The R116C mutation in αA-crystallin diminishes its protective ability against stress-induced lens epithelial cell apoptosis

αA-crystallin is a small heat-shock protein expressed preferentially in the lens and is detected during the early stages of lens development. Recent work indicates that the expression of αA-crystallin enhances lens epithelial cell growth and resistance to stress conditions. Mutation of the arginine 116 residue to cysteine (R1160 in αA-crystallin has been associated with congenital cataracts in humans. However, the physiological consequences of this mutation have not been analyzed in lens epithelial cells. In the present study, we expressed wild type or R116C αA-crystallin in the human lens epithelial cell line HLE B-3. Immunofluorescence and confocal microscopy indicated that both wild type and R116C αA-crystallin were distributed mainly in the cytoplasm of lens epithelial cells. Size-exclusion chromatography indicated that the size of the αA-crystallin aggregate in lens epithelial cells increased from 500 to 600 kDa for the wild type protein to >2 MDa in the R116C mutant. When cells were exposed to physiological levels of UVA radiation, wild type αA-crystallin protected cells from apoptotic death as shown by annexin labeling and flow cytometric analysis, whereas the R116C mutant had a 4-to 10-fold lower protective ability. UVA-irradiated cells expressing the wild type protein had very low TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining, whereas cells expressing R116C mutant had a high level of TUNEL staining. Factin was protected in UVA-treated cells expressing the wild type αA-crystallin but was either clumped around the apoptotic cells or was absent in apoptotic cells in cultures expressing the R116C mutant. Structural changes caused by the R116C mutation could be responsible for the reduced ability of the mutant to protect cells from stress. Our study shows that comparing the stress-induced apoptotic cell death is an effective way to compare the protective abilities of wild type and mutant αA-crystallin. We propose that the diminished protective ability of the R116C mutant in lens epithelial cells may contribute to the pathogenesis of cataract.