Rational Design of Human Fibroblast Growth Factor with Enhanced Stability

Abstract

Fibroblast growth factors (FGFs) constitute a large family of heparin-binding proteins which control the proliferation, differentiation, and migration of a variety of cell types. For its transduction of cellular signals, FGF must bind to the extracellular domains of fibroblast growth factor receptor (FGFR), a transmembrane tyrosine kinase receptor. The resulting FGF-FGFR complex leads to receptor dimerization which stimulates a phosphorylation cascade within the cell resulting in a variety of cellular outcomes. FGF also binds to a second class of receptors for which it has a lower affinity, heparan sulfate proteoglycans (HSPGs). Heparanase treatment of intact cells has shown that FGF fails to bind to FGFR resulting in non-proliferation of the cells, indicating the importance of HSPG-binding. Closely related to heparan sulfate is heparin, a structurally-similar proteoglycan which can bind to FGF isofunctionally. The role of heparin in FGF-FGFR interactions has been discussed greatly. Heparin is believed to play a role in FGF stability, protecting it from thermal denaturation and proteolytic degradation. It has also been proposed that HSPG will increase the binding affinity of FGF for FGFR and will stabilize the dimerization of FGFR. Some studies have shown, however, that heparin binding may not be essential to FGF-FGFR binding. The goal of this study was to define the role of heparin in human FGF-1 (hFGF-1) stability and cell proliferation activities. A His-tagged quadruple mutant with mutations made in the defined heparin-binding region of hFGF-1 at the N128K, Y139K, G140R, and Q141K positions (Quad-His) was created prior to the beginning of this study, and the mutant was compared against wild type hFGF-1 through a variety of biophysical techniques. Experiments were conducted with both heparin and sucrose octasulfate (SOS), an analog commonly used as a structural and functional mimic for heparin.