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J. Biol. Chem., Vol. 279, Issue 24, 25562-25566, June 11, 2004

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Identification and Kinetics Analysis of a Novel Heparin-binding Site (KEDK) in Human Tenascin-C^*

Jun-Hyeog Jang, Jung-Hee Hwang, Chong-Pyoung Chung, and Pill-Hoon Choung

From the Intellectual Biointerface Engineering Center, Seoul National University College of Dentistry, Seoul 110-749, Korea

The interaction between tenascin-C (TN-C), a multi-subunit extracellular matrix protein, and heparin was examined using a surface plasmon resonance-based technique on a Biacore system. The aims of the present study were to examine the affinity of fibronectin type III repeats of TN-C fragments (TNIII) for heparin, to investigate the role of the TNIII4 domains in the binding of TN-C to heparin, and to delineate a sequence of amino acids within the TNIII4 domain, which mediates cooperative heparin binding. At a physiological salt concentration, and pH 7.4, TNIII3–5 binds to heparin with high affinity (K_D = 30 nM). However, a major heparin-binding site in TNIII5 produces a modest affinity binding at a K_D near 4 µM, and a second site in TNIII4 enhances the binding by several orders of magnitude, although it was far too weak to produce an observable binding of TNIII4 by itself. Moreover, mutagenesis of the KEDK sequence in the TNIII4 domain resulted in the significant reduction of heparin-binding affinity. In addition, residues in the KEDK sequences are conserved in TN-C throughout mammalian evolution. Thus the structure-based sequence alignment, mutagenesis, and sequence conservation data together reveal a KEDK sequence in TNIII4 suggestive of a minor heparin-binding site. Finally, we demonstrate that TNIII4 contains binding sites for heparin sulfate proteoglycan and enhances the heparin sulfate proteoglycan-dependent human gingival fibroblast adhesion to TNIII5, thus providing the biological significance of heparin-binding site of TNIII4. These results suggest that the heparin-binding sites may traverse TNIII4–5 and thus require KEDK in TNIII4 for optimal heparin-binding.

Received for publication, March 22, 2004 , and in revised form, April 6, 2004.

^* This work was supported by the Korea Science and Engineering Foundation through the Intellectual Biointerface Engineering Center (IBEC) at Seoul National University. 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.

To whom correspondence should be addressed: IBEC, Seoul National University College of Dentistry, 28 Yongon-dong, Chongno-gu, Seoul 110-749, Korea. Tel.: 82-2-6228-1129; Fax: 82-2-740-8732; E-mail: juhjang{at}snu.ac.kr.

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