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

Authors

  • Jean-Philippe KLEMAN,

    1. Institute for Biology and Chemistry of Proteins, Centre National de la Recherche Scientifique, Unité Propre de Recherche 412, Lyon and Ecole Normale Supérieure de Lyon, France
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  • Daniel J. HARTMANN,

    1. Centre de radionanalyse, Institut Pasteur, Lyon, France
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  • Francesco RAMIREZ,

    1. Brookdale Center for Molecular Biology, Mt Sinai School of Medicine, New York, USA
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  • Michel van der REST

    Corresponding author
    1. Institute for Biology and Chemistry of Proteins, Centre National de la Recherche Scientifique, Unité Propre de Recherche 412, Lyon and Ecole Normale Supérieure de Lyon, France
      Correspondence to M. van der Rest, Ecole Normale Supérieure de Lyon, 46, Allée d'Italie, F-69364 Lyon Cedex 07, France
      Fax: +33 72 72 26 02.
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  • Note. This is article 108 from the Brookdale Centre for Molecular Biology at Mt Sinai, USA.

Correspondence to M. van der Rest, Ecole Normale Supérieure de Lyon, 46, Allée d'Italie, F-69364 Lyon Cedex 07, France
Fax: +33 72 72 26 02.

Abstract

The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of α1(XI) and α2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [α1(XI)]2α2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing α2(V) but not α1(XI) 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 β-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 β-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.

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