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J. Biol. Chem., Vol. 279, Issue 32, 33237-33245, August 6, 2004

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Identification of Prodomain Determinants Involved in ADAMTS-1 Biosynthesis^*

Jean-Michel Longpré and Richard Leduc

From the Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

The metalloprotease ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin type I motif), similarly to other members of the ADAMTS family, is initially synthesized as a zymogen, proADAMTS-1, that undergoes proteolytic processing at the prodomain/catalytic domain junction by serine proteinases of the furin-like family of proprotein convertases. The goals of this study were to identify residues of the prodomain that play an essential role in ADAMTS-1 processing and to determine the identity of the convertase required for zymogen processing. To gain insight into the putative roles of specific prodomain residues in ADAMTS-1 biosynthesis, we performed biosynthetic labeling experiments in transiently transfected human embryonic kidney 293 cells expressing wild-type and prodomain mutants of proADAMTS-1. Cells expressing wild-type ADAMTS-1 initially produced a 110-kDa zymogen form that was later converted to an 87-kDa form, which was also detected in the media. Although convertases such as PACE4 and PC6B processed proADAMTS-1, we found that furin was the most efficient enzyme at producing the mature ADAMTS-1 87-kDa moiety. Site-directed mutagenesis of the two putative furin recognition sequences found within the ADAMTS-1 prodomain (RRNR¹⁷³ and RKKR²³⁵) revealed that Arg²³⁵ was the sole processing site. Use of the Golgi disturbing agent, Brefeldin A, and monensin suggests that the cleavage of proADAMTS-1 takes place in the Golgi apparatus prior to its secretion. Conserved residues within the prodomain of other ADAMTS members hinted that they might act as maturation determinants. Replacement with alanine of selected residues Cys¹⁰⁶, Tyr¹⁰⁸, Gly¹¹⁰, Cys¹²⁵, and Cys¹⁸¹ and residues encompassing the 137-144 sequence significantly affected the biosynthetic profile of the enzyme. Our results suggest that conserved residues other than the furin cleavage site in the prodomain of ADAMTS-1 are involved in its biosynthesis.

Received for publication, December 2, 2003 , and in revised form, May 13, 2004.

^* This work was supported by the Canadian Institutes of Health Research (Grant MOP-13755) (to R. L.). 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.

A senior scholar of the Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed: Dept. of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Québec J1H 5N4, Canada. Tel.: 819-564-5413; Fax: 819-564-5400; E-mail: Richard.Leduc{at}USherbrooke.ca.

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