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Lucinda J. G. Robertson, Larry L. David, Michael A. Riviere, Phillip A. Wilmarth, Matthew S. Muir, James D. Morton; Susceptibility of Ovine Lens Crystallins to Proteolytic Cleavage during Formation of Hereditary Cataract. Invest. Ophthalmol. Vis. Sci. 2008;49(3):1016-1022. doi: 10.1167/iovs.07-0792.
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purpose. To produce two-dimensional electrophoresis (2-DE) maps for ovine crystallins and examine changes in ovine crystallins during cataract formation.
methods. Soluble and insoluble fractions were isolated from normal, whole lenses of 26-week-old sheep, the proteins separated by 2-DE, and the spots digested with trypsin and subjected to tandem mass spectral analysis. Spot identifications were made by using mass spectrometry data from each spot digestion and data from 2-DE maps of proteins from soluble and insoluble cortices of 10-month-old ovine lens. Ovine αA-, αB-, and βB3-crystallin cDNAs were sequenced, whereas other ovine crystallins were identified by using bovine sequences. Proteins were then isolated from whole lenses of 26-week-old lambs with mature hereditary cataracts, and the changes in the crystallins were determined by 2-DE. The masses of truncated crystallins were determined after elution from 2-DE gels.
results. The ovine lens contained the normal complement of crystallins and, similar to other mammalian lenses, underwent partial proteolysis of βB1-, βA3-, and βB3-crystallin during maturation. Cataract development was associated with enhanced truncation of α- and β-crystallins. C-terminal truncations of αA- and αB-crystallin and N-terminal truncation of βB2-crystallin were observed as well as a loss of γ-crystallin.
conclusions. These data provide the first 2-DE gel maps for ovine lens crystallins and indicated that ovine lens crystallins are truncated during lens maturation. The differences in proteolysis appearing in normal and cataractous lenses suggested that calpain isoforms may be differentially activated during lens maturation and cataract. The ovine hereditary cataract is a useful nonrodent model to study the role of calpain proteolysis in cataract formation.
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