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Articles by S Tsuzuki
Total Records ( 4 ) for S Tsuzuki
  K Kojima , S Tsuzuki , T Fushiki and K. Inouye

Matriptase is a type II transmembrane serine protease containing the non-catalytic domains (stem domain) and catalytic domain in the extra-cellular region. Our aim is to address the role of the stem domain in the interaction between matriptase and its physiological inhibitor, hepatocyte growth factor activator inhibitor type I (HAI-1). We prepared secreted variants of recombinant matriptase containing the entire extra-cellular domain (HL-matriptase) or only the catalytic domain (L-matriptase), and compared the inhibition activities of a cell membrane-anchored form of recombinant HAI-1 (maHAI-1) against the matriptase variants in the hydrolysis of peptidyl–4-methyl-coumaryl-7-amide (MCA) substrates. HL-matriptase and L-matriptase were inhibited by purified maHAI-1 with a similar extent when t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA (1) and acetyl-Lys-Thr-Lys-Gln-Leu-Arg-MCA (2) were used as substrates. However, HL-matriptase was inhibited more strongly than L-matriptase by maHAI-1 in the hydrolysis of Boc-[(2S)-2-amino-3-(benzyloxycarbonyl)propionyl]-Pro-Arg-MCA (3). These results show that the stem domain of matriptase facilitates the inhibitory interaction of this protease with maHAI-1 in the hydrolysis of substrate 3, although it has no effect in the hydrolysis of substrates 1 and 2. To our knowledge, this is the first evidence that the stem domain of matriptase can affect the interaction between this protease and HAI-1.

  Y Miyake , M Yasumoto , S Tsuzuki , T Fushiki and K. Inouye

Matriptase is a type II transmembrane serine protease. The activation (i.e. conversion of the single-chain pro-form to the disulphide-linked-two-chain active form) of this enzyme is known to occur via a mechanism requiring its catalytic triad. We reported previously that the activated enzyme was produced in the conditioned medium when full-length rat matriptase was expressed in monkey kidney COS-1 cells. The present study aimed to address when and where the matriptase activation occurs. COS-1 cells expressing matriptase were labelled with a membrane-impermeable biotin derivative and then solubilized with Triton. Both activated and non-activated matriptase molecules were detected in the avidin precipitants of Triton extracts, whereas only the non-activated molecules were detected in the flow-through fraction of avidin-precipitation procedure. Single-chain matriptase has been thought to have an inherent activity. Indeed, a secreted single-chain variant of recombinant matriptase bearing mutation at the activation-cleavage site was found to exhibit the activity in hydrolyzing a synthetic peptide substrate at pH 7.5. However, the variant had little activity at pH 5.5, as found in the lumen of post-Golgi secretory vesicles. Altogether, it is concluded that the activation of matriptase may occur when the enzyme reaches the cell surface.

  K Inouye , M Yasumoto , S Tsuzuki , S Mochida and T. Fushiki

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 ~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 ~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.

  S Mochida , S Tsuzuki , K Inouye and T. Fushiki

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 (His6t-S-CD) causes detachment and/or apoptosis of small-intestinal epithelial IEC-6 cells. His6t-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 His6t-S-CD decreased the attachment of cells, suggesting that the recombinant matriptase caused detachment through a mechanism involving a direct effect on laminin. His6t-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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