title: 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, Cys(340)-Pro(452)) likely contains the site(s) of interest. We also found that MAT undergoes cleavage between Lys(379) and Val(380) within CUB domain II and that the C-terminal residues after Val(380) are responsible for facilitating the interaction with HAI-1-58K. A synthetic peptide corresponding to Val(380)-Asp(390) markedly increased the matriptase-inhibiting activity of HAI-1-58K, whereas the peptides corresponding to Val(380)-Val(389) and Phe(382)-Asp(390) had no effect. HAI-1-58K precipitated with immobilized streptavidin resins to which a synthetic peptide Val(380)-Pro(392) 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.
authors.affiliation: From the Laboratory of Enzyme Chemistry, Sakyo-ku, Kyoto City 606-8502, Japan. Electronic address: inouye@kais.kyoto-u.ac.jp.
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
authors.affiliation: Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto City 606-8502, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tsuzuki
authors.affiliation: From the Laboratory of Enzyme Chemistry, Sakyo-ku, Kyoto City 606-8502, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Yasumoto
authors.affiliation: From the Laboratory of Enzyme Chemistry, Sakyo-ku, Kyoto City 606-8502, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kojima
authors.affiliation: Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto City 606-8502, Japan.
authors.forename: Seiya
authors.initials: S
authors.lastname: Mochida
authors.affiliation: Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto City 606-8502, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Fushiki
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D010455:Peptides; D011994:Recombinant Proteins; C504592:Spint1 protein, rat; D012697:Serine Endopeptidases; C485595:matriptase