The cataract-causing mutation G98R in human alphaA-crystallin leads to folding defects and loss of chaperone activity

The objective of this study is to understand the molecular basis of cataract that develops due to the mutation of the glycine-98 residue to arginine in alphaA-crystallin.

The glycine-98 residue was mutated to arginine by site-directed mutagenesis. The expression, structural and chaperone properties and thermal stability of the mutant, G98RalphaA-crystallin have been studied. The secondary and tertiary structure of the wild type and the mutant protein was studied using circular dichroism and fluorescence spectroscopy. The quaternary structure was studied by gel filtration chromatography and dynamic light scattering. Chaperone activity studies were carried out using DTT-induced aggregation of insulin.

Unlike the wild type protein, the heterologous expression of G98R alphaA-crystallin in E. coli results in the formation of inclusion bodies. Upon dissolving the inclusion bodies in 3 M urea and subjecting to refolding, it yielded a clear solution. The refolded mutant protein exhibits altered secondary, tertiary and quaternary structure, which lacks chaperone function, and is susceptible to heat-induced aggregation.

The G98R mutation in alphaA-crystallin results in altered folding and becomes aggregation-prone leading to formation of large oligomers lacking chaperone function. Tendency to aggregate and loss of chaperone activity could be contributing to turbidity and loss of transparency of the lens.