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J. Biol. Chem., Vol. 282, Issue 1, 142-150, January 5, 2007

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Substrate Conformation Modulates Aggrecanase (ADAMTS-4) Affinity and Sequence Specificity

SUGGESTION OF A COMMON TOPOLOGICAL SPECIFICITY FOR FUNCTIONALLY DIVERSE PROTEASES^*

Janelle L. Lauer-Fields, Dmitriy Minond, Thilaka Sritharan, Masahide Kashiwagi, Hideaki Nagase, and Gregg B. Fields¹

From the Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, Florida 33431-0991 and Kennedy Institute of Rheumatology Division, Imperial College London, 1 Aspenlea Road, Hammersmith, London W6 8LH, United Kingdom

Protease-substrate interactions are governed by a variety of structural features. Although the substrate sequence specificities of numerous proteases have been established, "topological specificities," whereby proteases may be classified based on recognition of distinct three-dimensional structural motifs, have not. The aggrecanase members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family cleave a variety of proteins but do not seem to possess distinct sequence specificities. In the present study, the topological substrate specificity of ADAMTS-4 (aggrecanase-1) was examined using triple-helical or single-stranded poly(Pro) II helical peptides. Substrate topology modulated the affinity and sequence specificity of ADAMTS-4 with K_m values indicating a preference for triple-helical structure. In turn, non-catalytic ADAMTS-4 domains were critical for hydrolysis of triple-helical and poly(Pro) II helical substrates. Comparison of ADAMTS-4 with MMP-1 (collagenase 1), MMP-13 (collagenase 3), trypsin, and thermolysin using triple-helical peptide (THP) and single-stranded peptide (SSP) substrates demonstrated that all five proteases possessed efficient "triple-helical peptidase" activity and fell into one of two categories: (k_cat/K_m)_SSP > (k_cat/K_m)_THP (thermolysin, trypsin, and MMP-13) or (k_cat/K_m)_THP (k_cat/K_m)_SSP and (K_m)_SSP > (K_m)_THP (MMP-1 and ADAMTS-4). Overall these results suggest that topological specificity may be a guiding principle for protease behavior and can be utilized to design specific substrates and inhibitors. The triplehelical and single-stranded poly(Pro) II helical peptides represent the first synthetic substrates successfully designed for aggrecanases.

Received for publication, May 31, 2006 , and in revised form, November 2, 2006.

^* This work was supported by National Institutes of Health Grants AR 39189 (to H. N.) and CA 77402, CA 98799, and EB 000289 (to G. B. F.), the Wellcome Trust (Reference Number 057508 (to H. N.)), a Glenn/American Federation for Aging Research (AFAR) Scholarship (to J. L. L.-F.), and the Florida Atlantic University Center of Excellence in Biomedical and Marine Biotechnology. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7.

¹ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Rd., Boca Raton, FL 33431-0991. Tel.: 561-297-2093; Fax: 561-297-2759; E-mail: fieldsg{at}fau.edu.

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