Cataract-causing αAG98R mutant shows substrate-dependentchaperone activity

[摘要] Purpose: The G98R mutation in human αA-crystallin isassociated with autosomal dominant cataract (presenile type). Thereasons for cataract development in αAG98R individuals are notfully understood. Therefore we undertook this study to analyze thestability, structural changes and chaperone function of αAG98Rprotein.Methods: Site-directed mutagenesis was employed to generate αAG98R mutant protein. Human αA-crystallin cDNA cloned into thepET23d vector was used as the template. The recombinant proteins wereexpressed in E. coli and purified using chromatographic methods.Both the wild-type and mutant proteins were characterized by SDS-PAGE,transmission electron microscopy, static and dynamic light scattering,and spectroscopic analysis. The chaperone-like function of the mutantprotein was compared with wild-type protein using different substrates.Results: The G98R mutant protein formed larger oligomers compared tothe wild-type αA-crystallin. Circular dichroism studies showedaltered secondary and tertiary structure whereas bis-ANS binding studiesshowed a gain of surface hydrophobicity in the αAG98R protein. TheαAG98R protein displayed a substrate-dependent chaperone-likeactivity. The mutant protein appeared to have diminished chaperone-likeactivity toward aggregating α-lactalbumin, whereas citrate synthaseand alcohol dehydrogenase were efficiently protected from aggregation.Conclusions: The present results reveal that the G98R mutationcauses conformational changes in αA-crystallin and that withcertain substrates the mutant protein forms complexes that are prone toprecipitate over time. The accumulation of mutant protein-substratecomplexes may be the reason for cataract development in individualscarrying the G98R mutation in αA-crystallin.

[学科分类] 生物化学/生物物理