Characterization of recombinant human type IX collagen. Association of alpha chains into homotrimeric and heterotrimeric molecules.
As type IX collagen is a minor cartilage component, it is difficult to purify sufficient amounts of it from tissues or cultured cells to study its structure and function. Also, the conventional pepsin digestion used for fibrillar collagens cannot be utilized for purifying type IX collagen, because it contains several interruptions in its collagenous triple helix. A baculovirus expression system was used here to produce recombinant human type IX collagen by coinfecting insect cells with three viruses containing full-length cDNAs for the alpha1(IX), alpha2(IX), and alpha3(IX) collagen chains together with a double promoter virus for the alpha and beta subunits of human prolyl 4-hydroxylase. Correctly folded recombinant type IX collagen was secreted, consisting of the three alpha chains in a 1:1:1 ratio and showing the expected biphasic thermal melting profile. When the individual alpha chains were expressed, disulfide-bonded homotrimers and homodimers of the alpha chains were observed. When the cells were coinfected with the viruses for all three alpha chains, heterotrimers of alpha1(IX), alpha2(IX), and alpha3(IX) were detected in cell culture medium, and the other possible combinations were less prominent. When any two of the alpha chains were co-expressed, in addition to the homodimers and homotrimers, only alpha1(IX) and alpha3(IX) chains were disulfide-bonded. The results thus suggest that the most favored molecular species is an alpha1(IX)alpha2(IX)alpha3(IX) heterotrimer, but the chains are also able to form disulfide-bonded heterotrimers of alpha1(IX) and alpha3(IX) chains and (alpha1(IX))(3), (alpha2(IX))(3), and (alpha3(IX))(3) homotrimers.
Pihlajamaa
T
T
Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland.
Perälä
M
M
Vuoristo
M M
MM
Nokelainen
M
M
Bodo
M
M
Schulthess
T
T
Vuorio
E
E
Timpl
R
R
Engel
J
J
Ala-Kokko
L
L
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002356:Cartilage / Q000737:chemistry; D003094:Collagen / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides; D015870:Gene Expression; D006801:Humans; D009036:Moths / Q000166:cytology / Q000821:virology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D013438:Sulfhydryl Compounds
D000596:Amino Acids; D004220:Disulfides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003094:Collagen