Biological Function of Type VIII Collagen

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Since several amino-terminal amino acid sequences determined from peptides isolated from Descemet's membranes were found within deduced amino acid sequence from the cDNA clones we isolated, we designated the polypeptide chain as alpha1(VIII) chain. We have isolated genomic DNA encoding the rabbit alpha1(VIII) chain. Intriguingly, the gene contains three small and one large exons. The largest exon contains a short NC2 domain, the entire COL and NC1 domains and 3' UTR. The human gene, COL8A1, has been assigned to chromosome 3q11.1-q13.2. The question here was whether the molecule contains another alpha chains. A set of PCR primers was designed and the PCR products were screened by various probes. The amino acid sequence deduced from a candidate clone contained several collagenous amino acid sequences obtained from Descemet's membrane that were not found in the alpha1(VIII) chain, therefore we identified the collagenous polypeptide as the second chain, alpha2(VIII), that joins the type VIII collagen molecule. The gene coding for the alpha2(VIII) chain was assigned to chromosome 1p32.3-1p34.3. Mouse cDNA and genomic DNA clones were isolated and the expression in various tissues and cells were demonstrated by Northern-blot analyzes and in situ hybridization. The results showed that the alpha1(VIII) mRNA is predominantly expressed in keratinocytes, corneal endothelial and epithelial cells, lens epithelial cells as well as mesenchymal cells surrounding cartilage and calvarial bone and in the meninges surrounding the brain.In order to know the biological function of type VIII collagen molecule, we are now trying to create transgenic animals. The first experiment we have done so far was to use presumptive regulatory region of type VIII collagen gene joined to Lac Z gene. Several lines of transgenic mice we obtained did not show typical expression features of the Lac Z gene. Upstream region of type VIII collagen gene needs to characterized further.