Abstract
alphaA-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 alphaA-crystallin enhances lens epithelial cell growth and resistance to stress conditions. Mutation of the arginine 116 residue to cysteine (R116C) in alphaA-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 alphaA-crystallin in the human lens epithelial cell line HLE B-3. Immunofluorescence and confocal microscopy indicated that both wild type and R116C alphaA-crystallin were distributed mainly in the cytoplasm of lens epithelial cells. Size-exclusion chromatography indicated that the size of the alphaA-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 alphaA-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. F-actin was protected in UVA-treated cells expressing the wild type alphaA-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 alphaA-crystallin. We propose that the diminished protective ability of the R116C mutant in lens epithelial cells may contribute to the pathogenesis of cataract.
Highlights
␣-Crystallin is a major lens protein and belongs to the small heat-shock protein family of molecular chaperones
We propose that the diminished protective ability of the R116C mutant in lens epithelial cells may contribute to the pathogenesis of cataract
Expression of Wild Type and R116C ␣A-crystallin in Lens Epithelial Cells—To compare their relative ability to protect lens epithelial cells from UVA radiation-induced stress, wild type or R116C␣A-crystallin were expressed in the human lens epithelial cell line HLE B-3
Summary
␣-Crystallin is a major lens protein and belongs to the small heat-shock protein family of molecular chaperones. The expression of ␣-crystallin in a variety of tissues outside the lens and its ability to prevent thermal aggregation of proteins in a manner similar to molecular chaperones suggest that it has general cellular functions over and above its role in light refraction [1, 2]. Lens epithelial cells derived from mice lacking ␣A-crystallin have a 50% slower growth rate in vitro, whereas lens epithelial cells derived from ␣B-crystallin knockout mice demonstrate hyperproliferation and genomic instability [5, 6] These findings have led to the suggestion that ␣A- and ␣Bcrystallin may have distinct functions in regulating cellular growth. In lens fiber cells exposed to thermal stress, ␣B-crystallin was shown to associate with lens-specific intermediate filament proteins phakinin and filensin [14]. Recent work suggests that the protein structure of ␣B-crystallin is stabilized by complex formation with ␣A-crystallin [22]
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