemokines of CCR2 and CCR5. Specific binders will be identified using our GAG compositional analysis. Chemokines will be expressed and purified in house. Collisional cross section measurements will be made for any of the non-covalent complexes identified, thus providing information about conformation change upon binding. Additional Surface Plasmon resonance and MS kinetic binding studies along with thermal calorimetry will provide important information about the binding strength. The intent is that a detailed understanding of these interactions will allow for effective targeting of the chemokine system for therapeutic benefit. The major areas proposed are interwoven in such a way that methods development, biotechnology and application to biologically relevant systems are the cohesive elements that bind the proposed research together.

Public Health Relevance

This proposal involves the analysis of non-covalent complexes and interactions of chemokine proteins with sulfated carbohydrates, as well as possible ternary interactions with the N-terminal G-protein coupled receptors. Ion Mobility mass Spectrometry will be used to measure these interactions and to obtain collisional cross section measurements of the binding partners.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047356-19
Application #
8197802
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
1992-05-01
Project End
2013-08-31
Budget Start
2011-12-01
Budget End
2013-08-31
Support Year
19
Fiscal Year
2012
Total Cost
$247,684
Indirect Cost
$76,166
Name
University of California Davis
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Seo, Youjin; Andaya, Armann; Bleiholder, Christian et al. (2013) Differentiation of CC vs CXC chemokine dimers with GAG octasaccharide binding partners: an ion mobility mass spectrometry approach. J Am Chem Soc 135:4325-32
Wei, Wei; Miller, Rebecca L; Leary, Julie A (2013) Method development and analysis of free HS and HS in proteoglycans from pre- and postmenopausal women: evidence for biosynthetic pathway changes in sulfotransferase and sulfatase enzymes. Anal Chem 85:5917-23
Seo, Youjin; Andaya, Armann; Leary, Julie A (2012) Preparation, separation, and conformational analysis of differentially sulfated heparin octasaccharide isomers using ion mobility mass spectrometry. Anal Chem 84:2416-23
Ninonuevo, Milady R; Leary, Julie A (2012) Ion mobility mass spectrometry coupled with rapid protein threading predictor structure prediction and collision-induced dissociation for probing chemokine conformation and stability. Anal Chem 84:3208-14
Holley, Rebecca J; Deligny, Audrey; Wei, Wei et al. (2011) Mucopolysaccharidosis type I, unique structure of accumulated heparan sulfate and increased N-sulfotransferase activity in mice lacking ?-l-iduronidase. J Biol Chem 286:37515-24
Seo, Youjin; Schenauer, Matthew R; Leary, Julie A (2011) Biologically Relevant Metal-Cation Binding Induces Conformational Changes in Heparin Oligosaccharides as Measured by Ion Mobility Mass Spectrometry. Int J Mass Spectrom 303:191-198
Wei, Wei; Ninonuevo, Milady R; Sharma, Anish et al. (2011) A comprehensive compositional analysis of heparin/heparan sulfate-derived disaccharides from human serum. Anal Chem 83:3703-8
Jen, Connie H; Leary, Julie A (2010) A competitive binding study of chemokine, sulfated receptor, and glycosaminoglycan interactions by nano-electrospray ionization mass spectrometry. Anal Biochem 407:134-40
Schenauer, Matthew R; Leary, Julie A (2009) An Ion Mobility-Mass Spectrometry Investigation of Monocyte Chemoattractant Protein-1. Int J Mass Spectrom 287:70-76
Meissen, John K; Sweeney, Matthew D; Girardi, Matthew et al. (2009) Differentiation of 3-O-sulfated heparin disaccharide isomers: identification of structural aspects of the heparin CCL2 binding motif. J Am Soc Mass Spectrom 20:652-7

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