A comparative study of the impact of calcium ion on structure,aggregation and chaperone function of human αa-crystallin and its cataract- causing R12C mutant

Background: the chaperone activity of α-crystallin (α-cry) plays an important role in maintenance of eye lens transparency. various mutations in the α-cry genes have been indicated to cause cataract diseases in human. also,the calcium imbalance has been shown to induce aggregation in α-cry. we investigated the impact of calcium ion on structure,chaperone activity of the recombinant wild-type and mutant r12c αa-cry. we suggested that the raise of calcium level in eye lens is an additional contributory factor accelerating the development of cataract diseases in patients with r12c mutation. objectives: the main objective of this study was to investigate the impact of calcium ion on structure,chaperone activity and amyloidogenic properties of the recombinant wild-type and mutant r12c αa-cry,in a comparative study. methods: the mutagenesis was performed on confirmed αa-cry cdna in pet-28b (+) which applied as a template to generate r12c mutant,using polymerase chain reaction (pcr) and a quick change lightning multi site-directed mutagenesis kit (stratgene). both wild-type and mutant plasmids were chemically transformed into e.coli bl21 (de3) and the respective recombinant proteins over-expressed in lb broth. the protein purification was done using q-sepharose anion exchange and sephacryl s-300 gel filtration chromatography. the purified αa-cry samples were incubated with different concentrations of calcium ion (0-40 mm) at 37 °c for 1 week. the secondary and tertiary structural analyses of each protein were performed by far-uv cd and try/trp and ans fluorescence assessments,respectively. the assessment of chaperone activity was done spectrophotometrically in both thermal and chemical-induced aggregation systems using γ-cry and bovine pancreatic insulin as the substrate proteins,respectively. also,the amyloidogenic properties of proteins was investigated by cr absorption and tht fluorescence measurements. results: the results of fluorescence and cd assessments suggested the significant secondary and tertiary structural alterations upon r12c mutation. r12c mutant αa-cry demonstrated preserved secondary and tertiary structures in the presence of calcium. the chaperone activity of wild-type and mutant r12c αa-cry was reduced in the presence of calcium. also,the extent of chaperone activity reduction was significantly higher for r12c αa-cry. both wild-type and mutant r12c αa-cry revealed slight amount of aggregation when incubated with different calcium concentrations for 1 week,at 37 °c. however,the susceptibility- of both proteins for aggregation was significantly increased in the presence of 40 mm calcium,at the elevated temperature (60 °c). also,the mutant protein exhibited extensive disulfide bridge cross-linking as indicated by gel electrophoresis. moreover,the mutant r12c αa-cry significantly resists against amyloid fibril formation in the presence of calcium ion compared to the wild-type protein as indicated by cr and tht assessments. conclusion: our data suggested that αa-cry conformational changes occurring upon r12c mutation and further functional damages induced by calcium may play an important role in the pathomechanism of the cataract development by this mutant protein. © 2017 bentham science publishers.