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J. Biol. Chem., Vol. 282, Issue 22, 16146-16154, June 1, 2007

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Regulation of ADAMTS9 Secretion and Enzymatic Activity by Its Propeptide^*

Bon-Hun Koo, Jean-Michel Longpré¹, Robert P. T. Somerville, J. Preston Alexander^¶, Richard Leduc², and Suneel S. Apte³

From the Department of Biomedical Engineering and Orthopaedic Research Center, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the Department of Pharmacology, Faculty of Medicine and Health Sciences, Universitéde Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada, and the ^¶Triple Point Biologics, Forest Grove, Oregon 97116

ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg⁷⁴ and Arg²⁸⁷) the ADAMTS9 propeptide was also furin-processed at Arg²⁰⁹. Substitution of Ala for Arg⁷⁴, Arg²⁰⁹, and Arg²⁸⁷ resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface.

Received for publication, October 31, 2006 , and in revised form, March 29, 2007.

^* This work was supported in part by National Institutes of Health Grant AR49930 (to S. A.) and a grant from the Canadian Institutes of Health Research (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.

¹ Received a Ph.D. studentship from the Fonds de la Recherche en Santédu Québec (FRSQ).

² A Chercheur National of the FRSQ.

³ To whom correspondence should be addressed: Dept. of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic Foundation (ND20), 9500 Euclid Ave., Cleveland OH 44195. Tel.: 216-445-3278; Fax: 216-444-9198; E-mail: aptes{at}ccf.org.

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