The streptococcal collagen-binding protein CNE specifically interferes with alphaVbeta3-mediated cellular interactions with triple helical collagen

• Published in: The Journal of biological chemistry Vol. 285; no. 46; p. 35803
• Main Authors: van Wieringen, Tijs, Kalamajski, Sebastian, Lidén, Asa, Bihan, Dominique, Guss, Bengt, Heinegård, Dick, Farndale, Richard W, Rubin, Kristofer
• Format: Journal Article
• Language: English
• Published: United States 12.11.2010
• Subjects: Amino Acid Sequence; Animals; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Becaplermin; Binding, Competitive; Calorimetry, Differential Scanning; Cell Adhesion - drug effects; Cell Line; Cells, Cultured; Collagen - chemistry; Collagen - metabolism; Collagen - ultrastructure; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - metabolism; Fibronectins - genetics; Fibronectins - metabolism; Humans; Integrin alphaVbeta3 - genetics; Integrin alphaVbeta3 - metabolism; Microscopy, Electron; Myoblasts - cytology; Myoblasts - drug effects; Myoblasts - metabolism; Oligopeptides - metabolism; Oligopeptides - pharmacology; Platelet-Derived Growth Factor - pharmacology; Protein Binding; Protein Conformation; Protein Structure, Secondary; Proto-Oncogene Proteins c-sis; Receptors, Collagen - genetics; Receptors, Collagen - metabolism; RNA Interference; Streptococcus - genetics; Streptococcus - metabolism; Transfection
• ISSN: 1083-351X
• DOI: 10.1074/jbc.M110.146001

Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin α(V)β(3)-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited α(V)β(3)-dependent cell-mediated collagen gel contraction, PDGF BB-induced and α(V)β(3)-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding β(1) integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited α(V)β(3)-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced α(V)β(3)-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation.