Molecular Features of the Collagen V Heparin Binding Site

Abstract

A heparin binding region is known to be present within the triple helical part of the ␣1(V) chain. Here we show that a recombinant ␣1(V) fragment (Ile 824 to Pro 950 ), referred to as HepV, is sufficient for heparin binding at physiological ionic strength. Both native individual ␣1(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 ␣1(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 ␣1(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 glycosaminoglycanscollagen V interactions, but the different affinities observed likely modulate cell and matrix interactions between collagen V and heparan sulfate proteoglycans in tissues.Collagen V is a fibrillar collagen that plays an important role in fibrillogenesis, and it also 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). Collagen V interacts with matrix proteoglycans such as the two small proteoglycans decorin and biglycan (2), the proteoglycan form of macrophage colony-stimulating factor (3), the cell surface proteoglycan syndecan-1 (4, 5), and as shown recently, the membrane spanning proteoglycan NG2 (6). Some of these interactions are mediated by the core proteins, but others depend on the glycosaminoglycan chains such as the heparan sulfate chains.Apart from in vitro binding of collagen V to membranespanning proteoglycans, the suggestion that heparan sulfate interacts with collagen V was supported by inhibition experiments showing a reduction of cell attachment to collagen V in the presence of heparin (7). It has been shown already that cell focal adhesion on fibronectin requires the cooperation of both cell transmembrane proteoglycans and integrin receptors (8).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....

Unraveling the Amino Acid Sequence Crucial for Heparin Binding to Collagen V

“…Indeed, direct evidence was obtained from collagen V gene mutations that provoke obvious alteration of fibril aggregates (1)(2)(3)(4). Aside from its role in collagen fibril formation, collagen V interacts specifically with a variety of macromolecules in the extracellular matrix (5) and with several cell-surface receptors such as integrins (6,7), tyrosine kinase receptors (8,9), and proteoglycans (10,11). Such interactions are important in regulating cell behavior and fibril formation during development and physiological events.…”

“…Using a recombinant approach, we have narrowed this region down to a 12-kDa fragment referred to as HepV. Interestingly, the recombinant fragment HepV expressed in Escherichia coli was shown to support heparindependent cell adhesion (11).…”

“…The binding site is exclusive to this chain since the two other chains, viz. ␣2(V) and ␣3(V), which can also be part of collagen V molecules, have no affinity for heparin (11)(12)(13). Using a recombinant approach, we have narrowed this region down to a 12-kDa fragment referred to as HepV.…”

Le syndrome d’Ehlers-Danlos : l’architecture matricielle en question

“…Tout d'abord, rien ne permet d'exclure que les formes molé-culaires du collagène V trouvées dans la cornée soient rigoureusement identiques à celles présentes dans le tendon. Il existe différentes isoformes du collagène V qui pourraient assumer des rôles différents, comme le suggèrent nos travaux [36][37][38]. Compte tenu du profil d'expression de chacune des isoformes, l'hypothèse d'une fonction dépendante du tissu paraît également plausible.…”

“…Les maladies matricielles doivent donc aussi se concevoir en intégrant l'aspect cellulaire. Plusieurs récepteurs cellulaires ont été identifiés pour le collagène V, mais leur rôle in vivo reste à élu-cider [37,[41][42][43]. La connaissance des causes moléculaires responsables de l'EDS a beaucoup progressé, mais est loin d'être exhaustive.…”