Interaction with Heparin and Matrix Metalloproteinase 2 Cleavage Expose a Cryptic Anti-adhesive Site of Fibronectin

Abstract

We recently found that fibronectin (FN) had a functional site [YTIYVIAL sequence in the heparin-binding domain 2 (Hep 2)] that was capable of suppressing the integrin-mediated cell adhesion to extracellular matrix. However, our 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 anti-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 fragment), 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 fragment from a more compact form to an unfolded one. Incubation of the Hep 2 fragment with heparin also induced similar conformational changes and expression of anti-adhesive activity. Additionally, both the urea and heparin treatments made the Hep 2 fragment and intact FN much more accessible to the polyclonal antibody (alphaIII14A), with a recognition site near the anti-adhesive site of FN. Specific cleavage of either the Hep 2 fragment or intact 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-adhesive site on the amino-terminal region. Thus, the cryptic anti-adhesive activity of FN can be expressed upon conformational change and proteolytic cleavage of Hep 2 domain.

Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing

“…42,43 Increased wound expression of fibronectin in castrated rats may contribute to the marked enhancement of repair in such animals. Moreover, it may be linked directly to the parallel decreases in wound gelatinase activities, since fibronectin has been identified as a substrate of both MMP-2 14 and MMP-9. 15 In venous ulceration, the serine protease neutrophil elastase contributes to the uncontrolled degradation of wound bed fibronectin.…”

“…Gelatinase A (MMP-2) also is expressed by the epidermis following injury and, along with gelatinase B (MMP-9), 12 by infiltrating macrophages. It is worth noting also that MMP-2 and MMP-9 possess elastolytic 13 and fibronectin-cleaving capabilities, 14,15 suggesting that neodermal elastin and fibronectin may be susceptible to degradation by locally secreted gelatinases. Controlled cleavage of provisional neomatrix constituents by MMP-2 contributes to connective tissue remodeling.…”

Gelatinase-mediated migration and invasion of cancer cells

“…MMP-2 can cleave fibronectin in the heparin-binding domain. Curiously, this cleavage exposes an antiadhesive site for integrins, which suppresses cell adhesion (Watanabe et al, 2000).…”

Inhibition of Angiogenesis by a Tenascin-C Peptide which Is Capable of Activating Beta1-Integrins

“…After a 90 min-incubation, the number of cells adhered on the FN substrate was counted as described previously. 14) Each point represents the meanϮS.E. of triplicate determinations.…”

“…13) These cryptic sites appear to become functionally exposed by processing the FN and TN-C molecules with matrix metalloproteinase-2 (MMP-2). 13,14) It may be expected that modulation of b1-integrin function contributes to modulation of cellular processes, such as cell proliferation and migration. These integrin function regulatory peptides, FNIII14 and TNIIIA2, may be useful for control of angiogenesis.…”