Matriptase is a transmembrane serine protease that is strongly expressed in epithelial cells. The single-chain zymogen of matriptase is considered to have inherent activity, leading to its own activation (i.e. conversion to the disulphide-linked-two-chain form by cleavage after Thr-Lys-Gln-Ala-Arg614). Also, there is growing evidence that the activation of zymogen occurs at the cell surface and in relation to the acidification and lowering of ionic strength within cell-surface microenvironments. The present study aimed to provide evidence for the involvement of zymogen activity in its activation in physiologically relevant cellular contexts. For this purpose, the activity of a pseudozymogen form of recombinant matriptase (HL-matriptase zymogen) was examined using acetyl-l-Lys-l-Thr-l-Lys-l-Gln-l-Leu-l-Arg-4-methyl-coumaryl-7-amide as a substrate. HL-matriptase zymogen exhibited optimal activity toward the substrate pH approximately 6.0. The substrate hydrolysis at the pH value was hardly detected when NaCl was present at a concentration of 145 mM. In a buffer of pH 6.0 containing 5 mM NaCl, the activity of HL-matriptase zymogen was only approximately 30-times lower than that of the respective two-chain form. These findings suggest that the in vivo activation of matriptase zymogen occurs via a mechanism involving the zymogen activity.

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 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.Matriptase (also known as epithin, membrane-type serine protease 1, SNC19, suppression of tumorigenicity 14) is a transmembrane serine protease (1-7). This protease belongs to the type II transmembrane serine protease group, which is characterized by an N-terminal cytoplasmic domain, a signal anchor transmembrane domain, and an extracellular C-terminal serine protease catalytic domain (8,9). Matriptase is first synthesized as a zymogen comprising 855 amino acid residues in the human, mouse, and rat (7,10,11). Interaction between the zymogens appears to result in the generation of disulfidelinked two-chain molecules with an N-terminal Val 615 (activated matriptase molecules) (Fig. 1A) (10, 12-16). In addition, matriptase appears to function both as a plasma membraneassociated form and as a shed (soluble) form (1, 15, 17). The two-chain matriptase purified from human milk exhibits activity with trypsin-like specificity and is known to cleave and thus activate single-chain urokinase-type plasminogen activator (scuPA) 2 and pro-hepatocyte growth factor (pro-HGF) (18). Bacterially expressed variants of the recombinant (r-) catalytic domain of matriptase also cleaved to activate sc-uPA (19) and pro-HGF (20). Another r-matriptase catalytic domain cleaved to activate the precursor form of prostasin, a glycosylphosphatidylinositol-linked serine protease (3, 21). Together with the abundant expression in epithelial cells and in keratinocytes (7,22,23), these characteristics lead to the proposition that matriptase plays a key role in the maintenance of epithelial integrity and homeostasis. Studies in genetically manipulated mice indicate the importance of matriptase for epithelial and epidermal barrier function (24 -27).In general, members of the type II transmembrane serine protease group have an extracellular noncatalytic stem domain comprising various structural motifs (8, 9). For instance, the stem domain of matriptase comprises a sea-urchin sperm protein-enteropeptidase-agrin (SEA) domain, followed by two tandem repeats of complement proteases C1r/C1s-u...

Matriptase is a type-II transmembrane serine protease that is expressed strongly in the epithelial elements of various organs. In the small intestine, it is expressed prominently at the villus tip where aged epithelial cells undergo shedding and/or apoptosis. This observation, together with the ability of matriptase to cleave laminin (a basement membrane component critical for epithelial cell attachment), prompted us to hypothesize that it plays an important part in the removal of aged epithelial cells in the small intestine. We tested this hypothesis by determining whether a recombinant catalytic domain of rat matriptase (His(6)t-S-CD) causes detachment and/or apoptosis of small-intestinal epithelial IEC-6 cells. His(6)t-S-CD caused detachment of cells attached to laminin-coated plates but did not detach cells attached to fibronectin- or type-IV collagen-coated plates. Pre-treatment of laminin-coated plates with His(6)t-S-CD decreased the attachment of cells, suggesting that the recombinant matriptase caused detachment through a mechanism involving a direct effect on laminin. His(6)t-S-CD was also found to induce apoptosis in the cells cultured on laminin-coated plates, as assessed by annexin-V staining, DNA fragmentation and caspase-3 activity assays. These findings support our hypothesis regarding the role of matriptase in the small intestine.

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