Asp 58 modulates lens αA‐crystallin oligomer formation and chaperone function

Senile cataract onset is caused by insolubilization of lens proteins. The lens crystallin protein family correctly orders the formation of homo‐ or hetero‐oligomers in lens fiber cells. Because lens fiber cells do not divide, covalent post‐translational modifications, such as isomerization of aspartate residues, accumulate with aging. Although many isomerization sites of αA‐crystallin have been reported, their structural and functional contributions have never been identified. In this study, αA‐crystallin was extracted from aged human lens and separated into each oligomeric state by size exclusion chromatography and electrophoresis. The novel combination methodology of in‐solution/gel tryptic digestion with liquid chromatography equipped with mass spectrometry ( LC ‐ MS / MS ) was used to evaluate the isomerization of Asp 58. The contributions of isomerization to assembly, solubility, and chaperone functions of αA‐crystallin were estimated using a series of mutations of Asp 58 in αA‐crystallin. The results indicated that the isomerization of Asp 58 depended on the oligomer size and age of the lens. The substitution of Asp 58 for hydrophobic residues increased αA‐crystallin oligomer size and decreased solubility. All substitutions decreased the chaperone function of αA‐crystallin for aggregates of bovine βL‐crystallin and alcohol dehydrogenase. The data indicated that Asp 58 in αA‐crystallin was critical for intermolecular interactions in the lens. Our results also suggested that LC ‐ MS / MS ‐based isomerization analyses of in‐gel‐digested products could be useful for investigating the isomerization of Asp residues in oligomeric states. This method could also be used to analyze d / l ratios of amino acid residues in soluble protein aggregates.