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
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.