Identification of the Matriptase Second CUB Domain as the Secondary Site for Interaction with Hepatocyte Growth Factor Activator Inhibitor Type-1

Matriptase is a type II transmembrane serine protease comprising 855 amino acid residues. The extracellular region of matriptase comprises a noncatalytic stem domain (containing two tandem repeats of complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein (CUB) domain) and a catalytic serine protease domain. The stem domain of matriptase contains site(s) for facilitating the interaction of this protease with the endogenous inhibitor, hepatocyte growth factor activator inhibitor type-1 (HAI-1). The present study aimed to identify these site(s). Analyses using a secreted variant of recombinant matriptase comprising the entire extracellular domain (MAT), its truncated variants, and a recombinant HAI-1 variant with an entire extracellular domain (HAI-1–58K) revealed that the second CUB domain (CUB domain II, Cys340–Pro452) likely contains the site(s) of interest. We also found that MAT undergoes cleavage between Lys379 and Val380 within CUB domain II and that the C-terminal residues after Val380 are responsible for facilitating the interaction with HAI-1–58K. A synthetic peptide corresponding to Val380–Asp390 markedly increased the matriptase-inhibiting activity of HAI-1–58K, whereas the peptides corresponding to Val380–Val389 and Phe382–Asp390 had no effect. HAI-1–58K precipitated with immobilized streptavidin resins to which a synthetic peptide Val380–Pro392 with a biotinylated lysine residue at its C terminus was bound, suggesting direct interaction between CUB domain II and HAI-1. These results led to the identification of the matriptase CUB domain II, which facilitates the primary inhibitory interaction between this protease and HAI-1.