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Journal of Biochemistry Advance Access originally published online on April 26, 2009
Journal of Biochemistry 2009 146(2):273-282; doi:10.1093/jb/mvp066
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Activation of a Membrane-Bound Serine Protease Matriptase on the Cell Surface

Yuka Miyake1, Makoto Yasumoto1, Satoshi Tsuzuki2, Tohru Fushiki2 and Kuniyo Inouye1,*

1Laboratory of Enzyme Chemistry; and 2Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto City 606-8502, Japan

*To whom correspondence should be addressed. Tel: +81-75-753-6266, Fax: +81-75-753-6265, E-mail: inouye{at}kais.kyoto-u.ac.jp

Received March 11, 2009; Accepted April 16, 2009


  Abstract

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.

Key Words: cell surface, enzyme activation, intracellular trafficking, matriptase, membrane-type serine protease

Abbreviations: CHO, Chinese hamster ovary; CTF, C-terminal fragment; ER, endoplasmic reticulum; HAI-1, hepatocyte growth factor activator inhibitor type I; HGF, hepatocyte growth factor; HRP, horseradish peroxidase; NTF, N-terminal fragment; PAR-2, protease-activated receptor-2; rEK, recombinant enterokinase


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