Glycosaminoglycan-protein interactions : the fibroblast growth factor paradigm

Abstract

Specific interactions between heparan sulfate glycosaminoglycans (HSGAGs) and proteins are central to a wide range of biological processes such as anticoagulation, angiogenesis and growth factor activation. The specificity involved in the HSGAG-protein interactions stems from the structural heterogeneity of HSGAGs, which are highly acidic biopolymers associated on the cell surface and in the extracellular matrix. It is believed that structural specificity in the HSGAG-protein interactions determines the biological functions mediated by HSGAG-binding proteins such as basic fibroblast growth factor (FGF2). A number of models have been proposed to account for the mode of FGF-FGFR interactions and the role of HSGAGs in modulating FGF2 signaling. It was hypothesized that one role played by HSGAGs was to stabilize FGF2 oligomers in a "side-by-side" or cis fashion for presentation to fibroblast growth factor receptor (FGFR). In this thesis research, we systematically examined different proposed modes of FGF2 dimerization and showed that extensive oligomerization of a FGF2 mutant protein could be achieved by oxidatively crosslinking. Heparin, a highly sulfated form of HSGAGs, was demonstrated to increase the extent of oligomerization. Therefore, the results reported here were consistent with the hypothesis that HSGAGs promoted FGF2 oligomerization in a "side-by-side" mode. The functional significance of a FGF2 dimer was tested using a genetically engineered dimeric FGF2 (dFGF2). Biochemical and biophysical properties of dFGF2, such as protein folding, heparin affinity and receptor

(cont.) binding, were assayed. dFGF2 was found to exhibit higher activities in stimulating cell proliferation and cell survival in vitro compared with the monomeric wildtype. An in vivo rat cornea pocket model further corroborated the in vitro findings. The functional role of HSGAGs derived from the cell surface was studied here. It was found that distinct HSGAG fragments released by heparinase treatment were capable of modulating FGF2-stimulated cell proliferation depending on the expression of FGFR isoforms. This founding is consistent with the proposal that structural specificity of distinct HSGAG fragments dictated the interaction of HSGAGs with FGF and FGFR. The role of heparinase-generated HSGAG fragments in inhibiting cell proliferation was investigated. B16 melanoma cells treated with heparinase III were found to exhibit biochemical and morphological hallmarks of apoptosis. Conditioned medium derived from heparinase-treated cells was shown to be effective in suppressing cell growth. Gene array experiments and caspase activity assays further suggested that apoptotic cell death was mediated through a caspase 8-, death receptor-dependent pathway. Thus, the present study lends further credence to the proposal that cell surface HSGAGs plays a critical role in orchestrating cell phenotype. This thesis work provides the framework for understanding the molecular mechanism of growth factor activation and the structure-function relationship of HSGAG-mediated cell signaling. Results from this study may potentially be useful for therapeutic protein engineering and carbohydrate-based drug discovery.