Title: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
VOLUME: 24 ISSUE: 11
Author(s):Sadaf Saba, Maryam Ghahramani and Reza Yousefi*
Affiliation:Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz
Keywords:αA-crystallin (αA-Cry), cataract, mutation, calcium, chaperone, aggregation.
Abstract: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.
