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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 DTT on γD-crystallin thermal aggregation.(A) Temperature-dependence of γD-crystallin thermal aggregation monitored by turbidity. (B) Time-course aggregation of γD-crystallin at 80°C. The samples were prepared in 10 mM PBS buffer, pH 7, with or without the addition of 2 mM DTT.
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pone-0020564-g009: Effect of DTT on γD-crystallin thermal aggregation.(A) Temperature-dependence of γD-crystallin thermal aggregation monitored by turbidity. (B) Time-course aggregation of γD-crystallin at 80°C. The samples were prepared in 10 mM PBS buffer, pH 7, with or without the addition of 2 mM DTT.

Mentions: A previous study has shown that one cataract-linked mutation R14C introduces an additional reactive thiol group, which contributes to the intermolecular cross-link of the γD-crystallin molecules and leads to aggregation therefore [50]. To investigate whether the cataract caused by the G61C mutation followed this molecular mechanism, we studied the effect of DTT on the thermal aggregation behavior of the WT and G61C γD-crystallin (Figure 9). At temperatures above 80°C, although the addition of DTT could decrease the turbidity at each given temperature, the whole process was not significantly affected. To avoid errors raised from the temperature adjustment, time-course aggregation was monitored by heating the protein solutions continuously at 80°C. As shown in Figure 9B, no significant changes were observed between the samples with and without the addition of DTT. Similarly, no significant effect of DTT was also observed for γD-crystallin aggregation induced by dilution-initialed refolding and acid-induced fibrilization (data not shown). These observations suggested that the reactive –SH group was not significantly altered by the G61C mutation and that the intermolecular disulfide formation was not a major cause of γD-crystallin aggregation.


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 DTT on γD-crystallin thermal aggregation.(A) Temperature-dependence of γD-crystallin thermal aggregation monitored by turbidity. (B) Time-course aggregation of γD-crystallin at 80°C. The samples were prepared in 10 mM PBS buffer, pH 7, with or without the addition of 2 mM DTT.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020564-g009: Effect of DTT on γD-crystallin thermal aggregation.(A) Temperature-dependence of γD-crystallin thermal aggregation monitored by turbidity. (B) Time-course aggregation of γD-crystallin at 80°C. The samples were prepared in 10 mM PBS buffer, pH 7, with or without the addition of 2 mM DTT.
Mentions: A previous study has shown that one cataract-linked mutation R14C introduces an additional reactive thiol group, which contributes to the intermolecular cross-link of the γD-crystallin molecules and leads to aggregation therefore [50]. To investigate whether the cataract caused by the G61C mutation followed this molecular mechanism, we studied the effect of DTT on the thermal aggregation behavior of the WT and G61C γD-crystallin (Figure 9). At temperatures above 80°C, although the addition of DTT could decrease the turbidity at each given temperature, the whole process was not significantly affected. To avoid errors raised from the temperature adjustment, time-course aggregation was monitored by heating the protein solutions continuously at 80°C. As shown in Figure 9B, no significant changes were observed between the samples with and without the addition of DTT. Similarly, no significant effect of DTT was also observed for γD-crystallin aggregation induced by dilution-initialed refolding and acid-induced fibrilization (data not shown). These observations suggested that the reactive –SH group was not significantly altered by the G61C mutation and that the intermolecular disulfide formation was not a major cause of γD-crystallin aggregation.

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