The cataract-causing mutation G98R in human αA-crystallinleads to folding defects and loss of chaperone activity

[摘要] Purpose: The objective of this study is to understand the molecularbasis of cataract that develops due to the mutation of the glycine-98residue to arginine in αA-crystallin.Methods: The glycine-98 residue was mutated to arginine bysite-directed mutagenesis. The expression, structural and chaperoneproperties and thermal stability of the mutant, G98RαA-crystallinhave been studied. The secondary and tertiary structure of the wild typeand the mutant protein was studied using circular dichroism andfluorescence spectroscopy. The quaternary structure was studied by gelfiltration chromatography and dynamic light scattering. Chaperoneactivity studies were carried out using DTT-induced aggregation ofinsulin.Results: Unlike the wild type protein, the heterologous expressionof G98R αA-crystallin in E.coli results in the formation ofinclusion bodies. Upon dissolving the inclusion bodies in 3 M urea andsubjecting to refolding, it yielded a clear solution. The refoldedmutant protein exhibits altered secondary, tertiary and quaternarystructure, which lacks chaperone function, and is susceptible toheat-induced aggregation.Conclusions: The G98R mutation in αA-crystallin results inaltered folding and becomes aggregation-prone leading to formation oflarge oligomers lacking chaperone function. Tendency to aggregate andloss of chaperone activity could be contributing to turbidity and lossof transparency of the lens.

[学科分类] 生物化学/生物物理