Comparison of the Water–Soluble Proteomes From Keratocytes and Serum–Cultured Corneal Fibroblasts

Abstract

Abstract: : Purpose: To compare the water–soluble protein expression profiles of freshly–isolated keratocytes versus serum–cultured corneal fibroblasts to obtain a better understanding of the biochemical processes regulating corneal transparency and haze development. Methods: Keratocytes were isolated from porcine corneas by collagenase digestion for 12 hours. Serum–cultured corneal fibroblasts were obtained using explant technique and grown to third passage in 10% FBS. Both phenotypes were lysed by sonication and the water–soluble proteomes were compared using 2D gel electrophoresis and analysed by mass spectrometry. Results: The proteome analysis revealed distinct differences in the protein expression profiles of keratocytes and serum–cultured corneal fibroblasts. The "phenotype–specific" proteins are involved in many cellular processes including cell metabolism and protection from oxidative stress. Specifically, the expression of the enzyme–crystallins, aldehyde dehydrogenase 3A1 and transketolase, are dramatically reduced in serum–cultured corneal fibroblasts, as verified by immunoblotting. Conclusions: This proteomic study reveals distinct differences in the protein expression profiles of keratocytes versus the serum–cultured fibroblast phenotype. Together with enzyme–crystallins, these "phenotype–specific" proteins may be involved in the regulation of corneal cellular transparency.

Keywords: cornea: stroma and keratocytes • proteomics • crystallins