| Metallothioneins (MTs) belong to a superfamily of intracellular metal-binding proteins, present in virtually all living organisms. Typically, MTs have low molecular weight (<7000Da), high metal content comprising predominantly Zn, Cu or Cd, highly conserved18-23cysteine residues and no aromatic amino acids or histidine. In mammals, MTs are thought to be primarily involved in copper sequestration and in the protection from reactive oxygen species. MTs are currently classified into15families. Tetrahymena MTs belong to the7th family.In T. thermophila,5MT isoforms (MTT1, MTT2, MTT3, MTT4and MTT5) were identified, but their functions are not clear. To learn the functions of different MTs from T. thermophila, MTT1and MTT2were analysed and identified, the following results are obtained:1. Bioinformatics analysis of MTT1and MTT2MTT1consists of162amino acids with a predicted molecular weight of approximately16.76kDa; MTT2consists of108amino acids with a predicted molecular weight of approximately11.17kDa. MTT1mainly contains Cys-Cys-Cys and Cys-Cys motifs, similar to Cys arrange pattern of mammalian MTs a-domain; MTT2mainly contains Cys-X-Cys motif, which is similar to Cys arrange pattern of mammalian MTs β-domain.2. Expression analysis of MTT1and MTT2exposed to Cd2+, Cu2+, NaCl, SO2derivatives and Tris-HClExpression of MTT1and MTT2was analysed using real-time quantitative-PCR. The MTT1gene was more sensitive than MTT2when it was exposed to Cd2+, NaCl, SO2derivatives and Tris-HCl. By contrast, the MTT2gene was more sensitive than MTT1when it was exposed to Cu2+3. Expression and purification of MTT1and MTT2MTT1and MTT2were cloned from the T. thermophila macronucleus genome. Original TAG and TAA triplet (encodes Gln) were substituted by CAG and CAA in MTT1sequence. In order to do spectral analysis, Tryptophan substituted the original fourth Valine in the MTT1Tryptophan substituted the original third threonine in the MTT2. Mutated MTT1and MTT2were expressed in E.coli BL21and purified by Ni-NTA and FPLC using a Superdex75gel filtration column.4. Secondary structure analysis of MTT1and MTT2According to the results of fluorescence quenching experiments, MTT1bind16Cd2+and MTT2bind11Cd2+. CD results showed apoMTT1and apoMTT2mainly consist of random coils. Cd16-MTT1complex mainly consists of α-helix and β-turns. In contrast, Cd11MTT2complex mainly consists of random coils.5. Antioxidant capacity of Cd16-MTT1and Cd11-MTT2complexAfter reaction of1mM Cd16-MTT1and Cd11-MTT2complex with0.5mM nitric oxide, disulfide bond formation was found in the intramolecular of both Cd,6-MTT1and Cd11-MTT2complex. FTIR showed that both Cd16-MTT1and Cd11-MTT2complex have antioxidant ability when they exposed to reactive oxygen species.6. Chelation capacity of MTT1and MTT2to Cd2+, Hg2+, Zn2+and Cu2+The stability constants of MTT1and MTT2with Cd2+, Hg2+and Zn2+were determined respectively by the UV-Visible. The results was as follows: Kcd-MTT1=1.45X1020.45, KHg-MTT1=2.47X1019.43, KZn-MTT1=1.41×1016.60. KCd-MTT2=2.19×1018.10, KHg-MTT2=8.46×107.79, KZn-MTT2=1.41×1010.98. In general, the stability constants of MTT1with metal ions are higher than those for MTT2. When Cd16-MTT1and Cd11-MTT2complex were titrated with Cu2+, Cd2+in Cd16-MTT1complex can not been replaced with Cu2+, but Cd2+in Cd11-MTT2complex can be replaced with Cu2+7. The impact of La3+on MTT1and MTT210-80μ M La3+can promote Tetrahymena cells proliferation and La3+can enter T. thermophila. Real-time Quantitative PCR showed that La3+induced the expression of MTT1and MTT2. Furthermore, fluorescence spectrum analysis showed La3+bind to MTT1and MTT2. 8. Construction of MTT1and MTT2gene knockout strainsBioinformatics showed that MTT1and MTT3, which contain consistent Cys pattern, were tandem arranged on the chromosome; and MTT2and MTT4, which also contain consistent Cys pattern, were tandem arranged on the chromosome. In order to learn biological function of MTT1and MTT2gene, A MTT1-MTT3and A MTT2-MTT4gene knockdown strains were constructed by homologous recombination, respectively. A MTT1-MTT3gene knockout strains proliferate faster than the wild type and they are sensitive to Cd2+;△MTT2-MTT4gene knockout strains proliferate lower than the wild type and they are sensitive to Cu2+In the study, MTT1and MTT2which have different characteristics of primary structure were expressed, purified and analyzed by spectroscopy methods in vitro for the first time. The results showed that the affinity of MTT1for Cd2+is stronger than for Hg2+, Zn2+and Cu2+; and the affinity of MTT2for Cu2+is stronger than for Cd2+."CCC" motif and "CXC" motif showed different metal ion binding character:MTT1containing exclusive "CCC" motif has preference for Cd2+, and MTT2containing "CXC" motif which was widespread in various organisms has preference for Cu2+. MTT1plays a major role in the detoxification of heavy metal ions, and MTT2participate in the homeostasis of copper ions. We also showed that trivalent rare earth elements La3+can promote Tetrahymena cells proliferation, and induce expression of MTT1and MTT2. MTT1and MTT2bind La3+via O atom from asparagines and glutamic acids. Furthermore, the MTTl and MTT2gene knockout cells showed that they are engaged in different biological function in Tetrahymena.
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