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L.J. G. Robertson, L.L. David, J.D. Morton; Ovine Lens cDNA Sequences and Predicted Protein Masses Allow Confirmation of Increased Proteolysis and Deamidation of A–Crystallins During Hereditary Cataract . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3887.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine ovine αA and αB–crystallin sequences so that the modifications to these proteins can be determined by mass spectrometry following formation of hereditary cataract. Methods: cDNA was synthesised from ovine lens epithelium RNA using oligo dT primers. PCR was optimised using gene specific primers designed for the 5’ and 3’ UTR regions of the bovine αA and αB–crystallin mRNA sequences. The amplimers were then cloned and sequenced. Soluble and insoluble ovine lens proteins were isolated from 26–week old lambs with mature hereditary cataracts, separated by two–dimensional electrophoresis, and alpha–crystallins eluted from gel slices and their masses determined by electrospray ionisation mass spectrometry. Results: Ovine αA and αB–crystallin cDNAs encoded proteins of 173 and 175 amino acids, with predicted masses of 19,818 and 20,109, respectively. These predicted masses matched the measured masses of the unmodified forms of αA– and αB–crystallins from normal control lenses. Formation of hereditary cataracts was associated with the appearance of both lower molecular weight and acidic forms of αA and αB. The measured masses of the modified forms of α–crystallins indicated that they were produced by a combination of proteolysis and deamidation. The measured masses indicated that both 5 and 11 residues were removed from the C–terminus of αA, and 5 and 12 residues from the C–terminus of αB. Additional acidic forms of truncated α–crystallins were also found that differed by less than 2 mass units from their accompanying less acidic forms. Deamidation was the only known modification that could cause this decrease in pI, since it introduces only a single unit increase in mass that was less than the experimental error in mass measurement (+/– 2 mass units). Conclusions: Determination of ovine α–crystallin sequences allowed confirmation of the cleavage sites occurring in these proteins during cataract formation. Furthermore, detection of more acidic species of α–crystallins with no significant change in mass suggested that increased deamidation occurred. Elevated lens calcium was previously reported in this hereditary cataract model. This may lead to activation of both calpains, which can cause the observed C–terminal truncation in α–crystallins, and transglutaminase, which can cause deamidation. Both these modifications may contribute to opacification in ovine cataracts by decreasing α–crystallin chaperone function.
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