1. The Collagen V Homotrimer[α1(V)]3Production Is Unexpectedly Favored over the Heterotrimer[α1(V)]2α2(V)in Recombinant Expression Systems
- Author
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Minna Männikkö, Efrat Kessler, Christelle Bonod-Bidaud, Eija-Riitta Hämäläinen, Muriel Roulet, Merja Välkkilä, Leena Ala-Kokko, Hélène Chanut-Delalande, and Florence Ruggiero
- Subjects
chemistry.chemical_classification ,biology ,Chemistry ,Health, Toxicology and Mutagenesis ,General Medicine ,Transfection ,Collagen trimer ,Molecular biology ,law.invention ,Cell culture ,law ,Complementary DNA ,Chaperone (protein) ,Heterotrimeric G protein ,Genetics ,biology.protein ,Recombinant DNA ,Molecular Medicine ,Glycoprotein ,Molecular Biology ,Biotechnology - Abstract
Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer[α1(V)]2α2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proα1(V) and proα2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of[α1(V)]3homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proα2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection withHsp47cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen Vα-chains showed that, contrary to fibroblasts, collagen Vα-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly.
- Published
- 2010