Recombinant Protein Of The First Two Ectodomains Of Cadherin 23 From Erl Mice Shows Impairment In Ca²⁺-dependent Proteolysis Protection

Objective:The erl mouse is a new model of nonsyndromic autosomal recessive deafness(DFNB12)on the C57BL/6J background.The erl allele is a missense mutation(S47P)in cadherin 23(Cdh23).The missense mutation causes the replacement of CDH23-EC1 amino acid(S47P),leading to the secondary structure changes of CDH23(such as a-helix.),thus affecting the higher structure of the CDH23 protein.The purpose of this study was to investigate the calcium binding properties of the first two ectodomains(EC 1+2)of the CDH23,and to explore the possible mechanism of hearing impairment in erl mice.Methods:This project was carried out firstly to express the first two ectodomains of cadherin 23(CDH23 EC 1+2)of erl mice in Escherichia coli and then to identify the Ca2+-binding ability of the recombinant proteins.DNA sequences of CDH23 EC 1+2 from wild type and erl mice were synthesized and cloned into pBV220 plasmids.Recombinant plasmids were transformed into Escherichia coli and the expression of CDH23 EC 1+2 was induced by increasing the temperature from 30 ? to 42 ?.The proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and the antigenicity of proteins was identified by Western Blot.Inclusion bodies were denatured in 8 M urea,purified by ion-exchange and gel filtration chromatography and refolded with dialysis in buffer containing 0.1%sarkosyl.The Ca2+-binding ability of CDH23 EC 1+2 was determined by Mag-Fluo4 which was used for Ca2'indicator and Ca2+-dependent proteolysis protection.Results:The results showed that the sizes and sequences of inserts in recombinant plasmids were consistent with the expectation.The recombinant proteins were mainly found in the form of inclusion bodies which maintain antigenicity.After refolding,the secondary structures of recombinant proteins were measured by circular dichroism(CD)spectra.Moreover,CDH23 EC 1+2 from the erl mice showed less Ca2+-dependent proteolysis protection comparing with that of the wild type control.Conclusions:We concluded that impairment of Ca2+-dependent protein interaction was likely involved in the progressive hearing loss in erl mice.The results may aid in understanding the mechanism of hearing loss in DFNB12.