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J. Biol. Chem., Vol. 282, Issue 29, 20991-20998, July 20, 2007

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TIMP-3 Inhibition of ADAMTS-4 (Aggrecanase-1) Is Modulated by Interactions between Aggrecan and the C-terminal Domain of ADAMTS-4^*

Gareth J. Wayne¹, Su-Jun Deng¹, Augustin Amour^¶1, Satty Borman^¶1, Rosalie Matico^||1, H. Luke Carter¹, and Gillian Murphy^**2

From the GlaxoSmithKline, New Frontiers Research Park, Harlow, Essex CM19 5AW, United Kingdom, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, ^¶GlaxoSmithKline, Stevenage, Hertfordshire SG1 2NY, United Kingdom, ^||GlaxoSmithKline, King of Prussia, Philadelphia, Pennsylvania 19406, and the ^**Department of Oncology, University of Cambridge and Cancer Research UK Cambridge Institute, Cambridge CB2 0RE, United Kingdom

ADAMTS-4 (aggrecanase-1) is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In this study, we developed a sensitive fluorescence resonance energy transfer peptide assay with a K_m in the 10 µM range and utilized this assay to demonstrate that inhibition of full-length ADAMTS-4 by full-length TIMP-3 (a physiological inhibitor of metalloproteinases) is enhanced in the presence of aggrecan. Our data indicate that this interaction is mediated largely through the binding of glycosaminoglycans (specifically chondroitin 6-sulfate) of aggrecan to binding sites in the thrombospondin type 1 motif and spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. The results of this study therefore indicate that the cartilage environment can modulate the function of enzyme-inhibitor systems and could have relevance for therapeutic approaches to aggrecanase modulation.

Received for publication, November 20, 2006 , and in revised form, April 19, 2007.

^* 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 Supported by GlaxoSmithKline.

² Supported by Cancer Research UK (C100). To whom correspondence should be addressed: Dept. of Oncology, University of Cambridge, Cancer Research UK Cambridge Inst., Robinson Way, Cambridge CB2 0RE, UK. Tel.: 44-1223-404-470; Fax: 44-1223-404-199; E-mail: gm290{at}cam.ac.uk.

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