Effect of a Novel Mutation in Alpha A-Crystallin Associated with Autosomal Dominant Cataract on Its Distribution and Function in Lens Epithelial Cells

Abstract

Abstract: : Purpose: A missense mutation resulting in the substitution of arginine to cysteine in αA-crystallin (R≷C), which lies outside of the α-crystallin domain has been associated with autosomal dominant cataract in humans (Mackay et al., ARVO 2002). In the present study, we analyzed the effect of the mutation on the cellular distribution and protective ability of αA-crystallin. Methods: HLE B-3 cells were transfected with a mammalian expression vector (pCIneo) encoding either wild type human αA-crystallin or the mutant protein. Cell lines stably expressing the wild type or mutant protein were generated and expression of the proteins was analyzed by western blotting. Intracellular distribution of αA-crystallin and αB-crystallin was examined by immunofluorescence and confocal microscopy and cryo-immunoelectron microscopy. Cells expressing wild type αA-crystallin or the mutant were exposed to the protein kinase C inhibitor staurosporine. The level of apoptosis was determined by annexin labeling and flow cytometric analysis. Results: Immunofluorescence and confocal microscopy showed that in 70% of the cells expressing the arginine mutant the protein was associated with the nucleus, and had a speckled appearance. αB-crystallin was associated with the mutant in the nuclear speckles. The speckled appearance and nuclear localization were not found in cells expressing wild type αA-crystallin. The nuclear distribution of the mutant was confirmed by cryo-immunoelectron microscopy and by western blotting of cell lysates. Cells expressing the mutant had a 50% lower growth potential. The basal level of apoptosis increased from 1.9% for wild type to 33% for cells expressing the mutant protein. Cells expressing wild type αA-crystallin or the mutant were exposed to the protein kinase C inhibitor staurosporine. Wild type αA-crystallin completely prevented apoptosis but in striking contrast, 67% of cells expressing the mutant were apoptotic. Conclusion: These studies indicate that expression of the mutant αA-crystallin results in abnormal nuclear distribution and cytotoxic effect on lens epithelial cells. The decreased protective ability of the mutant suggests that this conserved arginine residue which lies outside the α-crystallin domain of αA-crystallin plays a critical role in the structure and function of the protein. CR: None Support: NIH grants EY05681, EY12284, EY02687 and RPB.

Keywords: 343 chaperones • 378 crystallins • 338 cataract