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J. Biol. Chem., Vol. 282, Issue 52, 37316-37324, December 28, 2007

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An Arthropod Cuticular Chitin-binding Protein Endows Injured Sites with Transglutaminase-dependent Mesh^*

Yasuyuki Matsuda, Takumi Koshiba, Tsukasa Osaki¹, Haruka Suyama, Fumio Arisaka, Yoshihiro Toh, and Shun-ichiro Kawabata²

From the Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 and the Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

In mammals, the cornified cell envelope forms beneath the plasma membrane in epithelia and provides a vital physical barrier consisting of insoluble proteins cross-linked by transglutaminase (TGase). In the horseshoe crab Tachypleus tridentatus, TGase is stored in hemocytes and secreted in response to the simulation of bacterial lipopolysaccharides. Here we characterized a TGase substrate designated as caraxin that was identified in horseshoe crab cuticle. One of the homologs, caraxin-1, possessed a unique domain structure consisting of N-and C-terminal heptad repeats and a central domain with a tandem-repeated structure of a pentapeptide. Western blotting showed the specific localization of caraxin-1 in sub-cuticular epidermis. Moreover, we identified the pentapeptide motif to be a chitin-binding unit. Analytical ultracentrifugation revealed that caraxin-1 exists as an oligomer with 310–350 kDa, which is 20-mer based on the molecular mass of the monomer. The oligomers were cross-linked by TGase to form an elaborate mesh with honeycomb structures, which was electron-microscopically found to be different from the clotting mesh triggered by lipopolysaccharide-induced hemocyte exocytosis. We determined several cross-linking sites in the N-and C-terminal domains of caraxin-1. The replacements of Leu to Pro at positions 36 and 118 in caraxin-1 reduced the -helix content, which destroyed the TGase-dependent mesh, thus indicating the importance of the N-and C-terminal domains for the proper mesh formation. In arthropods, TGase-dependent protein cross-linking may be involved in the initial stage of host defense at the sub-cuticular epidermis, as in the case of mammalian skin.

Received for publication, July 12, 2007 , and in revised form, September 25, 2007.

^* This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Priority Area 839 to S. K., 18370045 to S. K., and 18770137 to T. K.) and The Kao Foundation for Arts and Sciences (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.

² To whom correspondence should be addressed. Tel.: 81-92-642-2632; Fax: 81-92-642-2632; E-mail: skawascb{at}mbox.nc.kyushu-u.ac.jp.

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