title: 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.
authors.affiliation: Nippi Research Institute of Biomatrix, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Watanabe
authors.forename: H
authors.initials: H
authors.lastname: Takahashi
authors.forename: Y
authors.initials: Y
authors.lastname: Habu
authors.forename: N
authors.initials: N
authors.lastname: Kamiya-Kubushiro
authors.forename: S
authors.initials: S
authors.lastname: Kamiya
authors.forename: H
authors.initials: H
authors.lastname: Nakamura
authors.forename: H
authors.initials: H
authors.lastname: Yajima
authors.forename: T
authors.initials: T
authors.lastname: Ishii
authors.forename: T
authors.initials: T
authors.lastname: Katayama
authors.forename: K
authors.initials: K
authors.lastname: Miyazaki
authors.forename: F
authors.initials: F
authors.lastname: Fukai
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004058:Diffusion; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D012542:Scattering, Radiation; D014508:Urea / Q000494:pharmacology
chemical_list: D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D016023:Integrins; D010446:Peptide Fragments; D014508:Urea; D006493:Heparin; D020778:Matrix Metalloproteinase 2