Impact of amino acid substitutions in domain 5 of high molecular weight kininogen on suppression of breast cancer cells invasion

Domain 5 (D5) of high molecular weight kininogen represent significant anti-invasion properties towards tumor cells, and His-Gly-Lys (HGK) in D5 is the core motif for inhibition of invasion. In an attempt to identify the core amino acid of HGK in the process of tumor invasion, PCR-mediated site-directed mutagenesis technology was use to generate 3 mutant constructs, pGEX-4T-1/D5 (H485A), pGEX-4T-1/D5 (G486A) and pGEX-4T-1/D5 (K487A). These mutants fusion protein was expressed in E. coli. We further detect the effect of natural D5H and the mutants on tumor invasion and adhesion, and the expression level intracellular adhesion molecule-1 (ICAM-1) in human breast cancer cells. Our study demonstrates that Lys of HGK at 487 sit plays an important role in invasion inhibition. The results also provide a potent tool to clarify the molecular mechanisms involved in D5 invasion inhibition.