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      The Streptococcal Collagen-binding Protein CNE Specifically Interferes with α Vβ 3-mediated Cellular Interactions with Triple Helical Collagen*

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          Abstract

          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 Vβ 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 Vβ 3-dependent cell-mediated collagen gel contraction, PDGF BB-induced and α Vβ 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 Vβ 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 Vβ 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.

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          Fibroblast biology in three-dimensional collagen matrices.

          Research on fibroblast biology in three-dimensional collagen matrices offers new opportunities to understand the reciprocal and adaptive interactions that occur between cells and surrounding matrix in a tissue-like environment. Such interactions are integral to the regulation of connective tissue morphogenesis and dynamics that characterizes tissue homeostasis and wound repair. During fibroblast-collagen matrix remodeling, mechanical signals from the remodeled matrix feed back to modulate cell behavior in an iterative process. As mechanical loading (tension) within the matrix increases, the mechanisms used by cells to remodel the matrix change. Fibroblasts in matrices that are under tension or relaxed respond differently to growth factor stimulation, and switching between mechanically loaded and unloaded conditions influences whether cells acquire proliferative/biosynthetic active or quiescent/resting phenotypes.
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            Measurement of cell numbers by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens.

            By using a chromogenic substrate for an ubiquitous lysosomal enzyme, hexosaminidase to estimate cell numbers, a sensitive and simple procedure has been developed in which microtiter reaction wells are directly scanned in a spectrophotometer. This method has been adapted to several cell biological assays. Quantitation of the biological activities of T cell growth factor and interferon can be performed on large numbers of samples. Adhesion of dispersed solid tissue cells to fibronectin coated substrates may be quantitated with little expenditure of reagents. By use of a panning procedure in microtiter plates a sensitive and very simple assay for the binding of monoclonal antibodies to cell surface antigens has been developed.
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              Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions.

              Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. We have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha2beta1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where O is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.
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                Author and article information

                Journal
                J Biol Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                12 November 2010
                13 September 2010
                13 September 2010
                : 285
                : 46
                : 35803-35813
                Affiliations
                From the []Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-751 23 Uppsala, Sweden,
                the [§ ]Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway,
                the []Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom,
                the []Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden, and
                the [** ]Department of Clinical Sciences Lund, BMC C12 Lund University, SE-221 84 Lund, Sweden
                Author notes
                [1 ] To whom correspondence should be addressed. Tel.: 46-18-4714116; Fax: 46-18-4714673; E-mail: Kristofer.Rubin@ 123456imbim.uu.se .
                Article
                M110.146001
                10.1074/jbc.M110.146001
                2975204
                20837478
                336cfa12-36dd-45ea-8e1f-f1b0ead7a26d
                © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 19 May 2010
                : 10 August 2010
                Categories
                Microbiology

                Biochemistry
                fibronectin,pdgf-bb,inflammation,cne,extracellular matrix,integrin,collagen
                Biochemistry
                fibronectin, pdgf-bb, inflammation, cne, extracellular matrix, integrin, collagen

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