Protein Expression, Localization And Function Prediction Of GJB2 Related Mutations

Objective:In the early stage of the experiment,we screened patients with non-syndromic hearing impairment and obtained several new mutation sites of GJB2 gene.In this study,we mainly performed cell expression and location of these mutation sites,and predicted the protein structure by molecular modeling in order to analyze the pathogenicity of these mutation sites and explore the molecular genetic mechanism of the pathogenic sites.Methods:Firstly,we predicted the pathogenicity of the new mutation site of GJB2 gene through bioinformatics,and established the cell model of the mutation with high conservation and high pathogenicity for pathogenicity research: Targeted the wild type GJB2 gene and mutations(P.W24 R,p.V43 L,p.Q57 H,p.H67 N,p.M93V)to construct the GFP-tagged fusion expression vector,and constructed expression vector for three previously reported pathogenic mutants(p.M34 T,p.T55 N,p.S85P).Transfected the constructed plasmids into HEK 293 T cells and HeLa cells,used Western blot analyze the expression differences of the protein and used immunofluorescence technology observe the localization of the protein;then we used the online software Swiss-Model and PDB-Viewer to predict the possible influence of protein conformation change caused by mutation on gap junction channel;finally,we constructed the co-expression vector to further study the function of the target protein and the pathogenic mechanism of the mutant protein in the future.Results:1.Bioinformatics analysis of p.W24 R,p.V43 L,p.Q57 H,p.H67 N,p.M93 V,and p.I107 V mutants showed that all the mutants except p.I107 V had high conservation and high pathogenicity.2.Eight mutant fusion expression vectors of the GJB2 gene were successfully constructed through site-directed mutagenesis.Western Blot results showed that the p.W24 R mutant protein had a smaller molecular weight than the wild-type protein.It may be that the mutation caused some modification or the normality molecular weight has abnormally degraded,the molecular weight of the remaining mutant proteins is basically the same as that of the wild type.The results of cell localization showed that p.M34 T,p.V43 L,p.Q57 H,p.H67 N,p.S85 P,and p.M93 V mutations are similar to the wild type protein,and they were located on the cell membrane,but the p.W24 R and p.T55 N mutant proteins are diffused in the cytoplasm and consistent with the endoplasmic reticulum localization.3.The protein model constructed for the new mutations showed that p.W24 R,p.V43 L,p.H67 N,and p.M93 V mutations may disrupt the stability of hemispheric channels or affect the formation of hemispheric channels,while p.Q57 H may destroy the formation of gap junction channels by affect the interactions of hemispheric channels.4.The pEGFP-N1 vector was successfully transformed into the IRES-mCherry bicistronic vector,and the GJB2(WT/MT)-IRES-mCherry co-expression vector was successfully constructed for subsequent research.Conclusion:The p.W24 R mutation of GJB2 gene is associated with genetic sensorineural hearing loss.The p.W24 R mutation may change the transport of Cx26 in the cell,make accumulation of Cx26 in the cytoplasm,and prevent the formation of functional Cx26 gap junctions,and lead to the cochlear gap junction dysfunction and hearing loss.