We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mM lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII ␤-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.The extracellular matrix protein elastin is responsible for the elastic properties of tissues such as lung, skin, and large arteries (1-3). Due to its numerous cross-links and the extreme hydrophobicity of its tropoelastin chains, elastin is highly resistant to proteolysis. However, during inflammatory disorders, proteinases secreted from polymorphonuclear neutrophils, such as elastase, cathepsin G, and gelatinase B may cause significant elastolysis (4).It has been established that peptides derived from elastin or from the hydrophobic domains of tropoelastin interact with cells via a cell surface-resided 67-kDa elastin-binding protein identical to an enzymatically inactive, alternatively spliced form of ␤-galactosidase (5). The binding of elastin peptides to the elastin-binding protein (EBP) 1 has been shown to be responsible for chemotaxis to the peptides (6 -12), stimulation of cell proliferation (13-16), ions flux modifications (17, 18), vasorelaxation (19 -22), and enzymes secretion (23,24).Matrix metall...

The binding of elastin peptides on the elastin receptor complex leads to the formation of intracellular signals but how this is achieved remains totally unknown. Using pharmacological inhibitors of the enzymatic activities of its subunits, we show here that the elastin peptide-driven ERK1/2 activation and subsequent pro-MMP-1 production, observed in skin fibroblasts when they are cultured in the presence of these peptides, rely on a membrane-bound sialidase activity. As lactose blocked this effect, the elastin receptor sialidase subunit, Neu-1, seemed to be involved. The use of a catalytically inactive form of Neu-1 and the small interfering RNA-mediated decrease of Neu-1 expression strongly support this view. Finally, we report that N-acetyl neuraminic acid can reproduce the effects of elastin peptides on both ERK1/2 activation and pro-MMP-1 production. Altogether, our results indicate that the enzymatic activity of the Neu-1 subunit of the elastin receptor complex is responsible for its signal transduction, presumably through sialic acid generation from undetermined substrates.Elastin is the extracellular matrix protein responsible for the elasticity of tissues. It is more abundant in tissues where resilience is required, such as skin, lung, ligaments, or large arteries (1). Elastin is constituted of tropoelastin molecules covalently bound to each other by covalent cross-links (2) and its hydrophobic and highly cross-linked nature make of it a very durable polymer experiencing essentially no turnover in healthy tissues (3).The biological role of elastin was originally thought to be restricted to this mechanical function. However, when Senior et al. (4) demonstrated that elastin digestion products were chemotactic for neutrophils and macrophages, it became suddenly apparent that peptides derived from amorphous elastin could modulate cell physiology. In fact, it has been shown because that fibroblasts (5-12), smooth muscle cells (7, 13-15), endothelial cells (16 -19), leukocytes (20, 21), and lymphocytes (22) were sensitive to the presence of these peptides yielding a broad range of biological activities (see Ref. 23 for a review). A corollary of these observations was that those cells do express a receptor for elastin peptides.The elastin receptor complex is constituted of three subunits, one peripheral 67-kDa subunit, which actually binds elastin, and two membrane-associated proteins of 61 and 55 kDa, respectively (10). The 67-kDa elastin-binding protein (EBP) 2 binds the VGVAPG elastin sequence with high affinity. Additionally, EBP can be eluted from elastin affinity column by galactosugars suggesting that the elastin-EBP interaction could be regulated by galactosugars bound on a lectin site on EBP (10, 24). Consequently, galactosugars such as lactose are commonly used antagonists of EBP.Later, the nature of this subunit was revealed by the work of Privitera et al. (25) who have shown that EBP is an enzymatically spliced variant of lysosomal ␤-galactosidase (␤-Gal, EC 3.2.1.23). Consequently, it was hypot...

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....

Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human ␣1 chain of collagen V using an episomal vector. High yields (15 g/ml) of recombinant collagen were secreted in the culture medium. In presence of ascorbate, the ␣1(V) collagen is correctly folded into a stable triple helix as shown by electron microscopy and pepsin resistance. Circular dichroism data confirm the triple-helix conformation and indicate a melting temperature of 37.5°C for the recombinant homotrimer. The major secreted form is a 250-kDa polypeptide (␣1FL). N-terminal sequencing and collagenase digestion indicate that ␣1FL retains the complete N-propeptide but lacks the C-propeptide. However, ␣1FL might undergo a further N-terminal trimming into a form (␣1TH) corresponding to the main triple-helix domain plus the major part of the NC2 domain. This processing is different from the one of the heterotrimeric (␣1(V)) 2 ␣2(V) and could have some physiological relevance. Analysis of cell homogenates indicates the presence of a 280-kDa polypeptide that is disulfide--linked through its C-terminal globular domain. This C-propeptide is rapidly cleaved after secretion in the medium, giving the first evidence of a C-terminal processing of recombinant fibrillar collagens. Rotary shadowing observations not only confirm the presence of a globular domain at the N-terminal end of the molecule but reveal the presence of a kink within the triple helix in a region poor in iminoacids. This region could represent a target for proteases. Together with the thermal stability data, these results might explain the low amount of (␣1(V)) 3 recovered from tissues.Fibrillar collagens represent the most abundant structural proteins in the extracellular matrix. They all participate in the elaboration of the fibrillar network and thus to the extracellular matrix architecture (1). However, the minor collagens V and XI can be distinguished from the others by their capacity to control fibrillogenesis (2, 3). All fibrillar collagens are composed of a major triple-helix domain (COL1) flanked by two noncollagenous domains, namely the N-propeptide (NC2, COL2, and NC3) and the C-propeptide (NC1). Whereas collagens I, II, and III undergo a processing that reduces the molecule mainly to the triple-helix domain, collagen V retains a large part of the N-propeptide in the mature molecule (4, 5). This propeptide forms a globular domain that could dictate the fibril diameter by sterically inhibiting the accretion of collagen I within heterotypic fibrils (6). In addition to the importance of the Npropeptide retention, heterotypic fibrils were shown to be thinner in tissues where the amount of collagen V is particularly high (7-11), and conversely, a reduction in the proportion of collagen V molecules alters the regulation of fibrillogenesis (12). Significantly, genetic alteration of collagen V molecules impairs the control of matrix assembly (13-16). Therefore, despite being a quantitatively minor collagen, collagen V is involved in fundamental processes such a...

Background:The interaction of the peptide VGVAPG with the elastin binding protein is critically involved in aneurysm progression.Results: A molecular model of this interaction is proposed and explored using a site-directed mutagenesis strategy. Conclusion: Three residues, Leu-103, Arg-107, and Glu-137, of elastin binding protein are critical players in this interaction. Significance: Our data now allow the design of antagonists of VGVAPG.