Journal
FEBS JOURNAL
Volume 285, Issue 12, Pages 2263-2277Publisher
WILEY
DOI: 10.1111/febs.14475
Keywords
cataract; crystallin; d-amino acid; post-translational modification; protein misfolding
Categories
Funding
- JSPS KAKENHI [JP17K13221, JP15K16514]
- Future Development Funding Program of Kyoto University Research Coordination Alliance
- Grants-in-Aid for Scientific Research [17K13221] Funding Source: KAKEN
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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 A-crystallin have been reported, their structural and functional contributions have never been identified. In this study, A-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 A-crystallin were estimated using a series of mutations of Asp 58 in A-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 A-crystallin oligomer size and decreased solubility. All substitutions decreased the chaperone function of A-crystallin for aggregates of bovine L-crystallin and alcohol dehydrogenase. The data indicated that Asp 58 in A-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.
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