A novel mutation (F71L) in alphaA-crystallin with defective chaperone-like function associated with age-related cataract

Abstract

Age-related cataract (ARC) is a multifactorial disease and the leading cause of blindness worldwide. Genetic predisposition in association with other etiological factors may contribute to ARC. However, gene mutation studies on ARC are scanty. In the present work, we identified a genetic variation (F71L) in the exon-2 of CRYAA (alphaA-crystallin) gene in three unrelated female sporadic cases among 711 ARC patients but not in 265 normal non-cataractous controls by SSCP and RFLP analysis. By comparing human recombinant wild-type and F71L-alphaA-crystallin, we characterized the functional significance of this missense mutation. Chromatography, fluorescence and far- and near-UV CD studies indicated that F71L missense mutation did not significantly affect the apparent molecular mass, secondary and tertiary structures and hydrophobicity of alphaA-crystallin. While the mutant alphaA-crystallin displayed significant (35-90%) loss of chaperone-like activity (CLA) in thermal aggregation of carbonic anhydrase, betaL- and gamma-crystallins, it showed moderate (10-50%) loss in CLA in DTT-induced aggregation of insulin and lysozyme. This is the first report of an alphaA-F71L mutation being associated with ARC and suggests that ARC in individuals carrying this mutation (F71L) might be due to the overall loss of in vivo chaperone activity due to interaction with other environmental factors.

Figure 1

Screening of mutations in CRYAA ex-2 in normal subjects and ARC patients. Panel A: SSCP gel showing the mobility shift pattern in the 3 patient samples of ARC (indicated by an arrow). Lanes 10 and 11 (with mobility shift) represent the samples from female patients with nuclear cataract and lane 13 represents sample from a female patient with mixed type of cataract on 12% non-denaturing PAGE gels. Lanes 3, 4, 7, 8 and 12 represent samples of non-cataractous healthy control subjects. Lanes 1, 2, 5, 6 and 9 represent samples from ARC patients (without mobility shift). Panel B: Sequence analysis of a partial fragment of the second exon of the CRYAA gene. Arrows indicate substitution of C>A at position 213 which resulted in the substitution of phenylalanine (TTC) to lLeucine (TTA) at amino acid position 71 (F71L).

Figure 2

Panel A-SDS-PAGE profile of wild-type and F71L-αA-crystallin at various stages of expression and purification. Lanes 1 and 2 represent before and after induction of wild-type, lanes 3 and 4 represent before and after induction of mutant αA-crystallin and lane M represents molecular weight marker. Panel B: Immunoblot analysis of fractions of F71L-αA-crystallin obtained from final gel purifications step using polyclonal αA-crystallin specific antibody.

Figure 3

Size-exclusion chromatography profile of wild-type and F71L-αA-crystallin on a TSK G-4000 SW size exclusion column.

Figure 4

Far- and near UV CD spectra of wild-type and and F71L-αA-crystallin. Panel A represents far-UV CD and panel B represents near-UV CD.

Figure 5

Tryptophan and ANS fluorescence spectra of wild-type and and F71L-αA-crystallin. Panel A represents tryptophan fluorescence and panel B represents ANS fluorescence.

Figure 6

Chaperone-like activity of wild-type and and F71L-αA-crystallin in heat induced aggregation of β- and γ-crystallin. Panel A: Representative graph of chaperone activity of αA-crystallin in suppressing heat-induced aggregation of βL-crystallin at 60°C. Solid curve-0.25 mg/ml of β-crystallin; dashed curve-0.25 mg/ml of β-crystallin with 0.05 mg/ml of wild-type αA-crystallin and dotted curve, 0.25 mg/ml of β-crystallin with 0.05 mg/ml of F71L-αA-crystallin. Panel B: Relative chaperone activity (percentage protection) of wild-type and F71L-αA-crystallin in β-crystallin assay. Percentage protection was determined considering aggregation of βL-crystallin in the absence of α-crystallins as 100%. Data are mean ± SD (n=4). Panel C: Relative chaperone activity of wild-type and F71L-αA-crystallin in γ-crystallin assay. Percentage protection was determined considering aggregation of γ-crystallin (0.2 mg/ml) in the absence of α-crystallins as 100%. Concentration of wild-type and F71L--αA-crystallin was 0.025 mg/mL. Data are mean ± SD (n=4).

Figure 7

Chaperone-like activity of wild-type and and F71L-αA-crystallin in insulin and carbonic anhydrase (CA) aggregation assays. Panel B: Relative chaperone activity (percentage protection) of wild-type and F71L-αA-crystallin in insulin assay. Percentage protection was determined considering DTT-induced aggregation of insulin (0.4 mg/ml) in the absence of α-crystallins at 37°Cas 100%. Concentration of wild-type and F71L--αA-crystallin was 0.3 mg/mL. Data are mean ± SD (n=4). Panel B: Relative chaperone activity of wild-type and F71L-αA-crystallin in CA assay. Percentage protection was determined considering heat-induced aggregation of CA (0.2 mg/ml) in the absence of α-crystallins at 60°C as 100%. Concentration of wild-type and F71L--αA-crystallin was 0.025 mg/mL. Data are mean ± SD (n=4).