Comparative proteomics analysis of degenerative eye lenses of nocturnal rice eel and catfish as compared to diurnal zebrafish

Purpose: The aim of this study was to determine the lens crystallin diversity of degenerative eyes from the rice eel (Monopterus albus) and walking catfish (Clarias batrachus) as compared to that of zebrafish (Danio rerio) by using comparative proteomics methodologies. We endeavored to investigate the evolution of vertebrate lenses particularly concerning the functional loss of lenses in degenerative eyes of rice eels and catfishes living under an environment of perpetual darkness. Methods: Fish lenses were collected and homogenized to extract total soluble proteins. The protein mixtures were separated by one- and two-dimensional gel electrophoresis (1D or 2D gel), plus the newer gel-free shotgun proteomic strategy, followed by in-gel digestion and subjection of the digested protein bands or spots to liquid chromatography coupled with tandem mass spectrometry. The proteomics data were analyzed and compared based on the proteomics databank of zebrafish. The soluble lens protein solutions of three piscine species were also processed by gel-filtration chromatography and 1D sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the comparison and validation of various crystallin families, e.g., α-, β-, and γ-crystallins. Results: In zebrafish eye lenses, γ-crystallin constituted about 71% and α- and β-crystallins comprised 30% of total lens proteins. In rice eel lenses, very little or almost no α-crystallins were detected and β- and γ-crystallins comprised more than 98% of total lens proteins. In catfish lenses, α- and β-crystallins comprised about 40% and γ-crystallin constitutes 60% of total lens proteins. It was of interest to find that α-crystallin was totally absent in the rice eel in contrast to the presence, albeit with very low amounts, of α-crystallin in similarly nocturnal catfish. The ratio of α-crystallin subunits (αA/αB) was found to be about 20:1 for the catfish lens, in great contrast to the ratio of about 3:1 found for most mammalian lenses. In contrast, β- and γ-crystallins were more abundant in lenses of these three piscine species, similar to mammalian lenses. By proteomics analysis, the most abundant β-crystallins were found to comprise a diverse group of βA1a, βA1-2, βA2a, βA2-2, βA4, βB1, βB2, and βB3 subunit crystallins; the monomeric γ-crystallin class contains γB, γD, γM2, γM3, γM5, γM7, γN-A, γN-B, γS1, and γS2 crystallins. Conclusions: In cave or nocturnal animals, the eye is sometimes reduced or eliminated because of adaptation to life in visual darkness. The comparative proteomics analysis of degenerative and normal lenses forms a firm molecular basis to investigate further the evolution of piscine lenses in the future. The total numbers of α-, β-, and γ-crystallins in the three fish species as revealed by the current proteomics methodology clearly indicate the complexity and diversity of crystallin species present in the piscine class of vertebrates. The unexpected finding that α-crystallin is absent in the degenerative eye lenses of rice eel may have some bearing on the chaperone function of α-crystallin in regard to its protective role of preventing protein aggregation in diurnal vertebrate lenses to maintain functional transparency.