Type 1 metallothionein (ZjMT) is responsible for heavy metal tolerance in <Emphasis Type="Italic">Ziziphus jujuba</Emphasis>

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that are able to make cells to uptake heavy metals from the environment. Molecular and functional characterization of this gene family improves understanding of the mechanisms underlying heavy metal tolerance in higher organisms. In this study, a cDNA clone, encoding 74-a.a. metallothionein type 1 protein (ZjMT), was isolated from the cDNA library of Ziziphus jujuba. At the N- and C-terminals of the deduced amino acid sequence of ZjMT, six cysteine residues were arranged in a CXCXXXCXCXXXCXC and CXCXXXCXCXXCXC structure, respectively, indicating that ZjMT is a type 1 MT. Quantitative PCR analysis of plants subjected to cadmium stress showed enhanced expression of ZjMT gene in Z. jujuba within 24 h upon Cd exposure. Escherichia coli cells expressing ZjMT exhibited enhanced metal tolerance and higher accumulation of metal ions compared with control cells. The results indicate that ZjMT contributes to the detoxification of metal ions and provides marked tolerance against metal stresses. Therefore, ZjMT may be a potential candidate for tolerance enhancement in vulnerable plants to heavy metal stress and E. coli cells containing the ZjMT gene may be applied to adsorb heavy metals in polluted wastewater.     

Isolation of putative type 2 metallothionein encoding sequences and spatial expression pattern in the seagrass Posidonia oceanica

Metallothioneins (MTs) are a group of proteins with low molecular mass and high cysteine content that bind to heavy metals and are thought to play a role in their metabolism and detoxification. Genes encoding MT-like proteins have been isolated in a number of plants. In this work we isolated nine MT-like sequences from copper- or cadmium-exposed plants of the seagrass Posidonia oceanica, a marine Angiosperm playing a major role in maintaining infralittoral ecosystems in the Mediterranean sea. These sequences, together with two other MT genes previously isolated from this species, show high similarities with genes encoding type 2 MTs. Neighbour-joining analysis, at both deduced protein and 3′-UTR sequence level, indicates that at least two subgroups occur within Posidonia type 2 MTs, showing, however, a strong sequence uniformity. Southern analysis of two type 2 MT-encoding sequences (Pomt2b and Pomt2f) belonging to the two different subgroups showed distinct hybridisation patterns. For both type 2 MTs, we have determined, by in situ technique, the expression domain in Posidonia plants. The members of these two MT subgroups show differences in their histological expression, with Pomt2b associated with proliferative tissues whereas Pomt2f is associated with lignified or suberized cell wall.     

Isolation of putative type 2 metallothionein encoding sequences and spatial expression pattern in the seagrass Posidonia oceanica

Metallothioneins (MTs) are a group of proteins with low molecular mass and high cysteine content that bind to heavy metals and are thought to play a role in their metabolism and detoxification. Genes encoding MT-like proteins have been isolated in a number of plants. In this work we isolated nine MT-like sequences from copper- or cadmium-exposed plants of the seagrass Posidonia oceanica, a marine Angiosperm playing a major role in maintaining infralittoral ecosystems in the Mediterranean sea. These sequences, together with two other MT genes previously isolated from this species, show high similarities with genes encoding type 2 MTs. Neighbour-joining analysis, at both deduced protein and 3′-UTR sequence level, indicates that at least two subgroups occur within Posidonia type 2 MTs, showing, however, a strong sequence uniformity. Southern analysis of two type 2 MT-encoding sequences (Pomt2b and Pomt2f) belonging to the two different subgroups showed distinct hybridisation patterns. For both type 2 MTs, we have determined, by in situ technique, the expression domain in Posidonia plants. The members of these two MT subgroups show differences in their histological expression, with Pomt2b associated with proliferative tissues whereas Pomt2f is associated with lignified or suberized cell wall.     

Structure,organization and expression of the metallothionein gene family inArabidopsis

Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Recent results indicate that plants also possess functional metallothionein genes. Here we report the cloning and characterization of five metallothionein genes fromArabidopsis thaliana. The position of the single intron in each gene is conserved. The proteins encoded by these genes can be divided into two groups (MT1 and MT2) based on the presence or absence of a central domain separating two cysteine-rich domains. Four of the MT genes (MT1a,MT1c,MT2a andMT2b) are transcribed inArabidopsis. Several lines of evidence suggest that the fifth gene,MT1b, is inactive. There is differential regulation of the MT gene family. MT1 mRNA is expressed highly in roots, moderately in leaves and is barely detected in inflorescences and siliques. MT2a and MT2b mRNAs are more abundant in leaves, inflorescences and in roots from mature plants, but are also detected in roots of young plants, and in siliques. MT2a mRNA is strongly induced in seedlings by CUSO₄, whereas MT2b mRNA is relatively abundant in this tissue and levels increase only slightly upon exposure to copper.MT1a andMT1c are located within 2 kb of each other and have been mapped to chromosome 1.MT1b andMT2b map to separate loci on chromosome V, andMT2a is located on chromosome III. The locations of these MT genes are different from that ofCAD1, a gene involved in cadmium tolerance inArabidopsis.     

Heavy metal and abiotic stress inducible metallothionein isoforms from <Emphasis Type="Italic">Prosopis juliflora</Emphasis> (SW) D.C. show differences in binding to heavy metals in vitro

Prosopis juliflora is a tree species that grows well in heavy metal laden industrial sites and accumulates heavy metals. To understand the possible contribution of metallothioneins (MTs) in heavy metal accumulation in P. juliflora, we isolated and compared the metal binding ability of three different types of MTs (PjMT1-3). Glutathione S-transferase fusions of PjMTs (GSTMT1-3) were purified from Escherichia coli cells grown in the presence of 0.3 mM cadmium, copper or zinc. Analysis of metal bound fusion proteins using atomic absorption spectrometry showed that PjMT1 bound higher levels of all three heavy metals as compared to PjMT2 and PjMT3. A comparative analysis of the genomic regions (including promoter for all three PjMTs) is also presented. All three PjMTs are induced by H₂O₂ and ABA applications. PjMT1 and PjMT2 are induced by copper and zinc respectively while PjMT3 is induced by copper, zinc and cadmium. Variation in induction of PjMTs in response to metal exposure and their differential binding to metals suggests that each MT has a specific role in P. juliflora. Of the three MTs analyzed, PjMT1 shows maximum heavy metal sequestration and is thus a potential candidate for use in heavy metal phytoremediation.     

Evolution of metallotionein isoforms complexes in hepatic cells of Mus musculus along cadmium exposure

Characterization of Cd-binding proteins has great analytical interest due to the high toxicity of Cd to living organisms. Metallothioneins (MTs), as Cd(II)-binding proteins are of increasing interest, since they form very stable Cd chelates and are involved in many detoxification processes. In this work, inductively coupled plasma octopole reaction cell mass spectrometry and nanospray ionization time-of-flight mass spectrometry were used in parallel and combined with two-dimensional chromatography: size exclusion followed by reversed-phase high performance liquid chromatography, to study metal complexes of MT isoforms produced in hepatic cytosols of Mus musculus during exposure experiments to Cd. Exposure experiments were carried out by subcutaneous injection of a growing dose of the toxic element ranging from 0.1 to 1.0 mg of Cd per kg of body weight per day during 10 days. A control group and three exposure groups at days 2, 6 and 10 of exposure were studied, and different cadmium, copper and zinc complexes with MTs isoforms were isolated and characterized from the two most exposed groups. The results allow gaining insight into the mechanisms involved in metal detoxification by MTs, showing the changes in the stoichiometry of metal complexes–MTs along cadmium exposure.     

Genetic Design of Stable Metal-Binding Biomolecules,Oligomeric Metallothioneins

Metallothionein (MT) is a suitable model for investigating molecular interactions relating to the handling of metals in cells. However, the production of functional MT proteins in microorganisms has been limited because of the instability of MT—the thiol group of cysteine is easily oxidized and proteolysis occurs. To increase the binding ability and to stabilize MT, we designed genes for dimeric and tetrameric MT and the genes were successfully overexpressed in Escherichia coli to generate functional oligomeric MTs. A human MT-II (hMT-II) synthesized with prokaryotic codons, a linker encoding a glycine tripeptide, and Met-deficient hMT-II was ligated to create a dimeric MT, from which a tetrameric MT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. Cells of E. coli carrying the oligomeric MT genes showed resistance toward Zn and Cd toxicity. The oligomeric proteins formed inclusion bodies, which were dissolved with dithiothreitol, and the purified apo-MTs were reconstituted with Cd or Zn ions under reducing conditions. The dimeric and tetrameric MT proteins exhibited both Cd and Zn binding activities that were, respectively, two and four times higher than those of the hMT-II monomer protein. These stable oligomeric MTs have potential as a biomaterial for uses such as detoxification and bioremediation for heavy metals.     

Drosophila proteins interacting with metallothioneins: A metal‐dependent recognition

Metallothioneins (MTs) are ubiquitous, low‐molecular weight, cysteine‐rich proteins. Despite a well‐established protective role in metal excess detoxification, there is little data about their putative physiological functions, commonly assumed to be metal homeostasis and redox equilibrium. Protein–protein interactions should have provided useful information to unveil unsuspected functions, but reports on MT interactions are scarce. This is probably due to the MT metal‐dependent 3D structure, a fact that has been seldom taken into account when performing proteomic interaction assays. In the present work, we have detected that the two major D. melanogaster isoforms (MtnA and MtnB) interact with the peroxiredoxin (Prx) encoded by the gene Jafrac1, both in a clear metal‐dependent pattern. The MT–Prx interaction is further confirmed in Saccharomyces cerevisiae by assaying both yeast MTs (Crs5p and Cup1p) versus Tsa1p and Tsa2p, the Jafrac1 homologous Prxs in this organism. Thus, a new methodological approach to detect MT‐interacting proteins in different proteomes is established on the basis of assaying MTs in the form of different metal complexes. Furthermore, new perspectives to investigate the often hypothesized contribution of MTs to the redox physiological networks are open.     

Comparative genomics analysis of metallothioneins in twelve Drosophila species

Metallothioneins (MTs) are a superfamily of Cys-rich polypeptides that bind heavy metal ions, both for physiological and detoxification purposes. They are present in all organisms, but their origin is probably polyphyletic, so that MT evolutionary studies are rather scarce. We present a thorough search and analysis of the MT coding sequences in the 12 Drosophila genomes completely sequenced, taking as reference the features reported for D. melanogaster, where four isogenes (MtnA to MtnD) are known and deeply characterized. We include a fifth isoform in this study, named MtnE, and recently annotated. The MTs polymorphism pattern is essentially the same for the 12 Drosophila species. Invariably, a MtnA form and an MtnB-cluster, comprising the MtnB-to-MtnE forms in tandem array, are observed. The whole set of genes are kept in the same synteny element (Muller E), but implicated in rearrangement events (mainly inversions), encompassing all or some of the isogenes. Gene exon/intron architecture, and cDNA and protein sequences appear highly conserved through Drosophila speciation, concordantly with an essential function for MT isoforms in flies, even for those previously considered as minor products. Data presented here will be comprehensively analyzed to provide a valuable guide for future MT evolutionary, structure and function studies.     

Functional homologs of fungal metallothionein genes from Arabidopsis.

Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Two cDNAs encoding proteins with homology to animal and fungal MTs have been isolated from Arabidopsis. The genes represented by these cDNAs are referred to as MT1 and MT2. When expressed in an MT-deficient (cup1 delta) mutant of yeast, both MT1 and MT2 complemented the cup1 delta mutation, providing a high level of resistance to CuSO4 and moderate resistance to CdSO4. Although the MT-deficient yeast was not viable in the presence of either 300 microM CuSO4 or 5 microM CdSO4, cells expressing MT1 were able to grow in medium supplemented with 3 mM CuSO4 and 10 microM CdSO4, and those expressing MT2 grew in the presence of 3 mM CuSO4 and 100 microM CdSO4. In plants, MT1 mRNA was more abundant in roots and dark-grown seedlings than in leaves. In contrast, MT2 mRNA accumulated more in leaves than in either roots or darkgrown seedlings. MT2 mRNA was strongly induced in seedlings by CuSO4, but only slightly by CdSO4 or ZnSO4. However, MT1 mRNA was induced by CuSO4 in excised leaves that were submerged in medium. These results indicated that Arabidopsis MT genes are involved in copper tolerance. Plants also synthesized metal binding phytochelatins (poly[gamma-glutamylcysteine]glycine) when exposed to heavy metals. The results presented here argue against the hypothesis that phytochelatins are the sole molecules involved in heavy metal tolerance in plants. We conclude that Arabidopsis MT1 and MT2 are functional homologs of yeast MT.     

A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein

Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10–30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd²⁺-ions.     

Modularity in Protein Evolution: Modular Organization and De Novo Domain Evolution in Mollusk Metallothioneins

Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxification, and therefore, MTs have been crucial for the adaptation of the living beings to variable situations of metal bioavailability. The evolution of MTs is, however, not yet fully understood, and to provide new insights into it, we have investigated the MTs in the diverse classes of Mollusks. We have shown that most molluskan MTs are bimodular proteins that combine six domains—α, β1, β2, β3, γ, and δ—in a lineage-specific manner. We have functionally characterized the Neritimorpha β₃β₁ and the Patellogastropoda γβ₁ MTs, demonstrating the metal-binding capacity of the new γ domain. Our results have revealed a modular organization of mollusk MT, whose evolution has been impacted by duplication, loss, and de novo emergence of domains. MTs represent a paradigmatic example of modular evolution probably driven by the structural and functional requirements of metal binding.