The human rhabdomyosarcoma cell line A204 lays down a highly insoluble matrix composed mainly of α1 type‐XI and α2 type‐V collagen chains

Abstract

The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen typeV and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of al(X1) and a2(V) collagen chains in an apparent 2 : l ratio, suggesting the formation of heterotypic molecules [al(XI)lza2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing a2(V) but not al(X1) collagen chains.Electron microscopy analyses of 24-h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold-labelled anti-(type-V collagen) IgG, but not with anti-(type-XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of P-aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of P-aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti-(type-V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti-(type-XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa.These data indicate that type-V and type-XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.Collagen types V and XI are members of the collagens forming quarter-staggered fibrils (Woodbury et al., 1989;Kimura et al., 1989;Weil et al., 1987). Type-V collagen is commonly found as a quantitatively minor component in collagen-type-I-containing extracellular matrices such as those of placenta, skin and bone (Niyibizi et al., 1984;Birk et al., 1988;Niyibizi and Eyre, 1989a). Type-XI collagen is found in association with type I1 in cartilaginous matrices (Mendler et al., 1989). This led to the dichotomous view that fibrilforming collagens can be subdivided into two sub-groups, according to their tissue distribution, (a) collagens associated with collagen type I in non-cartilaginous tissues, in particular type V, (b) collagens associated with type I1 in cartilaginous matrices, such as type-XI collagen. Recent data indicate that collagen types V and XI are not so strictly partitioned as initially thought. First, they are thought to play a similar role in collagen fibrillogenesis (Morris and Bichinger, 1987;Morris et al., 1990), possibly by forming a core to the fibril (Birk et al., 1990;Fessler et al., 1985a;Mendler et al., 1989). These two molecules are both immunologically masked and require partial collagen fibril disruption for immunological detection (Birk et al., 1988). Furthermore, close homologies have been reported between type-V and type-XI collagen chains. The al(X1) a...

The bone morphogenetic protein 1/Tolloid-like metalloproteinases

“…Type XI collagen consists of α1(XI)α2(XI)α3(XI) heterotrimers (Morris and Bachinger, 1987). In addition, heterotrimers containing α chains of both type V and type XI collagens have been reported (Kleman et al, 1992;Mayne et al, 1993), suggesting that V and XI may represent a single collagen type with various α chains expressed differentially in a tissue-specific manner. BMP1/TLD-like proteinases cleave the Cpropeptides of proα2 (V) chains (Unsold et al, 2002), which are more closely related in protein domain structure to the chains of the major fibrillar procollagens than are the proα1(V), proα1 (XI), and proα2(XI) chains.…”

Diversity in the processing events at the N‐terminus of type‐V collagen

“…To clarify this point, we used a rhabdomyosarcoma cell line (A204) which was reported to A B synthesize [al(XI)],a2(V) collagen. All the intermediates of a2(V) chain processing are present in the culture media of these cells (Kleman et al, 1992). We compared by immunodetection on the same gel the migration of the intact a2(V) chain from human bone with these processed forms of a2(V) chain.…”

“…Collagen preparation from human A204 cell media (ATCC HTB 82) was performed as previously described (Kleman et al, 1992). The A204 cells were cultured to near confluency for 24 h in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 50 pg/ml sodium ascorbate.…”

“…The rabbit polyclonal antibodies against pepsin-digested human placental type-V collagen (a generous gift of Dr D. Hartmann, Institut Pasteur de Lyon), used as an IgG fraction (anti-V IgG), were described previously (Kleman et al, 1992).…”

The Transcription Factor CCAAT-binding Factor CBF/NF-Y and Two Repressors Regulate the Core Promoter of the Human Pro-α3(V) Collagen Gene (COL5A3)

“…Other forms of type V collagen include the [␣1(V)] 3 homotrimer that is synthesized in cultures of hamster lung cells (9) and the ␣1(V)␣2(V)␣3(V) heterotrimer that can be extracted from the placenta (10 -12). Cross-type heterotrimer composed of ␣2(V) and ␣1(XI) chains is present in the rhabdomyosarcoma cell line A204 and bovine vitreous (13,14). These findings suggest that type V and type XI chains constitute a single collagen type in which different combinations of chains associate in a tissue-specific manner.…”