Decreasing the homodimer interaction: a common mechanism shared by the ΔG91 mutation and deamidation in βA3-crystallin

PURPOSE: Cataracts can be broadly divided into two types: congenital cataracts and age-related cataracts. ΔG91 is a previously discovered congenital mutation in βA3-crystallin that impairs protein solubility. On the other hand, the deamidation of β-crystallin is a significant feature in aged and cataractous lenses. Several deamidation sites were also identified in βA3-crystallin. The present study is to compare the functional consequence of ΔG91 mutation and the deamidation of βA3-crystallin in terms of folding properties and protein–protein interaction. METHODS: Protein secondary structure and hydrophobic properties were investigated by in silica analysis of the wild type and mutants sequences. Full-length βA3-crystallin was cloned into a mammalian two-hybrid system in order to investigate protein–protein interactions. Deletion and deamidation were introduced by site-directed mutagenesis protocols. Both the Q85 and Q180 deamidation sites were substituted with glutamic acid residues to mimic deamidation. Different combinations of plasmid constructs were transfected in HeLa cells, and changes of protein–protein interactions were analyzed by the luciferase assay. RESULTS: Bioinformatics prediction suggested that ΔG91 mutation alters both the predicted secondary structure and hydrophobic character of βA3-crystallin, while deamidation only exhibits minimal effects. Mammalian two-hybrid results indicated that both ΔG91 mutation and Q85/Q180 deamidation could significantly decrease the interaction of the βA3-crystallin homodimer. CONCLUSION: Our results provided evidence that both mutations involved in congenital cataracts and deamidation in aged lenses commonly altered protein–protein interaction between human lens βA3-crystallins, which may lead to protein insolubilization and contribute to cataracts.