Effect of His-Gly-Lys motif derived from domain 5 of high molecular weight kininogen on suppression of cancer metastasis both in vitro and in vivo

We have demonstrated previously that kinin-free high molecular weight kininogen, its domain 5 (D5(H), Gly(402) Lys(502)), and peptides derived from D5(H) inhibited vitronectin-mediated migration and invasion of cancer cells in vitro ( Kamiyama, F., Maeda, T., Yamane, T., Li, Y. H., Ogikubo, O., Otsuka, T., and Ohkubo, I. ( 2001) Biochem. Biophys. Res. Commun. 288, 975 - 980). In this study, we found that the amino acid sequence His-Gly-Lys (HGK) in D5(H) is the core motif for inhibition of adhesion and invasion of MDA-MB-231 cells in vitro. P-5m ((484)GHGKHKNK(491), Gly(484) - Lys(491)), an octapeptide including the HGK motif derived from D5(H), and HGK, a tripeptide, inhibited both cell adhesion and invasion in vitro. However, an octapeptide designated P-5m (K487R), in which Lys(487) was changed to Arg, did not inhibit either cell adhesion or invasion, and peptides HGR and HGG also had no inhibitory effect. Recombinant GST-D5(H) expressed in Escherichia coli had a stronger inhibitory effect on cell adhesion and invasion in vitro than did GST-D5(H) (K487R) in which Lys(487) was changed to Arg. Furthermore, P-5m (Gly(484) - Lys(491)) peptide clearly suppressed lung metastasis in mice experimentally induced by using B16-F10 cells, but P-5m (G487R) had no effect. These data strongly indicate that both the HGK motif and lysine residue (Lys(487)) play essential roles in inhibition of cell adhesion and invasion in vitro and in prevention of metastasis of cancer cells in vivo. We tried to identify the HGK motif binding protein on the surface of cancer cells. A 95-kDa surface biotin-labeled membrane protein was specifically detached from GST-D5(H) by P- 5 ( His(479)-Lys(493)) peptide but not by P-1 (Gly(402)-Lys(420)) peptide originating from the N-terminal region of D5(H).