Characterization of recombinant human type IX collagen. Association of alpha chains into homotrimeric and heterotrimeric molecules.
Publication/Presentation Date
8-6-1999
Abstract
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.
Volume
274
Issue
32
First Page
22464
Last Page
22468
ISSN
0021-9258
Published In/Presented At
Pihlajamaa, T., Perälä, M., Vuoristo, M. M., Nokelainen, M., Bodo, M., Schulthess, T., Vuorio, E., Timpl, R., Engel, J., & Ala-Kokko, L. (1999). Characterization of recombinant human type IX collagen. Association of alpha chains into homotrimeric and heterotrimeric molecules. The Journal of biological chemistry, 274(32), 22464–22468. https://doi.org/10.1074/jbc.274.32.22464
Disciplines
Medicine and Health Sciences
PubMedID
10428821
Department(s)
Department of Pathology and Laboratory Medicine
Document Type
Article