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Title: Analysis of fibroblast growth factor-heparin interactions
Author: Xu, Ruoyan
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2012
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The functions of a large number (> 435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding controls their transport between cells and is required for the assembly of a high affinity signaling complex with the cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. In this thesis, a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18 and FGF-21) spanning five FGF sub-families were used to probe their specificities for HS/heparin at different levels: recombinant FGF proteins were expressed and purified and their biological activities tested in a DNA synthesis assay. Then, the proteins were tested for their heparin binding specificity using a variety of complementary approaches: 1. Measurement of the binding parameters of FGFs and a model heparin sugar in an optical biosensor or by microscale thermophoresis; 2. Identification of the heparin binding site (HBS) in the proteins using a Protect and Label strategy; 3. Determination of stability changes in FGFs when bound to different heparin sugars and related glycosaminoglycans employing differential scanning flurometry; 4. Measurement of the conformational changes in FGFs when binding to a variety of molar ratios of heparin and chemically modified heparins using synchrotron radiation circular dichroism (SRCD); 5. Measure directly the binding of FGF-2 to cellular HS using nanoparticles (NPs) to label the FGF-2 and transmission electron microscopy. For interaction with heparin, the FGFs have KDs varying between 38 nM (FGF-18) and 620 nM (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 M-1s-1, FGF-9, 130,000 M-1s-1). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9 and FGF-18 differs in its size and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. SRCD revealed conformational changes in FGFs induced by binding to heparin and the changes were distinct at different heparin concentrations. Moreover, there was evidence that the conformational changes of FGFs differed with chemically modified heparins, indicating that the conformational change caused by binding to heparin is related to the sulfation pattern. At the cellular level, FGF-2 labeled with nanoparticles allowed the distribution of FGF-2 to be determined in the pericellular matrix of Rama 27 fibroblasts. The results showed that the FGF-2-NPs were specifically bound to cellular HS and were clustered. Taken together, these data suggest that the differences in heparin binding sites in both the protein and the sugar are greatest between FGF sub-families and may be more restricted within a FGF sub-family in accord with the known conservation of function within FGF sub-families, which supports the idea that heparin binding of these proteins is specific, but in terms of consensus sites on the GAG chain, rather than precisely defined chemical structures.
Supervisor: Fernig, David; Yates, Ed Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available