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L.J. Robertson, L.L. David, T.R. Shearer, J.D. Morton, R. Bickerstaffe; Susceptibility of Ovine Lens Crystallins to Proteolytic Cleavage by Calpain During Cataract Formation: A 2-DE Approach . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2377.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify and map crystallins in the normal ovine lens and to describe crystallin truncations. Truncations subsequently form during cataractogenesis and may be attributed to ubiquitous calpain II and lens specific calpain Lp82. Methods: Two dimensional gel electrophoresis (2-DE) was performed on lens soluble and insoluble proteins from normal and cataractous sheep. Gels were stained, crystallin subunits were digested with trypsin within gel slices, and peptides were analysed by tandem mass spectrometry using electrospray ionization and an ion trap. Since there are no known sequences for ovine crystallins, proteins were identified by homology to the sequences of other mammalian crystallins. In the cataract lenses, the masses of truncated α-crystallins were determined by eluting the proteins from 2-DE gels and deconvoluting their electrospray ionization mass spectra. Results: A 2-DE map of ovine lens crystallins was created showing the normal complement of crystallins found in other mammals. Specific crystallin modifications were also noted, such as phosphorylation of αA-crystallin at residue 122. Insoluble ovine αA-crystallin, with a mass of 19,877, was extensively truncated in mature cataracts. Truncated species of ovine αA-crystallin with masses of 19,446 and 18,720 were observed. These decreases in mass were identical to the decreases observed in bovine and rat αA-crystallins after they were proteolyzed by calpain II and the lens specific calpain isoform Lp82. These truncated αA-crystallin species corresponded to masses expected when 5 or 11 residues were removed from the C-terminus by Lp82 and calpain II, respectively. This suggested that both Lp82 and calpain II were activated in ovine lenses developing mature cataracts. However, the 2-DE protein spot for the –5 Lp82 specific species was more abundant than the calpain II specific –11 species. Conclusion: Crystallins in the normal ovine lens appear similar to those of other mammals. Both calpain II and Lp82 cleavage sites on α-crystallin were found in the insoluble fraction of cataract lenses. However, the greater abundance of truncated αA-crystallin missing 5 residues from its C-terminus, suggested that Lp82 is preferentially activated in ovine lens. This data provides further support for calpain activation in the ovine cataract. It also provides the first evidence for increased Lp82 activation in a non-rodent species during cataract formation. These results require confirmation of Lp82 and calpain II cleavage sites on ovine crystallins in vitro.
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