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J. Biol. Chem., Vol. 277, Issue 12, 10178-10186, March 22, 2002
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From the
The R116C Mutation in
A-crystallin Diminishes Its Protective
Ability against Stress-induced Lens Epithelial Cell Apoptosis*
§¶,, and
Department of Ophthalmology and Visual
Sciences and the § Department of Biochemistry and Molecular
Biophysics, Washington University School of Medicine,
St. Louis, Missouri 63110
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 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 A-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 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. We
propose that the diminished protective ability of the R116C mutant in
lens epithelial cells may contribute to the pathogenesis of cataract.
*
This work is supported by NEI, National Institutes of
Health Grants R01EY05681 (to U. P. A.), Core Grant EY02687,
and Research to Prevent Blindness, Inc.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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