C. Lethias et al., Identification and characterization of a conformational heparin-binding site involving two fibronectin type III modules of bovine tenascin-X, J BIOL CHEM, 276(19), 2001, pp. 16432-16438
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.