Aoyama H, Naka D, Yoshiyama Y, Ishii T, Kondo J, Mitsuka M, Hayase T
Yokohama Research Center, Mitsubishi Chemical Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227, Japan.
Biochemistry. 1997 Aug 19;36(33):10286-91. doi: 10.1021/bi962700f.
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.
肝细胞生长因子（HGF）是一种对肝细胞有强大作用的促有丝分裂原。HGF的促有丝分裂活性是通过其与高亲和力受体c-Met结合来介导的。硫酸乙酰肝素因其在细胞表面相对丰富，是HGF的初始结合位点。HGF与肝素或类肝素分子的结合可能诱导HGF寡聚化，并促进依赖c-Met的有丝分裂发生[齐昂切克等人（1995年）《生物化学杂志》270卷，16871 - 16878页]。因此，肝素结合对HGF的生物学活性很重要。为了确定HGF的肝素结合位点，我们通过对重组人HGF（rhHGF）进行有限蛋白酶解和化学降解，分离出了对应该位点的片段肽。肽的肝素结合能力通过它们在NaCl梯度洗脱的肝素亲和柱层析上的洗脱位置来表示。因为本研究中获得的所有肝素结合肽都是从HGF的N端发夹环区域（PyrGlu32 - Asn127）分离出来的，所以该区域被确定为HGF的肝素结合位点。其中一个分离出的肽，Phe42 - Glu111，含有N端发夹环结构，被认为是HGF肝素结合位点的合适模型肽。通过圆二色光谱观察表明，该肽在与肝素结合后，二级结构从无规结构转变为β-折叠样结构。此外，通过动态光散射观察到在肝素存在下HGF发生了寡聚化。基于我们的证据，认为肝素结合位点的构象变化可能诱导HGF的寡聚化。
