Beta-ig. Molecular cloning and in situ hybridization in corneal tissues

To identify a protein that copurifies with type VI collagen from rabbit cornea and to determine its cell source in rabbit corneal tissues by in situ hybridization.

Type VI collagen was extracted from cornea with urea and purified by ammonium sulfate precipitation and gel chromatography. The purity of the collagen was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction with mercaptoethanol or dithiothreitol, the alpha chains of type VI collagen ran into the gel. In addition to the type VI collagen polypeptides, an extra 68-kDa protein band appeared, suggesting that this protein is present as a large molecular weight component before reduction. Amino acid sequencing indicated that protein was related to beta ig-h3 from humans. Western blot analysis was used to determine immunologic similarity to this human protein. A rabbit stromal cell cDNA library was screened with human beta ig-h3 cDNA probe. Positive clones were sequenced and analyzed for sequence homology. Oligonucleotide probes prepared from rabbit cDNA sequences were used for Northern blot analysis and in situ hybridization of corneal tissues.

Electroblotting of the SDS-PAGE and amino acid sequence analysis of the first 10 N-terminal amino acids of the 68-kDa band gave 100% homology with a known protein produced by human adenocarcinoma cells, beta ig-h3. This 68-kDa protein was identical immunologically to beta ig-h3 by Western blot analysis. Sequence analysis of a rabbit cDNA clone contained the whole coding region and had high identity with both human beta ig-h3 and mouse beta ig-m3. The deduced amino acid sequence had 92% identity with these species. An oligonucleotide probe from the rabbit cDNA sequence detected a single band of mRNA from cultures of stromal cells consistent in size with human beta ig-h3 mRNA. The authors refer to the rabbit form of beta ig-h3 as beta ig because the protein was obtained from normal rabbit cornea and the mRNA comes from primary cultures of rabbit stromal cells and not from a cloned cell line. In situ hybridization of rabbit corneal tissue indicated that the beta ig mRNA is located primarily in the epithelium of normal adult cornea, in fetal stromal cells, and both endothelium- and stroma-derived cells in healing corneal wounds. Normal adult endothelium and stroma did not show beta ig mRNA label.

The highly conserved amino acid sequence homology between the human, mouse, and rabbit proteins and the temporal expression of beta ig message during corneal healing and development suggest this protein plays an important role in the morphogenesis of corneal tissues.