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J. Biol. Chem., Vol. 279, Issue 23, 24265-24273, June 4, 2004

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Characterization of Recombinant Amino-terminal NC4 Domain of Human Collagen IX

INTERACTION WITH GLYCOSAMINOGLYCANS AND CARTILAGE OLIGOMERIC MATRIX PROTEIN^*

From the ^aNMR Laboratory, the ^eProtein Chemistry Laboratory, the ^jProgram in Cellular Biotechnology, and the ^hHelsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland, the ^cPeptide and Protein Laboratory, Department of Virology, Haartman Institute, University of Helsinki, FI-00014 Helsinki, Finland, the ^dCenter for Gene Therapy and ^kDepartment of Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, the ^fCollagen Research Unit, Biocenter and Department of Medical Biochemistry and Molecular Biology, University of Oulu, FI-90014 Oulu, Finland, the ^gCenter for Biochemistry, Medical Faculty, University of Cologne, D-50931 Cologne, Germany, the ⁱDepartment of Cell and Molecular Biology, Section of Molecular Pathogenesis, Lund University, S-22184 Lund, Sweden, and the ^lDepartment of Chemistry, Laboratory of Organic Chemistry, University of Helsinki, FI-00014 Helsinki, Finland

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K_d of 0.6 µM, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K_d of 3.6 nM. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 °C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.

Received for publication, March 15, 2004

^* This work was funded by grants from the Academy of Finland (to I. K. and L. A.-K.) and by Academy of Finland Grants 75963 (to T. P.) and 42739 (to A. P.) and was partially supported by the Louisiana Gene Therapy Research Consortium (New Orleans, LA) and HCA-The Health Care Company (Memphis, TN) and by National Institutes of Health Grant AR45982 (to L. A.-K.). 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.

^b To whom correspondence and reprint requests should be addressed: NMR Laboratory, Institute of Biotechnology, P.O. Box 65, University of Helsinki, FI-00014 Helsinki, Finland. Tel.: 358-9-19159544; Fax: 358-9-19159541; E-mail: Tero.Pihlajamaa{at}helsinki.fi.

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