Isolation and conformational analysis of fragment peptide corresponding to the heparin-binding site of hepatocyte growth factor

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes. The mitogenic activity of HGF is mediated by its binding to a high-affinity receptor, c-Met. Heparan sulfate if an initial binding site for HGF, based on its relative abundance on the cell surface. The binding of HGF to heparin or heparin-like molecules may induce oligomerization of HGF and facilitate c-Met-dependent mitogenesis [Zioncheck er al, (1995) J. Biol. Chem. 270, 16571-16878]. Thus, heparin binding is important for the biological activity of HGF. To identify the heparin-binding site of HGF, we isolated fragment peptides corresponding to the site by limited proteolysis and chemical degradation of recombinant human HGF (rhHGF). The heparin-binding ability of the peptides was expressed as their elution positions on heparin-affinity column chromatography with NaCl gradient elution, Because all of the heparin-binding peptides obtained in this study were isolated from the N-terminal hairpin-loop region (PyrGlu(32)-Asn(127)) of HGF, the region was identified as the heparin-binding site of HGF, One of the isolated peptides, Phe(42)-Glu(111), containing the N-terminal hairpin-loop structure, was considered a suitable model peptide for the heparin-binding site of HGF. From the observation using circular dichroism spectroscopy, it was indicated that the secondary structure of the peptide changed from a random structure to a beta-sheet-like structure upon heparin binding, In addition, oligomerization of HGF in the presence of heparin:was observed by dynamic light scattering, Based on our evidence, it is considered that the conformational change in the heparin-binding site may induce the oligomerization of HGF.