Study On Characterization And Mechanism Of Metal Binding Of ShCdMT And ShCuMT In The Freshwater Crab Sinopotamon Henanense

In the freshwater crab Sinopotamon henanense,there are two metallothionein?MT?isoforms which are Cd-specific binding metallothionein and Cu-specific binding metallothionein,and are defined as ShCdMT and ShCuMT,respectively.In this paper,metal binding characterization of ShCdMT and ShCuMT are completed to help to well understand the physiological function/ structure and evolution/ function relationship of ShCdMT and ShCuMT.Firstly,we characterized the metal tolerance of recombinant strains harboring ShCdMT from the freshwater crab S.henanense in vivo and metal binding properties of ShCdMT purified in vitro.The recombinant strains harboring ShCdMT were exposed to 0.1 mM Cd²⁺,0.3 mM Cu²⁺,0.5 m M Pb²⁺,and 0.8 mM Zn²⁺.The growth curves and spot assays of recombinant strains and the contents of heavy metal ions were analysed in the media supplemented with above metal ions provided to recombinant E.coli synthesis.The structural characteristics of the Cd-,Cu-,Pb-,and Zn-ShCdMT were determined through ultraviolet spectroscopy?UV-vis?,circular dichroism?CD?,and isothermal titration calorimetry?ITC?.Secondly,we analysed the stoichiometry of ShCdMT yielded in vivo and exchange reactions of the ZnShCdMT with Cd²⁺,Pb²⁺ and Cu²⁺ in vitro via electrospray ionization time-offlight mass spectrometry?ESI-TOF-MS?,circular dichroism?CD?spectroscopy,inductively coupled plasma mass spectrometry?ICP-MS?,and isothermal titration calorimetry?ITC?.Thirdly,The nucleic and amino acid sequence of sh Cumt gene were analyzed by DNAMAN software,meanwhile,phylogenetic trees and multiple sequence alignment of ShCuMT were established by MEGA7.0 and Bioedit software.Physicochemical property and secondary structural element were predicted by the database of Protparam of EXPASY and SOPMA on line.In addition,prokaryotic expression vector of p ET-28a-SUMO-sh Cumt was built and the purification of ShCuMT was completed by Ni-NTA affinity chromatography and gel chromatography.Finally,the metal binding feature of ShCuMT were characterized by the reaction between Cd-?Cu-?? Zn-ShCuMT and DTNB,CD spectroscopy,ITC and ESI-TOF-MS.The results showed that:1.The metal binding characterization of Cd-specific metallothionein in S.henanenseCompared to control strains,recombinant strains tolerated Cd²⁺,Cu²⁺,Pb²⁺,and Zn²⁺.Furthermore,the contents of Cd²⁺ and Pb²⁺ in media decreased substantially.In vitro and the Cd-ShCdMT had a higher degree of folding compactness in solution.5,5'-Dithiobis-?2-nitrobenzoic?acid?DTNB?reaction and ITC results demonstrated that ShCdMT yielded Cd_6-,Cu_7-,and Pb_6- ShCdMT.The binding stability order was Cu-ShCdMT > Cd-ShCdMT > PbShCdMT > Zn-ShCdMT.2.The spectrometry and thermodynamics of the Cd-specific metallothionein in S.henanense for its binding ability with different metalsThe results of ESI-TOF-MS analyses showed that metal-ShCdMT synthesized in vivo had three major forms,namely Zn_15-,Cd_9-,and Pb_5- ShCdMT.The ITC analyses of exchange reactions demonstrated that ZnShCdMT exhibited up to 6,6,and 7 binding sites for Cd²⁺,Pb²⁺ and Cu²⁺.By the analyses of the UV and CD spectra in the substitution experiments showed that the geometric structural stability of metal-ShCdMT could be influenced when excess of over 6,6,or 7 equivalents of Cd²⁺,Pb²⁺,or Cu²⁺ were added into Zn-ShCdMT.Although both the reconstructed apo-ShCdMT and substituted Zn-ShCdMT with three metal ions fitted the same M₆?-and M₇?-ShCdMT binding models for divalent and monovalent metals,the differences in their thermodynamic data suggested that discrepancies exit in their physiological functions.3.Clone,bioinformatics analysis of Cu-specific metallothionein gene c DNA and prokaryotic expression of Cu-specific metallothionein protein in S.henanenseThe full length of sh Cumt c DNA is 198 bp,and the nucleic acid sequence of the of sh Cumt c DNA have the higher similar to mt gene sequence of Callinectes sapidus,Scylla paramamosain and Portunus pelagicus.ShCuMT protein contain 65 amino acids and the number of cysteines is 21 which accounts for about 32.3% of the total amino acid content of ShCuMT.The cysteines in ShCuMT arrange to the three conservative CXC motifs,two CCC motifs,one CX5 CXC motif,one CX3 CXC motif and one CXCX2 C motif.Besides,there are no aromatic amino acids in amino acid sequences of ShCuMT,however,small molecular weight amino acids,ie.Serine,glycine and lysine,are rich.The theoretical molecular weight of ShCuMT is about 6293 Da and isoelectric point is 8.3.ShCuMT protein contains hardly any secondary structure elements and the proportion of irregular curling is 87.69%.In addition,the optimal inducing IPTG concentration and time are 1m M in and 8 h for the prokaryotic expression of SUMO-ShCuMT protein.4.The metal binding characterization of Cu-specific metallothionein in S.henanenseShCuMT showed strong metal ion binding properties.The stability order of Cd-,Cu-and Zn-ShCuMT was Cu-> Cd-> Zn-ShCuMT and ShCuMT showed the stability and uniqueness for the binding of Cu²⁺.The main species of Cd-,Cu-,and Zn-ShCuMT synthesized in vivo are Cd19-,Zn19-,and Cu32-ShCuMT,respectively.However,the results of titration of apo-ShCuMT with Cd²⁺,Zn²⁺,and Cu²⁺ showed that ShCuMT could coordinated 6 Cd²⁺,3 Zn²⁺,and 10 Cu²⁺ in vitro.Moreover,the scan of CD spectra of Cd-,and ZnShCuMT showed that there is a Gaussian band,respectively,at 250 nm and 254 nm,however,Cu-ShCuMT shows the excited coupling band at 270 nm,and the typical Cu-thionein absorption band between 300 and 320 nm.Conclusions:Firstly,the present study suggests that ShCdMT is a Cd-specific MT isoform,has a canonical invertebrate MT structure that exhibits a ?/ ? structural domain,forms M₆^?-ShCdMT with divalent metal ions and has a high affinity to monovalent metal ions.In summary,we demonstrated that ShCdMT serves the important physiological functions of Cd²⁺ detoxification and Zn²⁺ homeostasis regulation in S.henanense.Secondly,the result demonstrated that ShCdMT in S.henanense could largely stockpile Zn²⁺ and uptake Cd²⁺.And it should be suitable for ZnShCdMT using metal binding models of M6?-ShCdMT for Cd²⁺ and Pb²⁺ and M7?-ShCdMT for Cu²⁺.The exchange reactions corroborated that Zn-ShCdMT could release Zn²⁺ and uptake Cd²⁺,Cu²⁺,or Pb²⁺.Zn-ShCdMT could mediate the inter-regional Zn²⁺ transfer and serve as a detoxification biomolecule for heavy metal ions.ShCdMT possessed a higher homeostasis regulatory ability of Zn²⁺ and detoxification for non-essential metal ions.Thirdly,the full length of sh Cumt c DNA is 198 bp.ShCuMT protein contain 65 amino acids and the number of cysteines is 21 which accounts for about 32.3% of the total amino acid content of ShCuMT.The theoretical molecular weight of ShCuMT is about 6293 Da and isoelectric point is 8.3.ShCuMT protein contains hardly any secondary structure elements and the proportion of irregular curling is 87.69%.The analysis of phylogenetic trees suggested that ShCuMT protein may be a typical Cu-thionein MT isoform.Fourthly the Cu-ShCuMT fold in the solution had the stable and compact three-dimensional structure.The binding of ShCuMT with Cu²⁺ are stable,uniqueness and higher stoichiometry.ShCuMT exhibited the preference for the binding of Cu²⁺,so,ShCuMT could be defined a typical Cu-specific MT isoform.Thus,although ShCuMT shows stronger metal binding capacity,ShCuMT won't play a role in detoxification of non-essential metal elements,on the contrary,ShCuMT play an important role in regulating the Cu²⁺ homeostasis so that to warrant the normal synthesis of hemocyanin which is an important cytochrome in respiratory chain for S.henanense.