Identification of the matriptase second CUB domain as the secondary site for interaction with hepatocyte growth factor activator inhibitor type-1

Abstract

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-Asp 390 markedly increased the matriptase-inhibiting activity of HAI-1-58K, whereas the peptides corresponding to Val380-Val 389 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. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.