Mutations in the heparin-binding domains of human basic fibroblast growth factor alter its biological activity

Abstract

Eleven structural analogues of human basic fibroblast growth factor (bFGF) have been prepared by site-directed mutagenesis of a synthetic bFGF gene to examine the effect of amino acid substitutions in the three putative heparin-binding domains on FGF's biological activity. After expression in Escherichia coli, the mutant proteins were purified to homogeneity by use of heparin-Sepharose chromatography and analyzed for their ability to stimulate DNA synthesis in human foreskin fibroblasts. Recombinant human bFGF 1-146 and [Ala69,Ser87]bFGF, an analogue where two of the four cysteines had been replaced by alanine and serine, were equipotent to standard bovine basic fibroblast growth factor. Substitution of aspartic acid-19 by arginine in the first heparin-binding domain yielded a molecule that stimulated a higher total mitogenic response in fibroblasts as compared to bFGF. In addition, replacement of either arginine-107 in the second domain or glutamine-123 in the third domain with glutamic acid resulted in compounds that were 2 and 4 times more potent than bFGF. In contrast, substitution of arginine-107 with isoleucine reduced the activity of the molecule by 100-fold. Combination of domain substitutions to generate the [Glu107,123]bFGF and [Arg19,Lys123,126]bFGF mutants did not show any additivity of the mutations on biological activity. Alterations in the biological activity of the analogues was dependent on both the site of and the type of modification. Increased positive charge in the first domain and increased negative charge in the second and third domains enhanced biological potency. The altered activities of the derivatives appear to be due in part to changes in the affinity of the analogues for heparin. We conclude that changes in all three of the putative heparin-binding domains result in altered mitogenic activity and heparin interaction of basic fibroblast growth factor.

Silk fibroin sponges with cell growth‐promoting activity induced by genetically fused basic fibroblast growth factor

“…(A)]. At higher concentrations, the bioactivity of the bFGF‐fused fibroin from PSGs was suppressed, which is consistent with reports from other research groups that an overdose of the growth factor inhibits cell proliferation . Based on a comparison of the effects of bFGF‐fused fibroin and rhbFGF [Fig.…”

Direct and Allosteric Inhibition of the FGF2/HSPGs/FGFR1 Ternary Complex Formation by an Antiangiogenic, Thrombospondin-1-Mimic Small Molecule

“…Residues showing significant CSP are R129 and K144 (Figure 1B, C), thus clearly identifying the sm27 binding site. This region, located in the long b10–b12 loop, is part of the reported heparin binding site [12], [15], [16], [18], [19]. 1 H and 15 N chemical shift deviations were also analyzed separately to finely describe both effects of direct ligand interaction and local structure rearrangements correlating with the binding process [20].…”

“…The observed antiangiogenic effects of sm27 could be due to a direct binding of the inhibitor to one of the two distinct binding sites identified for FGFR1 and for heparin/HSPGs [12], [13], [14], [15], [16], [17] or through an indirect perturbation of the conformational properties of the FGF2 sub-structures mostly involved in complex formation with FGF2 receptors. To clarify the molecular details underpinning sm27 inhibition mechanisms, we have set out to characterize the structural properties of the FGF2-sm27 complex and to investigate the perturbative effects of sm27 on the global FGF2 dynamical properties.…”

Mechanisms of FGF gradient formation during embryogenesis

“…Pioneering in vitro studies first suggested the role of HSPGs as co-receptors in FGF signaling [33–36,72,73]. In this role, HSPGs function by physically approximating FGF ligands to FGF receptors and by simultaneously binding FGF ligands and receptors to induce a conformational change required for FGF signal activation.…”