Efficient expression of chicken α1 (VI) collagen chain in transiently transfected mammalian cells

P. Bonaldo, M. T. Mucignat, A. Colombatti

Research output: Contribution to journalArticle

Abstract

Type VI collagen is a component of the extracellular matrix made of three subunits, α1 (VI) and α2(VI) (M(r) = 140,000), and α3 (VI) (M(r) = > 300,000). Triple helical monomers assemble intracellularly into disulfide-linked dimers and tetramers, with the tetramers being the 'building blocks' that give rise to higher order extracellular structures by head-to-head association, the microfilaments. To study the pattern of assembly and the structure-function relationships of type VI collagen, we transfected mammalian cells with a full-length cDNA coding for chicken α1(VI) under the control of SV40 early and late promoters and assayed the expression, secretion, and assembly of the protein by immunoperoxidase and immunoprecipitation of metabolically labeled cells. First, conditions were determined that allowed efficient transfection both in African monkey kidney COS-1 and CV-1 cells and in mouse fibroblasts. In our hands the late promotor was most efficient in CV-1 cells; whereas the early promoter was efficient in L cells at three days post-transfection. Chicken α1(VI) could be isolated from cell extracts as well as from cell medium. Both the intracellular and the secreted forms of α1(VI) are present as a monomer polypeptide and as disulfide-linked dimers and trimers that migrate in SDS gels with apparent M(r) of about 130,000, 240,000 and 360,000, repectively. In L cells, endogenous mouse type VI collagen also was isolated by immunoprecipitation with specific antibodies. However, heterologous molecules made of the chicken α1(VI) chain and the mouse α2(VI) and α3(VI) chains were not detected in the present experiments. Digestion with pepsin of the non-reduced chicken α1(VI) polypeptides immunoprecipitated from the cell medium resulted in the disappearance of the bands, suggesting improper or non-stable assembly of α1(VI) homotrimers. These data support predictions from sequence analysis that type VI collagen heterotrimeric molecules are more stable than other assembly alternatives.

Original languageEnglish
Pages (from-to)139-147
Number of pages9
JournalMatrix
Volume10
Issue number3
Publication statusPublished - 1990

Keywords

  • COS cells
  • CV-1 cells
  • immunoprecipitated
  • L cells
  • transient transfection
  • type VI collagen

ASJC Scopus subject areas

  • Rheumatology

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    Bonaldo, P., Mucignat, M. T., & Colombatti, A. (1990). Efficient expression of chicken α1 (VI) collagen chain in transiently transfected mammalian cells. Matrix, 10(3), 139-147.