Identification and characterization of a conformational heparin-binding site involving two fibronectin type III modules of bovine tenascin-X

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 contiguou s 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 a nd affinity chromatography, we found that interaction with heparin was conf ormational and involved both domains 10 and 11, Construction of a three-dim ensional model of domains 10 and 11 led us to predict exposed residues that were then submitted to site-directed mutagenesis, In this way, we identifi ed the basic residues within each domain that are crucial for this interact ion, 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 i nvolving domains 10 and 11 in the whole molecule. Finally, the biological s ignificance of this site was tested by cell adhesion studies. Heparan sulfa te cell surface receptors are able to interact with proteins bearing domain s 10 and 11, suggesting that tenascin-X may activate different signals to r egulate cell behavior.