Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum.

Using a yeast expression vector system, we have expressed both wild type and six mutated Chinese hamster metallothionein coding sequences in a metal-sensitive yeast strain in which the endogenous metallothionein gene has been deleted. The mutant proteins have single or double cysteine to tyrosine replacements (C13Y, C50Y, and C13,50Y), single cysteine to serine replacements (C13S and C50S), or a single cysteine to alanine replacement (C50A). These proteins function in their yeast host in cadmium detoxification to differing extents. Metallothioneins which contain a cysteine mutation at position 50 (C50Y, C50S, C50A, and C13,50Y) conferred markedly less cadmium resistance than wild type metallothionein, or metallothionein with a single cysteine mutation at position 13 (C13Y and C13S). Wild type and three of the mutant Chinese hamster metallothioneins (C13Y, C50Y, and C13,50Y) were purified from yeast grown in subtoxic levels of either CdCl2 or 113CdCl2. All three of the mutant proteins bound less cadmium than the wild type protein when metal-binding stoichiometries were determined. The one-dimensional 113Cd NMR spectrum of the recombinant wild type Chinese hamster metallothionein was compared to the spectra of native rat and rabbit liver metallothioneins. The close correspondence between the 113Cd chemical shifts in these metallothioneins is consistent with the presence of two separate metal clusters, A and B, corresponding, respectively, to the alpha- and beta-domains, in the recombinant metallothionein. The one-dimensional 113Cd NMR spectra recorded on each of the three mutant metallothioneins, on the other hand, provide some indication as to the structural basis for the reduced, by one, metal stoichiometry of each of the mutant metallothioneins. For the C13Y mutant, it appears that the beta-domain now binds a total of two metal ions whereas with the C50Y mutant, the alpha-domain appears metal-deficient. For the double mutant, C13,50Y, the 113Cd resonances are indicative of major structural reorganizations in both domains.     

Molecular cloning and sequencing of metallothionein in squamates: New insights into the evolution of the metallothionein genes in vertebrates

Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian metallothioneins, we have cloned and sequenced the cDNAs encoding for metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of metallothioneins in reptiles. Phylogenetic analysis shows that reptilian metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of metallothionein genes along the major vertebrate lineages.     

Metallothionein primary structure in amphibians: Insights from comparative evolutionary analysis in vertebrates

Metallothioneins are cysteine-rich, low-molecular weight metal-binding proteins ubiquitously expressed in living organisms. In the last past years, the increasing amount of vertebrate non-mammalian metallothionein sequences available have disclosed for these proteins differences in the primary structure that have not been supposed before. To provide a more up-to-date view of the metallothioneins in non-mammalian tetrapods, we decided to increase the still scarce knowledge concerning the primary structure and the evolution of metallothioneins in amphibians. Our data demonstrate an unexpected diversity of metallothionein sequences among amphibians, accompanied by remarkable features in their phylogeny. Phylogenetic analysis also reveals the complexity of vertebrate metallothionein evolution, made by both ancient and more recent events of gene duplication and loss.     

Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches.

Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X(n)-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan alphabeta-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of alphabeta-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the alpha-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four alpha-domains and that lack a beta-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.     

Multiple isoforms of metallothionein are expressed in the porcine liver

Isogenes are highly homologous to each other and are often difficult to ascertain, as has been the case with metallothionein, a metal-binding protein rich in cysteines. Conventional separation of metallothionein isoforms relied on ion exchange chromatography of the proteins, or screening for the sequences from gene libraries. In this study, a combination of RT–PCR and partial protein sequencing is used in the identification of metallothionein isogenes expressed in porcine liver. By this approach, we have identified expressed coding sequences which constitute 10 new isogenes. Of the four known groups of metallothioneins (MT), phylogenetic analyses place these pig isogenes in the MT-1 group, except two which are identified as being closely related to MT-2, and none in groups 3 and 4. The isogenes are thus named pMT-1a to -1g, and pMT-2a and -2b. While each of the isogene sequences is unique, two isogenes, pMT-1e1 and pMT-1e2, share an identical amino acid sequence, differing only in specific codons. Two others, pMT-1b and pMT-1g, have a cysteine substituted by arginine, the first such sequence ever detected in MT. pMT-2a and pMT-2b are closely aligned with the MT-2 group of vertebrates, in spite of the absence of a characteristic acidic amino acid at position 10 or 11, common in other mammalian metallothioneins.     

Effect of mutation of cysteinyl residues in yeast Cu-metallothionein

Metallothioneins have been isolated from Saccharomyces cerevisiae CUP1 mutants generated by Wright et al. (Wright, C. F., Hamer, D. H., and McKenney, K. (1986) Nucleic Acids Res. 14, 8489-8499). In the mutant metallothioneins, pairs of cysteinyl residues have been converted to seryl residues. The mutant proteins differ only in the positions of the double substitutions; each mutant molecule contains 10 cysteinyl residues. Each mutant protein lacks the first 8 residues at the amino terminus from the decoded gene sequence of the CUP1 locus. Mutant molecules consist of 53 residues analogous to the wild-type metallothionein and are designated 9/11, 24/26, 36/38, and 49/50 (in reference to the sequence positions of the Cys----Ser conversions). The properties of the mutant metallothioneins are vastly different, and host cells harboring the different plasmid-encoded mutant molecules show marked differences in sensitivity to CuSO4. Growth inhibition was observed at CuSO4 concentrations up to mM in cells containing the 9/11, 24/26, and 36/38 molecules, but not for cells containing protein 49/50. A CuSO4 concentration of 5 mM was required to inhibit the growth of yeast containing either 49/50 or the wild-type metallothionein. In the purified proteins the copper binding stoichiometry of each molecule, except protein 24/26, was nearly 8 mol eq. Protein 24/26 bound 5.5 copper ions/molecule. The Cu(I) chelator bathocuproine disulfonate reacted with over 50% of the copper ions in proteins 9/11, 24/26, and 36/38, but less than 10% of the copper ions in proteins 49/50 and wild-type metallothionein were reactive. The thiolates in 9/11, 24/26, and 36/38 were also more reactive in a disulfide exchange reaction with dithiodipyridine compared with the sulfhydryls in 49/50 and the wild-type molecules. The four mutant copper proteins are luminescent and exhibit a similar quantum yield. The cluster structures contributing to the particular electronic transitions are markedly more sensitive to oxygen in proteins 9/11, 24/26, and 36/38 compared with 49/50 and the wild-type molecules. The air-sensitive proteins exhibit a tertiary fold not recognized by polyclonal antibodies directed to a conformational epitope on yeast Cu-metallothionein. Protein 49/50 cross-reacts with the antibody in a concentration-dependent fashion similar to the wild-type protein. Mutation of 2 cysteinyl residues in the carboxyl portion of metallothionein does not significantly alter properties of the molecule, whereas mutation of several cysteines in the amino-terminal portion of the molecule yields a different conformation.     

Structural and functional studies of the amino terminus of yeast metallothionein

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.     

Hepatic metallothionein synthesis in neonatal mottled-brindled mutant mice

Mottled-brindled mutant mice did not display the elevated hepatic metallothionein synthesis normally observed in 2- to 6-day-old wild-type mice. This difference between normal and mutant mice was not due to a decreased ability to synthesize metallothionein in the liver, since hepatic metallothionein synthesis was inducible in response to copper, cadmium, zinc, or hydrocortisone administration to neonatal mutant mice. Hydrocortisone treatment resulted in increased metallothionein synthesis in liver of mutant mice but had no ameliorative effect on the mottled-brindled disease.This work was supported by Contract DE AM03 76 SF00012 between the Department of Energy and the Regents of the University of California. JEP is the recipient of a National Research Service Award from the National Institutes of Health.     

Pigeon metallothionein consists of two species

Two isospecies of metallothionein, a cysteine-rich protein that binds metals, exist in all mammals examined, but only one in some invertebrates and lower animals. Lower vertebrates such as fish and birds have one or two metallothionein genes depending upon the organism. In this study, we show by amino acid sequence determinations that two isospecies of metallothionein, 75% homologous to each other, can be induced by zinc to accumulate in pigeon livers. This is in contrast to single isospecies found in chicken and duck. Each of these two sequences consists of 63 amino acids, with all 20 cysteines in positions held invariant in most if not all class I mammalian metallothioneins. One of these two pigeon isometallothioneins is terminated with histidine at the carboxyl end, which is apparently unique to avians. Its sequence differs from that of duck and chicken by only four substitutions and is the predominant isospecies that accumulates upon induction. The other pigeon metallothionein has lysine at its carboxyl terminus and is devoid of arginine. None of these isospecies carries any aromatic amino acid, which is also characteristic of all higher metallothioneins. As this is the first demonstration with sequence data that two isospecies of metallothionein indeed exist in birds, these results suggest that pigeon metallothionein genes evolved from an ancestral form through duplication and mutation upon specification.     

The molecular characteristics of two metallothioneins in Ostrinia furnacalis and expression under conditions of heavy metal stress

Metallothioneins are ubiquitously-expressed metal-binding proteins. Despite their potential ecological relevance, no prior reports have identified any metallothioneins in Ostrinia furnacalis or other Lepidoptera species. A better understanding of the molecular characteristics and regulatory dynamics of metallothionein genes in O. furnacalis under heavy metal stress conditions would enable future studies of the roles played by these proteins in the context of heavy metal detoxification. Herein, we identified and characterized two metallothionein (OfMT) genes in O. furnacalis, including the 147 bp OfMT1 gene encoding a 48 amino acid protein containing 10 cysteine residues, and the 141 bp OfMT2 gene encoding a 46 amino acid protein containing 12 cysteine residues. The expression of OfMT2 was found to be related to Cu and Cd concentrations in a dose-dependent manner but was unaffected by Zn exposure. Overall, these results indicate that OfMT genes likely encode metal-binding proteins consistent with their potential role in the maintenance of heavy metal homeostasis.     

Histidine ligands in bacterial metallothionein enhance cluster stability

The cyanobacterial metallothionein (MT) SmtA is the prototype for bacterial MTs and protects against elevated levels of zinc. In contrast to mammalian MTs, bacterial MTs coordinate to metal ions not only via cysteine sulfurs, but unusually for MTs, also via histidine nitrogens. To investigate whether histidine coordination in these metal-sulfur clusters provides advantages over S-coordination only, we mutated the two metal-binding histidine residues in the cyanobacterial MT SmtA from Synechococcus PCC7942 to cysteines. We show that the mutant proteins are still capable of binding up to four zinc ions as is the wild-type protein. However, the mutations perturb protein folding and metal-binding dynamics. Interestingly, several homologues of SmtA also show variations in these two residues. We conclude that histidine residues in Synechococcus PCC7942 SmtA have a stabilising effect due to electrostatic interactions that impact on protein folding and metal cluster charge, and are involved in fine-tuning the reactivity of the bound metal ions.     

大肠杆菌碱性磷酸酶的体外定向进化研究

大肠杆菌碱性磷酸酶（E.coli alkaline phosphatase, EAP, EC 3.1.3.1）是一个非特异性二聚体磷酸单酯酶. 采用易错聚合酶链反应(error prone PCR)的方法,在原有高活力突变株的基础上,对EAP远离活性中心催化三联体的区域进行定向进化,经两轮error prone PCR,获得催化活力较亲本D101S突变株提高3倍、较野生型酶提高35倍的进化酶4-186,并对该酶的催化动力学特征进行了分析. 进化酶基因的DNA测序表明4-186含两个有义氨基酸置换：K167R和S374C,二者既不位于底物结合位点,也不位于酶的金属离子结合位点.     

Cadmium-induced conformational changes in type 2 metallothionein of medicinal plant Coptis japonica: insights from molecular dynamics studies of apo,partially and fully metalated forms

Plants play an important role in the removal of excess heavy metals from soil and water. Medicinal plants can also have non-traditional use in phytoremediation technologies. Among the heavy metals, Cadmium (Cd) is the most abundant and readily taken up by the crop plants. Plant metallothioneins (MTs) are small proteins having cysteine-rich residues and appear to play key roles in metal homoeostasis. Plant metallothionein 2 (MT 2) from Coptis japonica (Gold-thread; CjMT 2) is a typical member of this subfamily and features two cysteine-rich regions containing eight and six cysteine residues, respectively, separated by 42 amino acids long linker region. In-silico analysis of MT 2 protein sequences of C. japonica was performed. In this study, ab initio methods were utilised for the prediction of three-dimensional structure of CjMT 2. After structure validation, heavy metal-binding sites were predicted for the selected modelled structures of CjMT 2. To obtain Cd_i-CjMT 2 (i = 1–7), metalated complex individual docking experiments were performed. The stability of the metalated docked structures was assessed by molecular dynamics (MD) simulation studies. Our study showed that CjMT 2 binds up to 4 Cd²⁺ ions in two distinct domains: a N-terminal β-domain that binds to 2 Cd²⁺ ions and a C-terminal α-domain that binds with 2 Cd²⁺ ions. Our analysis revealed that Cys residues of alpha and beta domain and some residues of spacer region of CjMT 2 protein might be important for the cadmium interaction. MD simulation studies provided insight into metal-induced conformational changes and mechanism of metalation of CjMT 2, an intrinsically disordered protein. This study provides useful insights into mechanism of cadmium-type 2 metallothionein interaction.     

Toward a property/function relationship for metallothioneins: histidine coordination and unusual cluster composition in a zinc-metallothionein from plants

Early cysteine labeled (E(C)) proteins are plant metallothioneins, which were first identified in wheat embryos and are thought to be seed-specific. An exhaustive analysis of expressed sequence tag (EST) entries reveals that homologs are expressed in embryos of both classes of flowering plants (monocotyledons and dicotyledons), but also occur in conifers (gymnosperms) and seed-free spike moss (lycophyta). Mass spectrometric and elemental analysis results indicate that, contrary to the widely propagated number of five, E(C) binds predominantly six zinc ions in at least two zinc-thiolate clusters. 1H and 111Cd NMR experiments suggest that, in contrast to the majority of previously characterized metallothioneins, two conserved histidine residues participate in metal binding. The collected data is consistent with the presence of clusters unprecedented in metallothioneins so far. This novel cluster composition is accompanied by metal-binding properties that are substantially different from other metallothioneins; thus wheat E(C) binds zinc less strongly than either mammalian or cyanobacterial MTs. The unique biochemical properties of wheat E(C) render it ideally suited for a role in zinc donation to nascent proteins during seed development, a role that has been suggested based on the fact that E(C) is induced by the plant hormone abscisic acid, but not by heavy metals. Our results provide a step further toward developing a property/function relationship for metallothioneins.     

Metal-binding proteins in eggs of various sea urchin species.

Metallothionein presence and amount were determined in the unfertilized eggs of six sea urchin species by silver saturation assay and gel-chromatography of cell extracts. The results showed high levels of metallothionein in the egg cytoplasm of the two Mediterranean species Paracentrotus lividus and Sphaerechinus granularis. No metallothionein was found either in the eggs of Arbacia lixula, or in those of the three Eastern species Strongylocentrotus intermedius, Temnopleurus hardwickii and Clypeaster japonicus. However, the extracts of the latter three species revealed the presence of zinc bound in a macromolecular form, thus suggesting the existence of metal-binding proteins distinct from metallothioneins.     

Isolation and partial characterization of metallothionein in the liver of the red-eared turtle (Trachemys scripta) following intreperitoneal administration of cadmium

Cadmium was distributed predominantly in the liver (42% of the body burden) after intraperitoneal injection with 10 mg Cd/kg/day for 6 days, although the kidney, spleen, heart, gonads and shell also contained substantial amounts of the metal. The major cadmium-containing fraction of the liver cytosol eluted in the position of mammalian metallothionein on a Sephadex G-75 column and was further resolved into two isoforms by reverse-phase HPLC. The two isoforms had high cysteine contents (17–22 residues/molecule) and lacked aromatic amino acids, a composition similar to that of other vertebrate metallothioneins. The turtle metal-binding protein had other properties characteristic of vertebrate metallothioneins including heat stability (85°C for 10 min), a relatively high absorbance at 245 nm, a low absorbance at 280 nm and a high metal content (approximately 6 nmol cadmium/nmol protein).     

cDNA cloning and nucleotide sequence comparison of Chinese hamster metallothionein I and II mRNAs

Polyadenylated RNA was extracted from a cadmium resistant Chinese hamster (CHO) cell line, enriched for metal-induced, abundant RNA sequences and cloned as double-stranded cDNA in the plasmid pBR322. Two cDNA clones, pCHMT1 and pCHMT2, encoding two Chinese hamster isometallothioneins were identified, and the nucleotide sequence of each insert was determined. The two Chinese hamster metallothioneins show nucleotide sequence homologies of 80% in the protein coding region and approximately 35% in both the 5' and 3' untranslated regions. Interestingly, an 8 nucleotide sequence (TGTAAATA) has been conserved in sequence and position in the 3' untranslated regions of each metallothionein mRNA sequenced thus far. Estimated nucleotide substitution rates derived from interspecies comparisons were used to calculate a metallothionein gene duplication time of 45 to 120 million years ago.