Cataract-causing αAG98R-crystallin mutant dissociates into monomers having chaperone activity

PURPOSE: The G98R mutation in αA-crystallin is associated with autosomal dominant cataract in humans. We have reported that mutant G98R protein has substrate-dependent chaperone activity. Further studies on this G98R mutant protein revealed that mutant protein shows reduced oligomeric stability and accelerated subunit dissociation at a low protein concentration. The purpose of present study was to investigate the chaperone function of dissociated subunits of αAG98R-crystallin. METHODS: Substitution of glycine with arginine at position 98 in human αA-crystallin was accomplished by site-directed mutagenesis. The recombinant protein was expressed in E .coli cells and purified by chromatographic techniques. Purified αAG98R-crystallin was diluted to a concentration of 0.1 mg/ml in 50 mM phosphate buffer containing 150 mM NaCl (pH 7.2) and incubated at 37 °C for 24 h. The monomeric subunits were isolated from the oligomers through 50 kDa cutoff filters. The monomers were analyzed by SDS–PAGE, mass spectrometry, and circular dichroism spectroscopy and characterized by multi-angle light-scattering methods. Chaperone activity was tested against four client proteins: citrate synthesis, alcohol dehydrogenate, bovine βB2-crystallin and ovotransferrin. RESULTS: Gel filtration studies showed that αAG98R-crystallin oligomers dissociate readily into monomers. Subunits of αAG98R-crystallin, isolated either by size exclusion chromatography or filtration showed chaperone activity against heat-denatured alcohol dehydrogenase, citrate synthase, bovine βB2-crystallin, and chemically denatured ovatransferrin. SDS–PAGE analysis of the mutant protein incubated at 37 °C for 12 days showed autolysis, which was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS/MS) analysis of αAG98R-crystallin fragments recovered after SDS–PAGE. CONCLUSIONS: The present study shows that the G98R mutation in αA-crystallin produces unstable oligomers that dissociate into active chaperone subunits. The chaperone activity of the dissociated subunits against four client proteins suggests that the αA-crystallin subunits are the minimal units of chaperone activity.