Pathopoiesis Mechanism Of Congenital Cataract-related γS-S39C Variant

Background and Objective: The congenital cataract is one of the leading causes of treatable blindness in children worldwide.At present,the effective treatment for congenital cataracts is surgery,but there are some indications and complications.Studying the pathogenic mechanisms of crystallin mutations may provide new strategies for finding non-surgical treatment methods of congenital cataracts.Therefore,in this study,we selected the reported S39 C mutation and investigated its effects on the structure and properties of γS-crystallin and clarified its pathogenic mechanism.Methods: We constructed prokaryotic plasmids of γS-crystallin wild-type and the S39 C mutant.The plasmids were transferred into E.coli Rosetta strains and induced to express the protein for purification.Fluorescence spectroscopy,circular dichroism spectroscopy,Raman spectroscopy and other biophysical methods were used to investigate the effects of S39 C mutation on γS-crystallin structure and the stabilities under heat shock,Gdn HClinduced denaturation,UV radiation and oxidative stress.Molecular dynamics simulations of the dimer structure of γS-crystallin wild-type and the S39 C mutant were performed to analyze the effects of the S39 C mutation on the disulfide-linked dimer structure of γScrystallin.Results: The results of intrinsic and extrinsic fluorescence,circular dichroism spectroscopy and Raman spectroscopy suggested that the S39 C mutation did not significantly change the secondary and tertiary structure of γS-crystallin.The solubility experiment showed that the mutation reduced the solubility of γS-crystallin.From the results of protein stability experiments,the S39 C mutation significantly reduced the stabilities of γScrystallin under heat shock,Gdn HCl-induced denaturation,UV radiation and oxidative stress.Under oxidative conditions,the S39 C mutation prevented γS-crystallin from forming stable disulfide-linked dimers and remarkably increased hydrophobicity.The S39 C mutation increased the sensitivity of the mutant to the oxidative stress and the propensities to aggregate and precipitate.The results of molecular dynamics simulations showed that the S39 C mutation altered the spatial interaction between the γS-crystallin subunits and prevented the formation of the disulfide-linked dimer.Conclusion: S39 C mutation disrupted the disulfide-linked dimer of γS-crystallin,reduced the solubility of γS-crystallin and the stabilities coping with environmental stress,increased the sensitivity to oxidative stress,and made the mutant protein more prone to aggregate and precipitate,which led to the occurrence of the congenital cataract.