Pathopoicsis Mechanism Of The Last Strand At The C-terminus In βB2-crystallin Of Congenital Hereditary Cataract-linked Mutants

Purpose:Congenital cataract is the leading cause of childhood blindness worldwide. Many factors have been linked to the onset of congenital cataract, while approximately1/3of the cases are caused by genetic alterations with the most common being the autosomal dominant form. Crystallins are the predominant structural proteins of vertebrate lens and constitute about90%of water-soluble proteins of the lens. βB2-crystallin is one of the most important protein constituents of the human lens and its high stability and high solubility are believed to be crucial to the functional requirements of the lens such as transparency and so on. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on PB2-crystallin structure and stability. Both V187and R188are located at the last strand of βB2-crystallin Greek-key motif4. Molecular mechanisms by which this mutation caused congenital cataract were detected. Method:Wild type βB2-crystallin genes were polymerase chain reaction (PCR) amplified from genomic DNA, then the amplifications were subcloned into pET28a. Site-directed mutagenesis was performed by overlap extension using PCR, and construct the mutant plasmid. Then the plasmids were transformed into E. coli Rosetta (DE3) for the production of the recombinant proteins. Then the wild type and the mutant PB2-crystallin were overexpressed and purified. We studied the effects of the V187M, V187E and R188H mutations on βB2-crystallin structure and stability using a combination of biophysical and molecular dynamic simulation analysis. Behavior and cellular distribution of the wild type and the mutated βB2-crystallin were examined by ligating the genes of the WT and mutated CRYBB2to the vector pEGFP-C3for overexpression in the HeLa cells. Protein distribution was observed by confocal fluorescence microscopy.Results:All of the three mutations promoted βB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected β B2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer.Conclusion:Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of β-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between P-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the β-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of (3-crystallins are achieved by the precisely regulated oligomeric equilibrium and provided more theoretical basis for the prevention and gene therapy of congenital cataracts.