Pathopoiesis Mechanism Of Congenital Hereditary Cataract-linked Mutants Of βB2-crystallin

βB2-Crystallin is one of major protein constituents of the human lens. Its high solubility and stability are critical for lens clarity and refraction. Many congenital cataract-linked mutations in βB2-crystallin gene (CRYBB2) have been reported. However, researches focused on the pathopoiesis mechanism of these mutations are deficient, thus there is a missing link between the influence of these mutations on the protein structure/function and the onset of cataract.In this research, we studied the mechanism underlying several mutations including the A2V mutation at the N-terminus and four tryptophan (Trp) related mutations (S31W, R145W, W59C, W151C). We cloned βB2-crystallin coding sequence from human lens cDNA library, inserted it in the expression plasmid pET28a, and PCR-based site-directed mutagenesis was performed to construct the mutant plasmids. Then the wild type βB2-crystallin and the mutants were overexpressed in E. coli and purified. The effect of mutations on βB2-crystallin structure and stability was investigated via biophysical methods such as circular dichroism, fluorescence and size-exclusion chromatography. The cell level study was conducted by overexpressing the WT and mutated βB2-crystallin gene in HeLa cells with expression plasmid pEGFP-C3or pcDNA3.1-flag to characterize the subcellular localization of target proteins, and analyze the effect of the mutant proteins on cell apoptosis.The results showed that the A2V mutation did not affect the secondary and tertiary structures, but retard the tetramerization of βB2-crystallin at high protein concentrations. The mutation also slightly decreased the thermal stability and promoted the thermal aggregation of βB2-crystallin. For the Trp related mutations, The W59C and W151C mutations affected the tertiary structure of βB2-crystallin by increasing the hydrophobic exposure and severely decrease the solubility of the mutants both in vitro and in the cells. The S31W and R145W mutations mainly influenced the stability of βB2-crystallin by promoting thermal aggregation. All four Trp mutations reduced the stability of βB2-crystallin against denaturation induced by chemical denaturants. When exposed under UV radiation, the mutants except R145W were more prone to aggregate than the wild type protein.