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The congenital cataract-linked G61C mutation destabilizes γD-crystallin and promotes non-native aggregation.

Zhang W, Cai HC, Li FF, Xi YB, Ma X, Yan YB - PLoS ONE (2011)

Bottom Line: The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding.The aggregation-prone property of the mutant was not altered by the addition of reductive reagent.These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

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Effect of the G61C mutation on the fibrilization of γD-crystallin under acidic condition.(A) ThT fluorescence of the WT and mutated proteins in 10 mM PBS buffer, pH 3, incubated for 48 h at 4°C, 25°C, 31°C and 37°C. The protein concentration was 5 mg/ml. The spectra recorded at 4°C or 25°C superimposed each other for the WT and mutated proteins. (B and C) The transmission EM picture of 0.5 mg/ml WT (B) and G61C γD-crystallin (C) samples incubated at 37°C for 72 h. The bar represents 200 nm.
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pone-0020564-g008: Effect of the G61C mutation on the fibrilization of γD-crystallin under acidic condition.(A) ThT fluorescence of the WT and mutated proteins in 10 mM PBS buffer, pH 3, incubated for 48 h at 4°C, 25°C, 31°C and 37°C. The protein concentration was 5 mg/ml. The spectra recorded at 4°C or 25°C superimposed each other for the WT and mutated proteins. (B and C) The transmission EM picture of 0.5 mg/ml WT (B) and G61C γD-crystallin (C) samples incubated at 37°C for 72 h. The bar represents 200 nm.

Mentions: Cataract is a disease caused by protein aggregation. To further study the effect of the mutation on γD-crystallin fibrilization, the WT and mutated proteins were incubated at a high protein concentration (5 mg/ml) at acidic conditions, and then ThT fluorescence was used to probe the existence of amyloid-like fibrils. As presented in Figure 8, neither the WT nor the mutated protein formed fibrils at temperatures below 25°C. At 31°C, the ThT fluorescence of the mutated protein was significantly increased, while that of the WT protein did not. At body temperature, the ThT fluorescence of the mutant was much higher than that of the WT protein. The fibrilization of the proteins was also visualized by transmission EM. At a low protein concentration of 0.5 mg/ml, the mutant was found to convert to considerable amounts of amyloid-like fibrils, whereas the WT protein was at the initial stage of aggregation and formed bead-like structures. These observations suggested that the mutant was more prone to form amyloid-like fibrils than the WT protein when subject to certain physiological stresses or pathological conditions.


The congenital cataract-linked G61C mutation destabilizes γD-crystallin and promotes non-native aggregation.

Zhang W, Cai HC, Li FF, Xi YB, Ma X, Yan YB - PLoS ONE (2011)

Effect of the G61C mutation on the fibrilization of γD-crystallin under acidic condition.(A) ThT fluorescence of the WT and mutated proteins in 10 mM PBS buffer, pH 3, incubated for 48 h at 4°C, 25°C, 31°C and 37°C. The protein concentration was 5 mg/ml. The spectra recorded at 4°C or 25°C superimposed each other for the WT and mutated proteins. (B and C) The transmission EM picture of 0.5 mg/ml WT (B) and G61C γD-crystallin (C) samples incubated at 37°C for 72 h. The bar represents 200 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105094&req=5

pone-0020564-g008: Effect of the G61C mutation on the fibrilization of γD-crystallin under acidic condition.(A) ThT fluorescence of the WT and mutated proteins in 10 mM PBS buffer, pH 3, incubated for 48 h at 4°C, 25°C, 31°C and 37°C. The protein concentration was 5 mg/ml. The spectra recorded at 4°C or 25°C superimposed each other for the WT and mutated proteins. (B and C) The transmission EM picture of 0.5 mg/ml WT (B) and G61C γD-crystallin (C) samples incubated at 37°C for 72 h. The bar represents 200 nm.
Mentions: Cataract is a disease caused by protein aggregation. To further study the effect of the mutation on γD-crystallin fibrilization, the WT and mutated proteins were incubated at a high protein concentration (5 mg/ml) at acidic conditions, and then ThT fluorescence was used to probe the existence of amyloid-like fibrils. As presented in Figure 8, neither the WT nor the mutated protein formed fibrils at temperatures below 25°C. At 31°C, the ThT fluorescence of the mutated protein was significantly increased, while that of the WT protein did not. At body temperature, the ThT fluorescence of the mutant was much higher than that of the WT protein. The fibrilization of the proteins was also visualized by transmission EM. At a low protein concentration of 0.5 mg/ml, the mutant was found to convert to considerable amounts of amyloid-like fibrils, whereas the WT protein was at the initial stage of aggregation and formed bead-like structures. These observations suggested that the mutant was more prone to form amyloid-like fibrils than the WT protein when subject to certain physiological stresses or pathological conditions.

Bottom Line: The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding.The aggregation-prone property of the mutant was not altered by the addition of reductive reagent.These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

Show MeSH
Related in: MedlinePlus