Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.706969
Title: Specificities of the interaction of fibroblast growth factor and heparan sulfate
Author: Li, Yong
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2015
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Abstract:
More than 883 extracellular proteins that bind heparan sulfate (HS) and heparin have been identified whose activities are regulated by their interactions with these polysaccharides. FGFs are heparin-binding proteins (the physiological ligand is HS) and the interactions of FGF with HS determine their transport between cells and the assembly of signalling complexes with their cognate receptor tyrosine kinases, FGFRs. The FGF family has expanded from two or three FGF ligands in the worm C. elegans and the fly Drosophila to 18 FGFs in vertebrates and mammals, which is directly linked to the more complex specifications required in the development for increasingly complex body parts. However, the level of specificity of the interaction of FGFs with HS is still debated. Previous work generally focused on just one or two FGFs and a limited repertoire of sugar structures, so a systematic investigation of the interaction of FGFs with heparin/HS is required to determine at what level, if any, there is specificity at the molecular level underlying these interactions. The strategy of this work was to use the evolutional relationship of the FGF family as a defined system to explore the specificity of interactions of FGFs and heparin/HS. Six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, and FGF20) from 4 subfamilies have been produced and purified as recombinant proteins, to investigate the interaction between FGFs and HS from two different perspectives. The polysaccharide structure required for binding to the FGFs was determined by differential scanning fluorimetry (DSF) using a library of chemically modified heparins and model glycosaminoglycans. The heparin binding sites on the FGFs were then identified by a lysine selective technique called ‘protect and label’. For systemic analysis of the interactions, all of the results obtained have been mapped on the FGF evolutionary tree deduced amino acid sequence alignment, alongside previous work. This shows a clear pattern: FGF members from the same subfamily have a similar preference for binding particular subsets of HS/heparin structures and model glycosaminoglycans, and share similar secondary heparin binding sites on their surface. In contrast, FGFs from different subfamilies have a more divergent preference for binding structures in the polysaccharide and secondary binding sites on their surface. The secondary heparin binding sites (HBS) of FGF2 were mutated to begin the characterization of their functions. The properties of the mutants of FGF2’s secondary HBSs (HBS2, HBS3) were measured in terms of their preference for binding structures in heparins, their ability to stimulate the phosphorylation of p42/44MAPK and cell proliferation. FGF2 (HBS2) mutant was found to be distinct to wild-type only in its interactions with low sulfated heparins where mutant FGF2 (HBS2) exhibited a stronger preference for N-sulfated heparin. For mutant FGF2 (HBS3), a larger sugar structure was required for binding than wild type FGF2. Finally, since both lysine and arginine residues in HBSs contribute to the interaction between protein and polysaccharide, an arginine targeted protect and label method was developed. Phenylglyoxal (PGO) was successfully used in the protection step and was demonstrated to be capable of achieving full arginine labelling. However, arginine labelling with 4-azidophenylglyoxal (APG) suffered from a ring expansion side reaction and this second step still needs to be optimized. Overall the thesis demonstrates that there is specificity in the interaction of FGFs and glycosaminoglycans. Although this is not a simple one-to-one code, it has clearly been subjected to the same natural selection that led to the expansion and diversification of the FGF family, the specificities of FGFs for particular isoforms of the FGFRs and selective activities of the FGF family in specifying the different structures and organs of the mammalian body.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.706969  DOI: Not available
Keywords: QD Chemistry ; QR Microbiology
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