Matriptase is an epithelial-derived type-II transmembrane serine protease. This protease is expressed prominently in the villus tip of small-intestinal epithelia at which senescent cells undergo shedding and/or apoptosis. The basement membrane of epithelial cells, including small-intestinal epithelial cells, contains extracellular matrix (ECM) proteins such as fibronectin and laminin. We found previously that high concentrations of a recombinant matriptase catalytic domain (r-MatCD) (e.g. 1 μM) caused an increased detachment of and increases in the activity of apoptotic effector caspase-3 in a rat small-intestinal epithelial IEC-6 line cultured on laminin-coated plates and proposed that at sites with its high level of expression, matriptase contributes to promoting shedding and/or detachment-induced death of epithelial cells through a mechanism mediating loss of cell-ECM adhesion. In this study, we found that even without increasing cell detachment, a high concentration of r-MatCD causes an increase in caspase-3 activity in IEC-6 cells cultured on fibronectin-coated plates, suggesting that the recombinant matriptase can cause apoptosis by a mechanism unrelated to cell detachment. Also, r-MatCD-treated IEC-6 cells on fibronectin were found to display spindle-like morphological changes. We suggest that r-MatCD causes apoptosis of IEC-6 on fibronectin by a mechanism involving the disruption of cell integrity.

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.

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 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.

Matriptase is a transmembrane serine protease expressed in vertebrates. This enzyme is synthesized as a zymogen form and is converted to an active form by cleavage at the N-terminus of the serine protease catalytic domain. In a mammalian cell-based expression system, we have produced pseudozymogen forms of recombinant matriptase (r-matriptase) that are activated by cleavage with a recombinant enterokinase (r-EK) in vitro. In the present study, four different pseudozymogen forms of r-matriptase containing a site for activation by r-EK and a hexahistidine tag (His6-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His6-tag at their C-termini formed multimers linked by intermolecular disulfide bonds. After treatment with r-EK, they exhibited no detectable hydrolytic activity toward a chromogenic substrate. A pseudozymogen form of matriptase catalytic domain with N-terminal His6-tag (designated His6t-S-CD) was secreted as a monomer. His6t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His6t-S-CD could be purified from culture medium in milligram quantities. The expression in the yeast offers an efficient method of producing larger amounts of r-matriptase.

Matriptase is a type II transmembrane serine protease containing one potential site for asparagine-linked glycosylation (N-glycosylation) on the catalytic domain (Asn772). It has been found that the activation of matriptase zymogen occurs via a mechanism requiring its own activity and that the N-glycosylation site is critical for the activation. The present study aimed to determine the underlying reasons for the site requirement using Madin-Darby canine kidney cells stably expressing recombinant variants of rat matriptase. A full-length variant with glutamine substitution at Asn772 appeared to be unable to undergo activation because of its catalytic incompetence (i.e., decreased availability of the soluble catalytic domain and/or of the correctly folded domain). This was evidenced by the observations that (i) a recombinant catalytic domain of matriptase with glutamine substitution at the site corresponding to matriptase Asn772 [N772Q-CD-Myc(His)(6)] was not detected in the medium conditioned by transfected cells but was on the cell surface and (ii) purified N772Q-CD-Myc(His)(6) exhibited markedly reduced activity toward a peptide substrate. It is concluded that N-glycosylation site at Asn772 of matriptase is required for the zymogen activation because it plays an important role in rendering this protease catalytically competent in the cellular environment.