Molecular Features of the Collagen V Heparin Binding Site

Frédéric Delacoux,Christophe Geourjon,Agnès Fichard,Florence Ruggiero,Robert Garrone

doi:10.1074/jbc.273.24.15069

Abstract

A heparin binding region is known to be present within the triple helical part of the alpha1(V) chain. Here we show that a recombinant alpha1(V) fragment (Ile824 to Pro950), referred to as HepV, is sufficient for heparin binding at physiological ionic strength. Both native individual alpha1(V) chains and HepV are eluted at identical NaCl concentrations (0.35 M) from a heparin-Sepharose column, and this binding can be inhibited specifically by the addition of free heparin or heparan sulfate. In contrast, a shorter 23-residue synthetic peptide, containing the putative heparin binding site in HepV, fails to bind heparin. Interestingly, HepV promotes cell attachment, and HepV-mediated adhesion is inhibited specifically by heparin or heparan sulfate, indicating that this region might behave as an adhesive binding site. The same site is equally functional on triple helical molecules as shown by heparin-gold labeling. However, the affinities for heparin of each of the collagen V molecular forms tested are different and increase with the number of alpha1(V) chains incorporated in the molecules. Molecular modeling of a sequence encompassing the putative HepV binding sequence region shows that all of the basic residues cluster on one side of the helical face. A highly positively charged ring around the molecule is thus particularly evident for the alpha1(V) homotrimer. This could strengthen its interaction with the anionic heparin molecules. We propose that a single heparin binding site is involved in heparin-related glycosaminoglycans-collagen V interactions, but the different affinities observed likely modulate cell and matrix interactions between collagen V and heparan sulfate proteoglycans in tissues.

Highlights

Collagen V is a fibrillar collagen that plays an important role in fibrillogenesis, and it acts as an adhesive substrate for a large variety of cells and binds to a number of extracellular components through its major triple helical domain [1]
Expression and Characterization of HepV—We designed a fragment, referred to as HepV, which encompasses the complete NH2-terminal part of the 30-kDa CNBr peptide defined by Yaoi et al [15] and the sequence around the endoproteinase Glu-C cleavage site which was found to be determinant for heparin binding (Fig. 1)
Binding of trypsin-released cells drastically affects cell adhesion to HepV, whereas adhesion to intact collagen V is only partially decreased. These results indicate that Chinese hamster ovary (CHO) cells possess at least two distinct cell surface receptors for collagen V: a trypsin-labile receptor that binds to the heparin binding site, HepV, and a trypsin-resistant receptor that binds to another region of the molecule

Summary

Introduction

Collagen V is a fibrillar collagen that plays an important role in fibrillogenesis, and it acts as an adhesive substrate for a large variety of cells and binds to a number of extracellular components through its major triple helical domain [1]. Because we have demonstrated already that cell-collagen V interactions involved integrins [9, 10], the binding of membrane-spanning proteoglycans could reinforce cell attachment to collagen V and, in that sense, would be of physiological importance. The homotrimer [␣1(V)]3 occurs in cultures of hamster lung cells and was suggested to be present in embryonic tissue [11,12,13] It was produced recently as a recombinant molecule [14]. The heterotrimeric [␣1(V)]2␣2(V) molecular form was shown previously to bind to heparin at physiological salt concentrations. This activity was attributed to a proteolytic NH2-terminal 30-kDa fragment of the ␣1(V) chain [15]

Methods

Results

Conclusion