Mechanism of Asparagine Deamidation During Human Senile Cataractogenesis

Abstract

Previous studies have demonstrated that asparagine-143 of gammaS crystallin is preferentially deamidated in human cataractous lenses. A possible mechanism of nonenzymatic deamidation involves the initial formation of L -succinimidyl and D -succinimidyl intermediates, followed by ring cleavage and production of the respective L - and D - forms of beta-aspartate and alpha-aspartate. To determine if this mechanism occurs during cataractogenesis of the human lens, synthetic peptide standards together with reverse phase HPLC were used to purify and quantitate tryptic peptides from cataractous lenses that contained the beta-aspartate and alpha-aspartate forms of residue 143. For all lenses analysed, the ratios of beta-aspartate/alpha-aspartate produced were consistent with a mechanism of deamidation involving succinimide intermediates. Surprisingly, the D / L ratios for all beta- and alpha-aspartates produced were very low, suggesting that the mechanism of deamidation preferentially involves the L -succinimidyl intermediate, with formation of the D -succinimidyl intermediate almost completely blocked by steric hindrance. Together, the results demonstrate that deamidation of asparagine-143 from gammaS crystallin of different human cataracts undergoes the same mechanism, which due to higher ordered structure of the native protein, results in the preferential production of one of two possible succinimidyl intermediates.