Inhibition of Membrane-Type Serine Protease 1/Matriptase by Natural and Synthetic Protease Inhibitors

Abstract

Membrane-type serine protease 1 (MT-SP1), identical to matriptase, is a re cently identified type II transmembrane serine protease. MT-SP1/matriptase is of consider able interest for the development, homeostasis, and cancer invasion and metastasis of ep ithelial tissues. The administration of inhibitors for MT-SP1/matriptase may be effective to suppress the development of tumors where the enzyme may be involved. In the present study, we produced a secreted form of recombinant MT-SP1/matriptase (ekMT-SP1s) that can be activated by enterokinase in vitro and investigated the inhibitory ability of various protease inhibitors toward the recombinant enzyme. The enterokinase-treated ekMT-SP 1s (active ekMT-SP1s) cleaved various peptidyl-4-methylcoumaryl-7-amide (MCA) substrates with arginine (or lysine) residue at position P1, and the best substrate was t-butyloxycar bonyl (Boc)-Gln-Ala-Arg-MCA. The specificity for the synthetic and natural substrates of the active ekMT-SP1s was in good agreement with that of the natural enzyme. Endogenous protease inhibitors tested, except for antithrombin III, showed no or little inhibition on the cleavage of Boc-Gln-Ala-Arg-MCA by the active ekMT-SP1s. Aprotinin showed strong in hibitory activity toward the cleavage. Food-derived inhibitors, such as soybean trypsin in hibitor, Bowman-Birk inhibitor, and lima bean trypsin inhibitor inhibited it, while chicken ovomucoid did not. Synthetic inhibitors tested inhibited it, and among them, the inhibitory effect of FOY 305 was strongest. The present findings provide important information for the suppression of cancer invasion and metastasis for which MT-SP1/matriptase is responsible.

Requirement of the activity of hepatocyte growth factor activator inhibitor type 1 for the extracellular appearance of a transmembrane serine protease matriptase in monkey kidney COS-1 cells

“…Aprotinin (a Kunitz-type protease inhibitor from ruminants) is known as a potent inhibitor of matriptase (Yamasaki et al 2003). In an in vitro assay, aprotinin inhibited HL-matriptase (Kojima et al 2008) with an IC 50 value of 3 nM (our unpublished work).…”

“…2a) is known to occur via a mechanism requiring its catalytic triad (Takeuchi et al 1999;Oberst et al 2003b;Désilets et al 2008;Miyake et al 2009). The activated two-chain protease cleaves to activate a number of substrates, including pro-HGF, possibly at the cell surface (Lee et al 2000;Satomi et al 2001;Yamasaki et al 2003;Kojima et al 2009a). Like HAI-1, the ectodomain of matriptase is released via cleavage probably with certain active MMPs (Kim et al 2005).…”

Protein Interaction Analysis of ST14 Domains and Their Point and Deletion Mutants

“…HAI-1, aprotinin, and ecotin inhibit the serine protease activity of ST14 by binding to this domain (5,10). The CUB and LDLR domains have been thought to be involved in protein-protein interactions or protein-ligand interactions (32,33), but no interactions were reported for these domains on ST14.…”

“…It has been suggested that TMEFF1 inhibits nodal signaling through binding to the nodal co-receptor Cripto (37). Although the Kazal_1 domain is known to inhibit a number of serine peptidases of the S1 family (35) that includes ST14 (38), Yamasaki et al (10) showed that pancreatic secretory trypsin inhibitor containing a Kazal_1 domain did not inhibit ST14. Similarly, the GST pull-down assays in our work also did not provide evidence to support the interaction between the SP domain and TMEFF1.…”

“…Friedrich et al illustrated the crystal structures of the catalytic domain of ST14 and its complex with a bovine pancreatic trypsin inhibitor (8). Kunitz type serine protease inhibitors, such as hepatocyte growth factor activator inhibitor-1 (HAI-1) (5, 9) and aprotinin (10), strongly inhibit ST14 through binding to its catalytic domain.…”