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Papers In Press, published online ahead of print December 27, 2001
Ophthalmology, Washington University, St. Louis, Missouri 63110
Corresponding Author: andley{at}vision.wustl.edu
Summary
J. Biol. Chem, 10.1074/jbc.M109211200
Submitted on September 24, 2001
Revised on December 27, 2001
Accepted on December 27, 2001
The R116 C mutation in
A-crystallin diminishes its protective ability against stress-induced lens epithelial cell apoptosis
A-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 A-crystallin enhances lens epithelial cell growth and resistance to stress conditions. Mutation of the arginine 116 residue to cysteine (R116C) in A-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 A-crystallin in the human lens epithelial cell line HLE B-3. Immunofluorescence and confocal microscopy indicated that both wild type and R116C A-crystallin were distributed mainly in the cytoplasm of lens epithelial cells. Size-exclusion chromatography indicated that the size of the A-crystallin aggregate in lens epithelial cells increased from 500-600 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 A-crystallin protected cells from apoptotic death as shown by annexin labeling and flow cytometric analysis, whereas the R116C mutant had a four- to ten-fold lower protective ability. UVA-irradiated cells expressing the wild type protein had very low TUNEL 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 A-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 A-crystallin.
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