The Molecular Mechanism Of Cataract-causing Mutations L45P And Y46D Of γC-crystallin

PurposeCongenital cataract is the leading cause of blindness among children worldwide.More than 50% of congenital cataracts caused by genetic mutations are caused by crystallins mutations.γC crystallin is one of the lens major structural protein,and its structure stability plays an important role in lens correct refractive gradient and transparency.Existing studies have reported a few pathogenic mutations of γC crystallin causing congenital cataract,and there are few studies on the pathogenic mechanism ofγC crystallin mutations.The human γC-crystallin structure has been reported in recent years,making it possible to explore the molecular mechanism throught molecular dynamics(MD)simulation.The molecular mechanism of congenital cataract pathogenic mutations is helpful to prevention and treatment of congenital cataract and development of cataract drugs.In this study,we explored the pathogenic and molecular mechanisms of γC crystallin mutations L45 P and Y46 D found in two Chinese families with congenital cataract at Eye Center.MethodsProkaryotic plasmids expressing γC-crystallin wild-type(γC-WT),γC-L45 P,andγC-Y46 D mutants were constructed,and Escherichia coli were used for protein expression and further protein purification.With biophysical methods,such as fluorescence spectroscopy,circular dichroic spectroscopy,ultraviolet spectroscopy and other technical methods,the structural differences between γC-L45 P or γC-Y46 D andγC-WT and the protein stability under different environmental stress conditions were detected.Through MD simulation,the structure comparation and trajectory calculation of γC-WT,γC-L45 P,and γC-Y46 D were used to explained the molecular mechanism causing congenital cataract.ResultsIn protein experiment in vitro,γC-L45 P and γC-Y46 D stayed stable at a low temperature,but showed structural differences with γC-WT at physiological temperature with protein denaturation and aggregation.L45 P and Y46 D mutations reduced the γC-crystallin stability under various environmental stresses,such as heat shock,ultraviolet radiation,oxidative environment,p H disorder,and chemical denaturation,and changed the unfolding process of γC-crystallin.γC-L45 P andγC-Y46 D had intermediate states in the unfolding process of heat denaturation and guanidine hydrochloride denaturation.MD simulation showed that L45 P and Y46 D mutations affected the formation of hydrogen bond network around tryptophan at the second Greek key motif of γC-crystallin,resulting in more hydrophobic amino acids exposured of γC-L45 P and γC-Y46 D mutants.Dynamics molecular simulation results explained the molecular mechanisjm of decreased protein stability and protein aggregation formation caused by L45 P and Y46 D mutations.ConclusionThe protein experiment results in vitro indicated that the L45 P and Y46 D pathogenic mutations of γC-crystallin were mainly due to the increased exposure ofγC-crystallin hydrophobic surface,resulting in decreased protein stability under environmental stress.MD results show that the L45 P and Y46 D mutations reduce the stability of γC-crystallin through affecting the hydrogen bond network formation around tryptophan at the second Greek key motif,leading to occurrence and development of congenital cataract.Combined the results of MD in vitro protein experiments,it showed that different molecular mechanisms affecting the hydrogen bond network around different tryptophans of the same Greek motif,causeing γC-L45 P and γC-Y46 D mutants to show similar tryptophan fluorescence change in the protein experiment.