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J. Biol. Chem., Vol. 282, Issue 38, 27847-27856, September 21, 2007

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Stomach-specific Calpain, nCL-2/Calpain 8, Is Active without Calpain Regulatory Subunit and Oligomerizes through C2-like Domains^*

Shoji Hata, Naoko Doi, Fujiko Kitamura, and Hiroyuki Sorimachi¹

From the Department of Enzymatic Regulation for Cell Functions (Calpain Project), The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo 113-8613 and CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan

Calpains constitute a family of intracellular Ca²⁺-regulated cysteine proteases that are indispensable in the regulation of a wide variety of cellular functions. The improper activation of calpain causes lethality or various disorders, such as muscular dystrophies and tumor formation. nCL-2/calpain 8 is predominantly expressed in the stomach, where it appears to be involved in membrane trafficking in the gastric surface mucus cells (pit cells). Although the primary structure of nCL-2 is quite similar to that of the ubiquitous m-calpain large subunit, the enzymatic properties of nCL-2 have never been reported. Here, to characterize nCL-2, the recombinant protein was prepared using an Escherichia coli expression system and purified to homogeneity. nCL-2 was stably produced as a soluble and active enzyme without the conventional calpain regulatory subunit (30K). Purified nCL-2 showed Ca²⁺-dependent activity, with half-maximal activity at about 0.3 mM Ca²⁺, similar to that of m-calpain, whereas its optimal pH and temperature were comparatively low. Immunoprecipitation analysis revealed that nCL-2 exists in both monomeric and homo-oligomeric forms, but not as a heterodimer with 30K or 30K-2, and that the oligomerization occurs through domains other than the 5EF-hand domain IV, most probably through domain III, suggesting a novel regulatory system for nCL-2.

Received for publication, April 16, 2007 , and in revised form, July 3, 2007.

^* This work was supported in part by MEXT.KAKENHI Grants 17780115 (to S. H.), 17028055, and 18076007 (to H. S.), by JSPS.KAKENHI Grant 18380085 (to H. S.), by a Sasagawa Scientific Research Grant from The Japan Science Society (to S. H.), a Research Grant (14B-4) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare, and a Takeda Science Foundation Research grant (to H. S.). 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 To whom correspondence should be addressed: Dept. of Enzymatic Regulation for Cell Functions, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Fax: 81-3-3823-2359; E-mail: sorimach{at}rinshoken.or.jp.

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