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M. Posner, R. W. Corbin, M. Hawke, C. LaCava, C. Prince; Proteomics of the Zebrafish (Danio rerio) Lens. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2045. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To quantify the relative proportions of crystallins in the zebrafish lens. These data will facilitate the use of zebrafish as a model for investigating crystallin function and lens development.
Zebrafish lenses were collected and homogenized in lysis solution to solubilize all lens protein. Homogenates were isoelectrically focused using 7 cm IPG strips (pH 5-8 or 3-10 nonlinear). Second dimension separation was performed on 12% SDS-PAGE gels with subsequent Coomassie staining. The intensities of individual spots were quantified to determine the proportion of each crystallin. The identity of spots was determined by western blot and MALDI-TOF MS analysis.
We distinguished 50 protein spots on 2D gels produced from zebrafish total lens homogenates reflecting the larger numbers of crystallins expressed in this species compared to mammals. Alpha crystallin expression was reduced compared to mammals. Alpha A, alpha Ba and alpha Bb crystallin comprised 2.8, 2.7 and 1.5% of total lens protein, respectively, producing a ratio of approximately 2:2:1.
The low percentage of alpha crystallin in the zebrafish lens (7%) compared to humans (40%) or rodents (20%) is similar to quantities found in other fish species. However, the 2:3 ratio of alpha A to total alpha B determined here differs markedly from a recent study of the catfish lens that reported a ratio of 19:1. Interestingly the ratio from our quantitation is more similar to ratios reported for humans (3:2 to 3:1). Previous work suggests that the functions of mammalian alpha B crystallin have been divided between the two zebrafish orthologs (alpha Ba and Bb). Therefore, the zebrafish lens may provide a valuable model for better understanding these functions.
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