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

Purpose:Congenital cataracts are one of the major causes of vision impairment in children,and about one quarter of them are genetically related.Lens crystallin is the most important structural protein of the lens,accounting for about 90%of the water-soluble protein of the lens.Among them,γS-crystallin is one of the important lens crystallins and plays an important role in maintaining the transparency and refraction of the lens.The study of the detailed mechanism of congenital cataracts helps to thoroughly understand the specific functions and pathogenic pathways of the related mutated genes,thus making it possible to completely prevent congenital cataracts in the gene or protein level.At present,there are relatively few reports about congenital cataract caused by mutation of γS-crystallin,and the specific mechanism of congenital cataract caused by γS-crystallin is not deep enough.Therefore,we have selected two recently reported γS-crystallin mutations reported from our eye center(G75V and G18D)to study the effect of mutations on the structure and stability of γS-crystallin,thereby providing insights into the specific molecular mechanisms of congenital cataract induced by γS-crystallin mutations.Methods:The prokaryotic expression plasmids of the wild-type and mutant γS-crystallin were constructed by polymerase chain reaction and transformed into Rosetta E.coli.A large number of wild-type and mutant proteins were induced and purified;Biophysical methods,such as circular dichorosim and fluorescence spectra,were used to study the effect of mutations on protein structure and stability.At the same time,the eukaryotic expression plasmids of wild-type and mutant γS-crystallin were constructed and transfected into human embryonic kidney epithelial cell Hek293T for expression.The localization of the fusion protein in the Hek293T cell line was evaluated under a confocal laser microscope.Results:Both mutations reduced the stability of γS-crystallin,but the effects of the two mutants on the structure of γS-crystallin were different:G75V mutation changed the secondary and tertiary structure of γS-crystallin to a certain extent,and significantly increased the hydrophobic area of the protein.And,in cell experiments,G75V did not cause intracellular protein accumulation.G18D mutant did not significantly change the secondary structure or tertiary structure of γS-crystallin,but caused intracellular protein aggregation.Conclusions:Our current study found that G75V mutant changed the secondary and tertiary structure of protein and increased the hydrophobic area.Meanwhile,we confirmed the high stability of wild-type γS-crystallin and found that the stability of G75V and G18D mutant proteins were decreased under thermal denaturation and GdnHCl degeneration.It may be the major cause of congenital cataracts.The phenotypes of these two mutational families are mainly progressive cortical cataracts.Cataracts in patients with mutations tend to get worse gradually under the pressure of the external environment and this phenomenon is consistent with the experimental results.