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J. Biol. Chem., Vol. 283, Issue 28, 19551-19560, July 11, 2008

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Mapping of SPARC/BM-40/Osteonectin-binding Sites on Fibrillar Collagens^*

Camilla Giudici, Nicolas Raynal^¶, Hanna Wiedemann, Wayne A. Cabral^||, Joan C. Marini^||, Rupert Timpl, Hans Peter Bächinger^**, Richard W. Farndale^¶1, Takako Sasaki¹², and Ruggero Tenni¹

From the Max-Planck-Institut für Biochemie, Am Klopferspitz 18, 82152 Martinsried, Germany, the Department of Biochemistry "A. Castellani," University of Pavia, via Taramelli 3b, 27100 Pavia, Italy, the ^¶Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, United Kingdom, the ^||Bone and Extracellular Matrix Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, and the ^**Shriners Hospitals for Children, Portland Research Center and the Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix A of the extracellular calcium-binding domain of SPARC and is partially masked by helix C. Previously, we found that the removal of helix C caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site 180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven 2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.

Received for publication, December 7, 2007 , and in revised form, May 5, 2008.

^* This work was authored, in whole or in part, by National Institutes of Health staff. This work was supported by Deutsche Forschungsgemeinschaft Grants Sa 1003/1 and 2 (to T. S.), European Community Project QLK3-CT2000-00084 (to R. T. and T. S.), and grants from the Shriners Hospital for Children (to H. P. B.), the Wellcome Trust (to R. W. F.), and by University of Pavia (to R. T.). 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–S4 and Table S1.

Deceased on October 20, 2003. We dedicate this paper to the memory of Rupert Timpl, who greatly contributed to the progress on the biochemistry and the structure of BM-40 (SPARC).

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Shriners Hospital for Children, 3101 SW Sam Jackson Park Rd., Portland, OR 97239. Fax: 503-221-3451; E-mail: txs{at}shcc.org.

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