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.

Human COL9A1 and COL9A2 genes. Two genes of 90 and 15 kb code for similar polypeptides of the same collagen molecule.

Here we report the complete structure for the human COL9A1 and the complete sequence for the human COL9A2 genes. The COL9A1 gene is about 90 kb and consists of 38 exons. The COL9A2 gene is only about 15 kb, and it contains 32 exons. Sequence analysis of the promoter regions for the human COL9A2, the mouse Col9a2 and the human COL2A1 genes identified a conserved 14 bp sequence. The data also indicated that the alternative exon 1* found in intron 6 of the COL9A1 gene is separated from exon 7 only by a short intron in the chick, human, mouse and rat genes probably explaining why transcripts from exon 1* are spliced directly to exon 8.

The mouse col11a2 gene. Some transcripts from the adjacent rxr-beta gene extend into the col11a2 gene.

Type XI collagen is present in small amounts in cartilage, together with small amounts of type IX and type V collagens and large amounts of type II collagen. Here, primers based on the nucleotide sequences of partial human cDNAs and mouse genomic DNAs that were analyzed by other investigators were used to isolate a cDNA for the mouse col11a2 gene. Cosmid clones for the mouse col11a2 gene were isolated, and 12.4 kb of the nucleotide sequences were defined. Analysis of the genomic sequences identified three exons in the mouse gene that were recently shown to undergo alternative splicing (Tsumaki and Kimura, J. Biol. Chem. 270, 2372-2378, 1995; Zhidkova et al., J. Biol. Chem. 270, 94886-9493, 1995). In addition, analysis of the cosmid clones revealed that the 5' end of the mouse col11a2 gene was located head-to-tail with the mouse retinoic X receptor beta gene. RT-PCR assays demonstrated that some transcripts from the retinoic X receptor beta gene extend into the col11a2 gene. Therefore, there may be coordinate expression of the two genes.