Interaction with heparin and matrix metalloproteinase 2 cleavage expose a cryptic anti-adhesive site of fibronectin

We recently found that fibronectin (FN) had a functional site [YTIYVIAL seq uence in the heparin-binding domain 2 (Hep 2)] that was capable of suppress ing the integrin-mediated cell adhesion to extracellular matrix. However, o ur results also indicated that this anti-adhesive site seemed to be usually buried within the Hep 2 domain structure because of its hydrophobic nature , raising a question as to the physiological significance of the cryptic an ti-adhesive activity of FN. The present study demonstrates that the cryptic anti-adhesive activity can be exposed through the physiological processes. A 30-kDa chymotryptic FN fragment derived from Hep 2 domain (Hep 2 fragmen t), which had no effect on adhesion of MSV-transformed nonproducer 3T3 cell line (KN(7)8) to FN, expressed the anti-adhesive activity after treatment with 6 M urea. Light scattering and circular dichroism measurements showed that the urea treatment induced the conformational change of the Hep 2 frag ment from a more compact form to an unfolded one. Incubation of the Hep 2 f ragment with heparin also induced similar conformational changes and expres sion of anti-adhesive activity. Additionally, both. the urea and heparin tr eatments made the Hep 2 fragment and intact FN much more accessible to the polyclonal antibody (alpha III14A), with a recognition site near the anti-a dhesive site of FN. Specific cleavage of either the Hep 2 fragment or intac t FN by matrix metalloproteinase 2 (MMP-2) released a 10-kDa fragment with the anti-adhesive activity, which was shown to have the exposed anti-adhesi ve site on the amino-terminal region. Thus, the cryptic anti-adhesive activ ity of FN can be expressed upon conformational change and proteolytic cleav age of Hep 2 domain.