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J. Biol. Chem., Vol. 279, Issue 48, 49835-49841, November 26, 2004

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Deletion of Epidermal Growth Factor-like Domains Converts Mammalian Tolloid into a Chordinase and Effective Procollagen C-proteinase^*

Laure Garrigue-Antar, Vincent François, and Karl E. Kadler¶

From the Wellcome Trust Centre for Cell-Matrix Research, the University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom and Institut de Génétique Humaine, CNRS/UPR 1142, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France

Bone morphogenetic protein (BMP)-1 and mammalian tolloid (mTld) are Ca²⁺-dependent metalloproteinases that result from alternative splicing of the bmp1 gene. They have different proteinase activities, e.g. BMP-1 effectively cleaves procollagen (an extracellular matrix protein) and chordin (a BMP antagonist), whereas mTld is a poor procollagen proteinase and will not cleave chordin in the absence of twisted gastrulation. This is perplexing because mTld (being the longer variant) might be expected to cleave all substrates cleaved by BMP-1. Studies have shown that the minimal structure for procollagen proteinase activity is proteinase-CUB1-CUB2 (BMP-1EC3) and therefore lacking the epidermal growth factor (EGF)-like domain thought to account for the Ca²⁺ dependence of BMP-1. In this study we generated three deletion mutants of mTld that lacked either one or both EGF-like domains (referred to as "mTld-EGF"). The mutated proteins were poorly but sufficiently secreted from 293-EBNA cells for in vitro assays of procollagen and chordin cleavage. Most surprisingly, the mTld-EGF mutants required Ca²⁺ for proteolytic activity, thereby showing that the EGF-like domains do not account for the Ca²⁺ dependence of BMP-1/mTld. Moreover, the mTld-EGFs are effective procollagen proteinases and cleave chordin. Furthermore, BMP-1EC3 cleaves chordin and requires Ca²⁺ for activity. Studies using nondenaturing gels showed that mTld molecules lacking EGF-like domains have a loose conformation such that in the presence of Ca²⁺ binding sites for chordin and procollagen on the "BMP-1-part" of the molecule are exposed. We propose that the EGF-like domains could hold CUB4/5 domains in locations that exclude substrates cleavable by BMP-1.

Received for publication, July 19, 2004 , and in revised form, September 7, 2004.

^* This work was supported by a research grant from The Wellcome Trust (to K. E. K.) and l'Association pour la Recherche sur le Cancer (to V. F.). 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.

^¶ To whom correspondence should be addressed. Tel.: 44-161-275-5086; Fax: 44-161-275-1505; E-mail: karl.kadler{at}man.ac.uk.

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