The collagen V homotrimer [alpha1(V)](3) production is unexpectedly favored over the heterotrimer [alpha1(V)](2)alpha2(V) in recombinant expression systems.
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 [alpha1(V)](2)alpha2(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 proalpha1(V) and proalpha2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of [alpha1(V)](3) homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proalpha2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection with Hsp47 cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen V alpha-chains showed that, contrary to fibroblasts, collagen V alpha-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.
Roulet
Muriel
M
Institut de Biologie et Chimie des Protéines, UMR CNRS 5086, Université Lyon 1, IFR128 Biosciences Gerland, 7 passage du Vercors, Lyon, France.
Välkkilä
Merja
M
Chanut-Delalande
Hélène
H
Hämäläinen
Eija-Riitta
ER
Kessler
Efrat
E
Ala-Kokko
Leena
L
Männikkö
Minna
M
Bonod-Bidaud
Christelle
C
Ruggiero
Florence
F
D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D024062:Collagen Type V / Q000096:biosynthesis* / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D050958:HSP47 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007313:Insecta / Q000166:cytology; D010843:Pichia / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry*
C530725:COL5A1 protein, human; D024062:Collagen Type V; D050958:HSP47 Heat-Shock Proteins; D011994:Recombinant Proteins