Mixed oligomer formation between human alphaA-crystallin and its cataract-causing G98R mutant: structural, stability and functional differences.
Mutation of the glycine 98 residue to arginine in alphaA-crystallin has been shown to cause presenile cataract in an Indian family. Our earlier study showed that the mutant protein exhibits folding defects that lead to aggregation and inclusion body formation in Escherichia coli. Despite the presence of a normal copy, the pathology is seen in the heterozygous individuals. Formation of mixed oligomers between wild-type and the mutant subunits might be crucial for manifestation of such dominant negative character. We have investigated the role of G98R mutation in alphaA-crystallin in its structural stability and subunit exchange. G98R alphaA-crystallin unfolds at lower concentrations of urea compared to wild-type alphaA-crystallin. The mutant protein is more susceptible to proteolysis than the wild-type protein and transiently populates fragments that are prone to aggregation. Subunit exchange studies using fluorescence resonance energy transfer show that the mutant protein forms mixed oligomers with the wild-type protein. The mutant protein is more susceptible to thermal aggregation, whereas mixed oligomer formation leads to a decreased propensity to aggregate. Co-expression of wild-type alphaA-crystallin with G98R alphaA-crystallin in E. coli rescues the mutant alphaA-crystallin from formation of inclusion bodies. These observations may underlie the molecular basis for the presenile onset, not congenital cataract, in spite of severe folding defect and aggregation of the mutant. Our study shows that the mixed oligomers of wild-type and G98R alphaA-crystallin exhibit properties dominated by those of the mutant protein in structural aspects, oligomeric size, urea-induced unfolding and, more importantly, the chaperone activity, which may provide the molecular basis for presenile cataract formation in affected individuals.
Singh
Devendra
D
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Raman
Bakthisaran
B
Ramakrishna
Tangirala
T
Rao
Ch Mohan
ChM
D002386:Cataract / Q000235:genetics*; D019281:Dimerization; D006801:Humans; D018832:Molecular Chaperones; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D018832:Molecular Chaperones; D038202:alpha-Crystallin A Chain