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J. Biol. Chem., Vol. 276, Issue 19, 16432-16438, May 11, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/19/16432    most recent M010210200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lethias, C. Articles by Garrone, R. Search for Related Content PubMed PubMed Citation Articles by Lethias, C. Articles by Garrone, R. Social Bookmarking                      What's this?

Identification and Characterization of a Conformational Heparin-binding Site Involving Two Fibronectin Type III Modules of Bovine Tenascin-X^*

Claire Lethias, Florent Elefteriou, Goetz Parsiegla, Jean-Yves Exposito, and Robert Garrone

From the Institut de Biologie et Chimie des Protéines, CNRS UMR 5086, Université Claude Bernard, 7 passage du Vercors, 69367 Lyon Cedex 07, France

Tenascin-X is known as a heparin-binding molecule, but the localization of the heparin-binding site has not been investigated until now. We show here that, unlike tenascin-C, the recombinant fibrinogen-like domain of tenascin-X is not involved in heparin binding. On the other hand, the two contiguous fibronectin type III repeats b10 and b11 have a predicted positive charge at physiological pH, hence a set of recombinant proteins comprising these domains was tested for interaction with heparin. Using solid phase assays and affinity chromatography, we found that interaction with heparin was conformational and involved both domains 10 and 11. Construction of a three-dimensional model of domains 10 and 11 led us to predict exposed residues that were then submitted to site-directed mutagenesis. In this way, we identified the basic residues within each domain that are crucial for this interaction. Blocking experiments using antibodies against domain 10 were performed to test the efficiency of this site within intact tenascin-X. Binding was significantly reduced, arguing for the activity of a heparin-binding site involving domains 10 and 11 in the whole molecule. Finally, the biological significance of this site was tested by cell adhesion studies. Heparan sulfate cell surface receptors are able to interact with proteins bearing domains 10 and 11, suggesting that tenascin-X may activate different signals to regulate cell behavior.

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