The streptococcal collagen-binding protein CNE specifically interferes with alphaVbeta3-mediated cellular interactions with triple helical collagen.
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.
van Wieringen
Tijs
T
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-751 23 Uppsala, Sweden.
Kalamajski
Sebastian
S
Lidén
Asa
A
Bihan
Dominique
D
Guss
Bengt
B
Heinegård
Dick
D
Farndale
Richard W
RW
Rubin
Kristofer
K
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D000077214:Becaplermin; D001667:Binding, Competitive; D002152:Calorimetry, Differential Scanning; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D020574:Proto-Oncogene Proteins c-sis; D034622:RNA Interference; D038981:Receptors, Collagen / Q000235:genetics / Q000378:metabolism*; D013291:Streptococcus / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D001426:Bacterial Proteins; D005353:Fibronectins; D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D038981:Receptors, Collagen; D000077214:Becaplermin; D003094:Collagen