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S.K. Swamynathan, W.G. Robison, Jr, M.A. Crawford, J. Piatigorsky; Gelsolin Is the Most Abundant Water-soluble Protein in the Corneal Epithelium of Anableps anableps, the Four-eyed Fish . Invest. Ophthalmol. Vis. Sci. 2003;44(13):908.
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Purpose: Anableps, the four eyed fish, live at the surface of the water with their ocular surface bisected horizontally by the waterline. Here, we have attempted to find out if there are differences in the ultrastructure and protein composition between the dorsal cornea exposed to air and the ventral cornea exposed to water. Further, we wanted to identify and characterize the abundant protein(s) present within the Anableps corneal epithelia. Methods: Adult Anableps from British Guyana were obtained from Nova Tropicals, Alexandria, VA. Methacrylate embedded eyes were sectioned and stained with hematoxylin and eosin for light microscopy. Ultra thin sections stained with uranyl acetate and lead citrate were used for transmission electron microscopy. Water-soluble proteins from the epithelia were separated by SDS-PAGE and stained with coomassie blue. Amino termini of the peptides derived by trypsin digestion of the abundant water-soluble protein in the corneal epithelia were sequenced by the Edman degradation method. RT-PCR, and 5’ and 3’ RACE were used to clone the corresponding full length cDNA for the most abundant protein. Immunohistochemistry was performed with sections from paraffin embedded eyes using anti-gelsolin antibodies. Results: The epithelium and the stroma were 1.2 mm and 1.0 mm thick in the dorsal cornea, and 0.45 mm and 1.4 mm thick in the ventral cornea, respectively. The radius of curvature was 1.76 mm and 1.50 mm for the dorsal and the ventral corneal surface, respectively. Electron microscopy showed that the basal epithelial cells were columnar in the dorsal cornea and cuboidal in the ventral cornea. The SDS-PAGE profiles of the water-soluble proteins from the dorsal and ventral corneal epithelia were similar, except for the relative amount of an 80 kDa protein which accounted for 38% and 21% of the water-soluble proteins in the dorsal and ventral corneal epithelia, respectively. This protein was identified by Edman sequencing and confirmed by immunoblotting as gelsolin. We have shown earlier that gelsolin represents approximately 50% of the water-soluble proteins of the zebrafish cornea. The 2284 bp Anableps gelsolin cDNA sequence was used to deduce the amino acid sequence, which was 55% identical to zebrafish gelsolin. Conclusions: We have identified differences in the structure and protein composition of Anableps cornea exposed to air and water. Gelsolin was recruited for abundant expression in the corneal epithelium in a common ancestor of Zebrafish and Anableps, which diverged more than 100 million years ago.
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