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Title: Chemical biology tools for structure-function studies on heparan sulphates : decoding specificity in FGF signalling
Author: Sery, E. C.
ISNI:       0000 0004 6422 6471
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Fibroblast growth factors (FGFs) regulate a broad spectrum of biological processes and aberrant FGF signalling has been linked to many disorders and diseases. Heparan sulphate (HS) is a highly sulphated glycosaminoglycan, which regulates the activity of its protein ligands, mainly through anionic interactions. The interactions between HS, FGF and FGF receptors (FGFRs) depend on the size and the spatial distribution of the anionic moieties of the saccharides. To test the effects of these on FGF signalling, the activities of well-defined HS oligosaccharides were screened in bioassays. The aim of this thesis was to study the structural determinants which underpin the specificity/selectivity in HS-FGF-FGFR interactions. Two approaches were initially explored to generate suitable saccharides for screening. Firstly, the preparation of libraries of HS oligosaccharides was attempted by enzymatic digestion of porcine mucosal HS using heparinase III. This was followed by the sequential fractionation using size exclusion chromatography, strong anion-exchange and cetyltrimethylammonium strong anion-exchange chromatography. However, with regards to preparing saccharides of sufficient purity and amounts, this was found to be a limiting factor for bioassay screening. Secondly, the dimeric modular synthesis of HS octasaccharides was attempted by means of in-solution multi-step synthesis. The synthesis of a small oligosaccharide library was partially successful and resulted in novel synthetic routes for generation of pentasaccharide precursors, but not sulphated saccharides for screening. An alternative strategy was taken to exploit nineteen existing fully defined chemically and chemoenzymatically prepared oligosaccharides. Screening in bioassays of specific FGF ligands, using BaF3 cells expressing single receptors, showed that both general and specific structural changes, as subtle as a single additional sulphate moiety, significantly impact the ability of saccharides to support FGF signalling in a ligand-receptor complex-dependent manner. The size of the saccharides, the presence/absence of 2-O-, 6-O- and N-sulphate groups were shown to be crucial factors for functional activity. Most notably, the presence of a single 3-O-sulphate group towards the non-reducing end of the saccharides was related to high levels of activation for FGF1-FGFR1c, FGF1-FGFR2b and FGF7-FGFR2b and was demonstrated for the first time. Overall, this thesis provides new evidence concerning structural determinants in HS that modulate FGF signalling, using fully defined saccharides for the first time. The results support the view that significant selectivity is involved, and contribute to a better fundamental understanding of how HS modulates FGF-FGFR-HS complex formation. Ultimately, this information could be relevant for the development of better targeted HS saccharide-based therapeutics.
Supervisor: Turnbull, J. E. ; Yates, E. A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral