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 is 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 et al. (1995) J. Biol.Chem. 270, 16871-16878]. 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 (PyrGlu32-Asn127) of HGF, the region was identified as the heparin-binding site of HGF. One of the isolated peptides, Phe42-Glu111, 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.
Aoyama
H
H
Yokohama Research Center, Mitsubishi Chemical Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227, Japan.
Naka
D
D
Yoshiyama
Y
Y
Ishii
T
T
Kondo
J
J
Mitsuka
M
M
Hayase
T
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation
D010446:Peptide Fragments; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D006493:Heparin