Synergistic effects of metal ion and the pre-senile cataract-causing G98R αA-crystallin: self-aggregation propensities and chaperone activity

Abstract

Purpose: αA- and αB-crystallins are abundantly present in the eye lens, belong to the small heat shock protein family, and exhibit molecular chaperone activity. They are also known to interact with metal ions such as Cu²⁺, and their metal-binding modulates the structure and chaperone function. Unlike other point mutations in aA-crystallin that cause congenital cataracts, the G98R mutation causes pre-senile cataract. We have investigated the effect of Cu²⁺ on the structure and function of G98R αA-crystallin. Methods: Fluorescence spectroscopy and isothermal titration calorimetry were used to study Cu²⁺ binding to αA- and G98R αA-crystallin. Circular dichroism spectroscopy was used to study secondary and tertiary structures, and dynamic light scattering was used to determine the hydrodynamic radii of the proteins. Chaperone activity and self-aggregation of the wild type and the mutant protein in the absence and the presence of the metal ions was monitored using light scattering. Results: Our fluorescence quenching and isothermal titration calorimetric studies show that like αA-crystallin, G98R α A-crystallin binds Cu²⁺ with picomolar range affinity. Further, both wild type and mutant aA-crystallin inhibit Cu2+-induced generation of reactive oxygen species with similar efficiency. However, G98R αA-crystallin undergoes pronounced self-aggregation above a certain concentration of Cu²⁺ (above subunit to Cu2+ molar ratio of 1:3 in HEPES-NaOH buffer, pH 7.4). At concentrations of Cu²⁺ below this ratio, G98R αA-crystallin is more susceptible to Cu²⁺-induced tertiary and quaternary structural changes than αA-crystallin. Interestingly, Cu²⁺ binding increases the chaperone-like activity of αA-crystallin toward the aggregation of citrate synthase at 43°C while it decreases the chaperone-like activity of G98R αA-crystallin. Mixed oligomer formation between the wild type and the mutant subunits modulates the Cu²⁺-induced effect on the self-aggregation propensity. Other heavy metal ions, namely Cd²⁺ and Zn²⁺ but not Ca²⁺, also promote the self-aggregation of G98R αA-crystallin and decrease its chaperone-like activity. Conclusions: Our study demonstrates that unlike wild type αA-crystallin, G98R αA-crystallin and its mixed oligomers with wild type protein are vulnerable to heavy metal ions. Our study provides insight into aspects of how environmental factors could augment phenotype(s) in certain genetically predisposed conditions.