The chaperone-like activity of αA-crystallin has an important role in maintaining lens transparency. Previously we identified residues 70-88 as a chaperone site in αA-crystallin. In this study, we deleted the chaperone site residues to generate αAΔ70-76 and αAΔ70-88 mutants and investigated if there are additional substrate-binding sites in αA-crystallin. Both mutant proteins when expressed in E. coli formed inclusion bodies, and on solubilizing and refolding, they exhibited similar structural properties, with a 2- to 3-fold increase in molar mass compared to the molar mass of wild-type protein. The deletion mutants were less stable than the wild-type αA-crystallin. Functionally αAΔ70-88 was completely inactive as a chaperone, while αAΔ70-76 demonstrated a 40-50% reduction in anti-aggregation activity against alcohol dehydrogenase (ADH). Deletion of residues 70-88 abolished the ADH binding sites in αA-crystallin at physiological temperature. At 45°C, cryptic ADH binding site(s) became exposed, which contributed subtly to the chaperone-like activity of αAΔ70-88. Both of the deletion mutants were completely inactive in suppressing aggregation of βL-crystallin at 53°C. The mutants completely lost the anti-apoptotic property that αA-crystallin exhibits while they protected ARPE-19 (a human retinal pigment epithelial cell line) and primary human primary lens epithelial (HLE) cells from oxidative stress. Our studies demonstrate that residues 70-88 in αA-crystallin act as a primary substrate binding site and account for the bulk of the total chaperone activity. The β3 and β4 strands in αA-crystallin comprising 70-88 residues play an important role in maintenance of the structure and in preventing aggregation of denaturing proteins.
Keywords: Aggregation; Apoptosis; Chaperone; Crystallin; Deletion; Structure-function.
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