Efficient expression of chicken alpha 1(VI) collagen chain in transiently transfected mammalian cells.
Type VI collagen is a component of the extracellular matrix made of three subunits, alpha 1(VI) and alpha 2(VI) (Mr = 140,000), and alpha 3(VI) (Mr = greater than 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 alpha 1(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 promoter was most efficient in CV-1 cells; whereas the early promoter was efficient in L cells at three days post-transfection. Chicken alpha 1(VI) could be isolated from cell extracts as well as from cell medium. Both the intracellular and the secreted forms of alpha 1(VI) are present as a monomer polypeptide and as disulfide-linked dimers and trimers that migrate in SDS gels with apparent Mr of about 130,000, 240,000 and 360,000, respectively. In L cells, endogenous mouse type VI collagen also was isolated by immunoprecipitation with specific antibodies. However, heterologous molecules made of the chicken alpha 1(VI) chain and the mouse alpha 2(VI) and alpha 3(VI) chains were not detected in the present experiments. Digestion with pepsin of the non-reduced chicken alpha 1(VI) polypeptides immunoprecipitated from the cell medium resulted in the disappearance of the bands, suggesting improper or non-stable assembly of alpha 1(VI) homotrimers. These data support predictions from sequence analysis that type VI collagen heterotrimeric molecules are more stable than other assembly alternatives.
Bonaldo
P
P
Divisione di Oncologia Sperimentale 2, Centro di Referimento Oncologico, Aviano, Italy.
Mucignat
M T
MT
Colombatti
A
A
D000818:Animals; D002460:Cell Line; D002645:Chickens; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005822:Genetic Vectors; D011233:Precipitin Tests; D011487:Protein Conformation; D014162:Transfection
D003094:Collagen