Another look at collagen V and XI molecules.

The fibrillar collagens are the most abundant proteins of extracellular matrices. Among them, collagens V and XI are quantitatively minor components which participate in the formation of the fibrillar collagen network. Since these collagens were discovered, studies have demonstrated that they may play a fundamental role in the control of fibrillogenesis, probably by forming a core within the fibrils. Another characteristic of these collagens is the partial retention of their N-propeptide extensions in tissue forms, an unusual observation in comparison to the other known fibrillar collagens. The tissue locations of collagens V and XI are different, but their structural and biological properties seem to be closely related. It has been shown that their primary structures are highly conserved at both the gene and protein levels, and that these conserved features are the bases of their similar biological properties. In particular, they are both resistant to mammalian collagenases, and surprisingly sensitive to trypsin treatment. Collagens V and XI are usually buried within the major collagen fibrils, although they have both cell adhesion and heparin binding sites which could be of crucial importance in physiological processes such as development and wound healing. It has became evident that several molecules are in fact heterotypic associations of chains from both collagens V and XI, demonstrating that these two collagens are not distinct types but a single type which can be called collagen V/XI.

Influence of glycosaminoglycans on neurite morphology and outgrowth patterns in vitro.

The neuritic growth patterns obtained on substrates made of several glycosaminoglycans (GAGs) bound to type I collagen were analysed and compared in primary cultures of chick embryo dorsal root ganglion grown in serum-free supplemented medium. In 2-day cultures grown on type I collagen or heparan sulphate (HS)-collagen surfaces, ganglionic explants exhibit a dense, symmetrical network of long, parallel neuritic processes and very few flat migrating non-neuronal cells. In contrast, on either dermatan sulphate (DS), chondroitin-6-sulphate (C6S) or hyaluronic acid (HA)-bound collagen substrates, neurons form irregular nerve fibre patterns; indeed, neurites follow convoluted paths and often, after abrupt turns, totally reverse their direction of extension. Experiments were carried out in which a choice was given to growing neural processes between collagen or GAG-collagen substrates. While growth cones elongating over type I collagen easily cross the border with HS-bound collagen surface and indiscriminately extend on this substrate, in contrast, neurites generally avoid surfaces coated with DS, C6S or HA and change their direction of growth in order to stay on collagen. The binding of DS, C6S or HA, but not HS, to type I collagen thus decreases its ability to promote neurite elongation. The interaction of neuronal cells with these extracellular matrix components by restricting neurites in their paths of extension may, therefore, play a role in the patterning of the nervous circuitry.

Effects of tunicamycin on the avoidance reaction of epidermis by sensory neurites in co-cultures.

In 7-day chick embryo dorsal root ganglia and epidermis or dermis co-cultures, nerve fibres establish contacts with dermis while avoiding epidermis. Previous results have indicated that factor(s) secreted by epidermis could be involved in this avoidance reaction. The present study demonstrates that the avoidance reaction is abolished when epidermal cells are treated by the N-linked glycoproteins synthesis inhibitor, tunicamycin. The same result is obtained after monensin treatment. The epidermal cell viability, development and total protein secretion are not significantly affected by tunicamycin, as demonstrated by trypan blue exclusion, electron microscopy and SDS-PAGE electrophoresis after 35S-methionine labelling. It has thus been concluded that the avoidance factor is glycoproteic in nature. It is also suggested that this factor possibly contains chondroitin-6-sulphate moieties.

Involvement of a chondroitin sulfate proteoglycan in the avoidance of chick epidermis by dorsal root ganglia fibers: a study using beta-D-xyloside.

In 7-day chick embryo dorsal root ganglia and epidermis cocultures, nerve fibers avoid the epidermis. Previous studies have indicated that glycoproteic factors, secreted by epidermis, could be involved in this phenomenon. Treatment of epidermis by beta-D-xyloside, a specific proteoglycan synthesis inhibitor, abolishes the avoidance reaction. The same result is obtained when anti-chondroitin sulfate antibodies are added to the culture medium. Using HPLC and 35SO4 labeling combined with chondroitinase and hyaluronidase treatment, it has been demonstrated that chondroitin sulfate is present in the epidermal conditioned medium. This suggests that a chondroitin sulfate proteoglycan secreted by the epidermis is implicated in the neurite avoidance reaction and that epidermis could therefore control its own "noninnervation". In vivo, inhibitory influences by local extracellular components may control the guidance of growth cones during nerve pattern formation.

Differentiation of pure chick embryo epidermis grown in primary serum-free culture.

The differentiation of precocious embryonic epidermis in serum-free primary culture was analyzed by light and electron microscopic methods. Explants of 7-day chick embryo epidermis were grown on collagen or poly-L-lysine substrates in the absence of dermal mesenchyme. The serum substitute consisted of a mixture of insulin, transferrin, putrescine and seleneous acid together with (or without) Nerve Growth Factor. These culture conditions were shown to support proliferation, growth and development (evaluated using morphological criteria) of the epidermal explants up to 4-5 days; during this period, the epidermis underwent stratification; well-developed desmosomes as well as tonofilaments were formed and the epidermis achieved a morphology close to that of 10-11 day epidermis in ovo. However long-term survival of the explants was not obtained as cellular death, starting on day 5, progressively led to the necrosis of most parts of the explant. This morphological study demonstrates that the early phases of epidermal growth and maturation can occur to some extent in the virtual absence of dermal elements and serum factors. Chick embryo epidermal cells may thus possess the intrinsic ability to go through, at least for short periods in vitro, their differentiation programme. Then, at the onset of epidermal keratinization (12 days in ovo), they require specific exogenous factors to fully differentiate in vitro.

Unraveling the amino acid sequence crucial for heparin binding to collagen V.

We have previously shown that a recombinant 12-kDa fragment of the collagen alpha1(V) chain (Ile(824)-Pro(950)), referred to as HepV, binds to heparin and heparan sulfate (Delacoux, F., Fichard, A., Geourjon, C., Garrone, R., and Ruggiero, F. (1998) J. Biol. Chem. 273, 15069-15076). No consensus sequence was found in the alpha1(V) primary sequence, but a cluster of 7 basic amino acids (in the Arg(900)-Arg(924) region) was postulated to contain the heparin-binding site. The contribution of individual basic amino acids within this sequence was examined by site-directed mutagenesis. Further evidence for the precise localization of the heparin-binding site was provided by experiments based on the fact that heparin can protect the alpha1(V) chain heparin-binding site from trypsin digestion. The results parallel the alanine scanning mutagenesis data, i.e. heparin binding to the alpha1(V) chain involved Arg(912), Arg(918), and Arg(921) and two additional neighboring basic residues, Lys(905) and Arg(909). Our data suggest that this extended sequence functions as a heparin-binding site in both collagens V and XI, indicating that these collagens use a novel sequence motif to interact with heparin.

Molecular features of the collagen V heparin binding site.

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.

Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer.

The processing of the fibrillar procollagen precursors to mature collagens is an essential requirement for fibril formation. The enzymes involved in these events are known as the procollagen N and C proteinases. The latter, which cleaves the C-propeptides of the fibrillar procollagens I-III, is identical to the previously described bone morphogenetic protein-1 (BMP-1). Surprisingly, unlike the other fibrillar collagens, the processing of the C-propeptide domain of the procollagen V homotrimer was found to be mediated by furin rather than BMP-1. However, the presence of putative BMP-1 cleavage sites in the alpha1(V) C-propeptide sequence prompted us to reconsider the procollagen V C-propeptide cleavage by BMP-1. Using a recombinant system to produce substantial amounts of the proalpha1(V) homotrimer, we have previously shown that the C-propeptide is spontaneously released in the culture medium. The trimeric C-propeptide fragment, resulting from the furin cleavage, still encompassed the predicted BMP-1 cleavage sites. It was purified and tested as a substrate for BMP-1. In parallel, the release of the C-propeptide in the culture medium was inhibited by the addition of a specific furin inhibitor, allowing the re-examination of BMP-1 activity on the intact molecule. We showed that BMP-1 does cleave both substrates at one of the two predicted C-proteinase cleavage sites. Our results favor a role for PCP/BMP-1 in physiological C-terminal processing of procollagen V and imply a general mechanism for fibrillar collagen C-terminal processing.

Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing.

Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human alpha1 chain of collagen V using an episomal vector. High yields (15 microgram/ml) of recombinant collagen were secreted in the culture medium. In presence of ascorbate, the alpha1(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 degrees C for the recombinant homotrimer. The major secreted form is a 250-kDa polypeptide (alpha1FL). N-terminal sequencing and collagenase digestion indicate that alpha1FL retains the complete N-propeptide but lacks the C-propeptide. However, alpha1FL might undergo a further N-terminal trimming into a form (alpha1TH) 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 (alpha1(V))2alpha2(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 (alpha1(V))3 recovered from tissues.

Control of heterotypic fibril formation by collagen V is determined by chain stoichiometry.

Although the collagen V heterotrimer is known to be involved in the control of fibril assembly, the role of the homotrimer in fibrillar organization has not yet been examined. Here, the production of substantial amounts of recombinant collagen V homotrimer has allowed a detailed study of its role in homotypic and heterotypic fibril formation. After removal of terminal regions by pepsin digestion, both the collagen V heterotrimer and homotrimer formed thin homotypic fibrils, thus showing that diameter limitation is at least in part an intrinsic property of the collagen V triple helix. When mixed with collagen I, however, various complementary approaches indicated that the collagen V heterotrimer and homotrimer exerted different effects in heterotypic fibril formation. Unlike the heterotrimer, which was buried in the fibril interior, the homotrimer was localized as thin filamentous structures at the surface of wide collagen I fibrils and did not regulate fibril assembly. Its localization at the fibril surface suggests that the homotrimer can act as a molecular linker between collagen fibrils or macromolecules in the extracellular matrix or both. Thus, depending on their respective distribution in tissues, the different collagen V isoforms might fulfill specific biological functions.