The Streptococcal Collagen-binding Protein CNE Specifically Interferes with αVβ3-mediated Cellular Interactions with Triple Helical Collagen

Tijs Van Wieringen,Sebastian Kalamajski,Åsa Lidén,Dominique Bihan,Bengt Guss,Dick Heinegård,Richard W Farndale,Kristofer Rubin

doi:10.1074/jbc.m110.146001

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 αVβ3-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited αVβ3-dependent cell-mediated collagen gel contraction, PDGF BB-induced and αVβ3-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 β1 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 αVβ3-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced αVβ3-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.

Highlights

Collagen fibrils packed in the quarter-staggered fashion expose specific domains that interact with other molecules or molecular assemblies of the interstitial matrix, or with cells
This effect of PDGF-BB was in turn dependent on ␣V␤3 integrin, because the contraction was blocked by a cyclic RGD peptide, which inhibits ␣V␤3 integrin function at the concentrations used here
Whereas peptide II-1 inhibited fibrillogenesis measured by change in turbidity of dilute collagen type I or II solutions incubated at 37 °C, peptide II-44 slightly stimulated fibrillogenesis. These findings suggest that the collagen region defined by peptide II-44, and not peptide II-1, is involved in ␣V␤3-mediated PDGF BB-induced contraction, whereas this region is not involved in fibrillogenesis

Summary

Introduction

Collagen fibrils packed in the quarter-staggered fashion expose specific domains that interact with other molecules or molecular assemblies of the interstitial matrix, or with cells (reviewed in Ref. 1). PDGF-BB induces integrin ␣V␤3-dependent collagen gel contraction by these cells [8].

Results

Conclusion