Localization of biologically uncommon D-β-aspartate-containing αA-crystallin in human eye lens

α-Crystallin is one of the major proteins in the vertebrate eye lens and its function has been indicated to be maintenance of lens transparency. α-Crystallin exists in the lens fiber cell cytoplasm as a polydisperse aggregate with an average molecular mass of approximately 800 kDa. The aggregate is composed of two kinds of polypeptides, αAand αB-crystallins, containing 173 and 175 amino acid residues, respectively. The amino acid sequences of αAand αB-crystallins are approximately 55% homologous [1,2]. Recently, it was demonstrated that α-crystallin is one of the small heat shock proteins [3] and functions as a molecular chaperone which protects other proteins against denaturation and eventual aggregation [4]. Proteins of the lens have very long half-lives, therefore, various posttranslational modifications such as deamidation [5,6], racemization [7,8], stereoinversion [9-12], isomerization [812], truncation [6,10,13-15], phosphorylation [6,16], oxidation [6,8,17], and an increase in intramolecular disulfide bonding [6,18] have been shown to take place during aging. These post-translational modifications may cause the formation of high molecular weight protein aggregates and may lead to the formation of cataract by affecting the close-packing of the crystallins or by reducing the chaperone-like activity of αcrystallins. We found racemization of amino acids in lens protein generated at specific sites of αAand αB-crystallin which could not be found in other crystallins (our unpublished data). Subsequently, we identified a biologically uncommon D-isomer at Asp-58 and Asp-151 [9,10] in αA-crystallin, and also at Asp-36 and Asp-62 in αB-crystallin, [8] from aged human lenses. D-Asp-151 of αA-crystallin was found to be present in the lenses of cattle [7], horse (our unpublished data), rat [19], and mouse [20]; however, the D-isomers of Asp-58 in αA-crystallin and Asp-36 and Asp-62 in αB-crystallin were restricted to human lenses. The results showed that the configuration of Asp-151 in αA-crystallin is stereochemically labile. Strikingly, the D isomer to native L ratio (D/L ratio) of Asp-151 of human αA-crystallin was higher than 1.0 [9-12]. Since racemization is defined as a reversible first order reaction, racemization is in equilibrium when the D/L ratio reaches 1.0. Therefore, the reaction which induces D-Asp predominantly (D/L>1.0) is not termed racemization but stereoinversion. There have been no reports of stereoinversion of amino acids in proteins except in human αA-crystallin. DAsp formation was also accompanied by isomerization from © 2000 Molecular Vision