**4.1 Structure and anti-invasive function of NK4**

NK4 was initially purified as a fragment from elastase-digested samples of recombinant human HGF (Date *et al*., 1997). The N-terminal amino acid sequence of NK4 and of the remnant fragment, assumed to be composed of an HGF β-chain, revealed that NK4 is cleaved between the 478th valine and the 479th asparagine. The N-terminal amino acid sequence of NK4 revealed that the N-terminal structure of NK4 is the same as undigested HGF (*i.e*., 32nd pyroglutamate), indicating that NK4 is composed of the N-terminal 447 amino acids of the α-chain of HGF and contains the N-terminal hairpin domain and four kringle domains (thus designated NK4) (**Fig. 2A**). The binding domains that are responsible for high-affinity binding to MET are the N-terminal hairpin and the first kringle domains in NK4 (and HGF). MET tyrosine phosphorylation occurs in A549 lung carcinoma within 10 minutes after HGF addition, while NK4 inhibits the HGF-mediated MET activation (**Fig. 2B**). Actually, NK4 functions as an HGF-antagonist: HGF induces invasion and migration of the gallbladder and bile duct carcinoma cells in ECM-based gels, while NK4 inhibits HGF-induced invasion in a dose-dependent manner (**Fig. 2C**) (Date *et al*., 1998). These anti-invasive effects of NK4 are seen in distinct types of cancer cells (Hiscox *et al*., 2000; Maehara *et al*., 2001; Parr *et al*., 2001), strengthening the common role of NK4 during cancer migration.
