BETA-IG - MOLECULAR-CLONING AND IN-SITU HYBRIDIZATION IN CORNEAL TISSUES

Purpose. To identify a protein that copurifies with type VI collagen f rom rabbit cornea and to determine its cell source in rabbit corneal t issues by in situ hybridization. Methods. Type VI collagen was extract ed from cornea with urea and purified by ammonium sulfate precipitatio n and gel chromatography. The purity of the collagen was assessed by s odium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). O n reduction with mercaptoethanol or dithiothreitol, the alpha chains o f type VI collagen ran into the gel. In addition to the type VI collag en polypeptides, an extra 68-kDa protein band appeared, suggesting tha t this protein is present as a large molecular weight component before reduction. Amino acid sequencing indicated that protein was related t o beta ig-h3 from humans. Western blot analysis was used to determine immunologic similarity to this human protein. A rabbit stromal cell cD NA library was screened with human beta ig-h3 cDNA probe. Positive clo nes were sequenced and analyzed for sequence homology. Oligonucleotide probes prepared from rabbit cDNA sequences were used for Northern blo t analysis and in situ hybridization of corneal tissues. Results. Elec troblotting of the SDS-PAGE and amino acid sequence analysis of the fi rst 10 N-terminal amino acids of the 68-kDa band gave 100% homology wi th a known protein produced by human adenocarcinoma cells. beta ig-h3. This 68-kDa protein was identical immunologically to beta ig-h3 by We stern blot analysis. Sequence analysis of a rabbit cDNA clone containe d the whole coding region and had high identity with both human beta i g-h3 and mouse beta ig-m3. The deduced amino acid sequence had 92% ide ntity with these species. An oligonucleotide probe from the rabbit cDN A sequence detected a single band of mRNA from cultures of stromal cel ls 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 obta ined from normal rabbit cornea and the mRNA comes from primary culture s of rabbit stromal cells and not from a cloned cell line. In situ hyb ridization of rabbit corneal tissue indicated that the beta ig mRNA is located primarily in the epithelium of normal adult cornea, in fetal stromal cells. End both endothelium- and stroma-derived cells in heali ng corneal wounds. Normal adult endothelium and stroma did not show be ta ig mRNA label. Conclusions. The highly conserved amino acid sequenc e homology between the human, mouse, and rabbit proteins and the tempo ral expression of beta ig message during corneal healing and developme nt suggest this protein plays an important role in the morphogenesis o f corneal tissues.