Vertebrate Metallothioneins Research Articles

The Solution Structure and Dynamics of Cd-Metallothionein from Helix pomatia Reveal Optimization for Binding Cd over Zn.

Metallothioneins (MTs) are cysteine-rich polypeptides that are naturally found coordinated to monovalent and/or divalent transition metal ions. Three metallothionein isoforms from the Roman snail Helix pomatia are known. They differ in their physiological metal load and in their specificity for transition metal ions such as Cd2+ (HpCdMT isoform) and Cu+ (HpCuMT isoform) or in the absence of a defined metal specificity (HpCd/CuMT isoform). We have determined the solution structure of the Cd-specific isoform (HpCdMT) by nuclear magnetic resonance spectroscopy using recombinant isotopically labeled protein loaded with Zn2+ or Cd2+. Both structures display two-domain architectures, where each domain comprises a characteristic three-metal cluster similar to that observed in the β-domains of vertebrate MTs. The polypeptide backbone is well-structured over the entire sequence, including the interdomain linker. Interestingly, the two domains display mutual contacts, as observed before for the metallothionein of the snail Littorina littorea, to which both N- and C-terminal domains are highly similar. Increasing the length of the linker motionally decouples both domains and removes mutual contacts between them without having a strong effect on the stability of the individual domains. The structures of Cd6- and Zn6-HpCdMT are nearly identical. However, 15N relaxation, in particular 15N R2 rates, is accelerated for many residues of Zn6-HpCdMT but not for Cd6-HpCdMT, revealing the presence of conformational exchange effects. We suggest that this snail MT isoform is evolutionarily optimized for binding Cd rather than Zn.

Evolutionary progress in the functions of metallothioneins in the ever changing ecosystems

Metallothioneins (MTs) exist in various org anisms ranging from some prokaryotes to eukaryotes and mammals. MTs are low molecular weight proteins (MW ranging from 500 to 14000 Da), highly rich in cysteine residues and effectively bind with metals. MTs have the capacity to bind both physiological (such as zinc, copper, selenium) and xenobiotic (such as cadmium, mercury, silver, arsenic) heavy metals through the thiol group of its cysteine residues, which represent nearly 30 % of its constituent amino acid residues. Their combination with heavy metals gives rise to metal-thiolate clusters. It has been suspected that the presence of cysteine in MT is necessary for its functioning and that MT itself is essential for life, modulating complex diseases and the immune system. All different types of MTs are classified with respect to so many factors mainly in groups I and II. There are four main distinguished sub families of the mammalian MT gene families; MT-1 (subtypes A, B, E, F, G, H, L, M, X), MT-2, MT-3, MT-4. They are synthesised primarily in the liver and kidneys. Their production is dependent on availability of the dietary minerals, as zinc, copper and selenium, and the amino acids histidine and cysteine. The rise of these sub families can be attributed to the different functions fulfilled by them. The absence of one defined primary common function could certainly be the reason for serial duplications that gave rise to the respective isoforms. The evolution of MT has been so important to science that they have been considered valid biomarkers in medicine and environmental studies. MT is not limited to the human system, it has also been found in other mammals of the animal kingdom vertebrates (such as the chicken, Gallus gallus, or the mammalian Mus musculus), in higher plants (such as Pisum sativum, Triticum durum, Zea mays, Quercus suber), in protozoa (ex. the ciliate Tetrahymena genera), in yeast (such as Saccharomyces cerevisiae, Candida albicans), in invertebrates (such as the nematode Caenorhabditis elegans, the insect Drosophila melanogaster, the mollusc Mytilus edulis, or the echinoderm Strongylocentrotus purpuratus and in many prokaryotes (such as the cyanobacteria Syneccococus spp). The MTs from this diverse taxonomic range represent a high- heterogeneity sequence (regarding molecular weight and number and distribution of Cys residues) and do not show general homology; in spite of this, homology is found inside some taxonomic groups (such as vertebrate MTs). In this review, we will be looking at the evolution of MTs in respective organisms and the different roles they perform in each of their respective locations in these organisms. We will specifically look at the following factors; evolution of the genetic constitution of MTs and their structural composition and functions.

Identification and expression of an atypical isoform of metallothionein in the African clawed frog Xenopus laevis

Exploiting the annotation of the western clawed frog Silurana tropicalis genome, we identified a new metallothionein (MT) gene, exhibiting all the features to be considered an active gene, but with an atypical coding region, showing only 17 cysteine residues instead of the canonical 20 cysteines of vertebrate metallothioneins and two anomalous cysteine triplets. However, the presence of a gene in the genome does not ensure its effective expression. By using conventional and Real-Time PCR analyses, we demonstrated that this atypical MT is constitutively expressed throughout the life cycle of the African clawed frog Xenopus laevis; moreover, this gene is highly expressed in the adult liver, the major site of MT expression and synthesis in vertebrates. To our knowledge, the X. laevis MT described in this paper is the first sequence of a vertebrate MT showing only 17 cysteine residues, arranged in two Cys–Cys–Cys motifs. Phylogenetic analyses also demonstrated that the atypical X. laevis MT merges in the anuran clade, but is the most derived sequence among tetrapods MTs. Finally, Tajima's Relative Rate Test suggested a different evolutionary rate between the canonical X. laevis MT and this novel isoform.

Molecular Evolution and Functional Divergence of the Metallothionein Gene Family in Vertebrates

The metallothionein (MT) gene superfamily consists of metal-binding proteins involved in various metal detoxification and storage mechanisms. The evolution of this gene family in vertebrates has mostly been studied in mammals using sparse taxon or gene sampling. Genomic databases and available data on MT protein function and expression allow a better understanding of the evolution and functional divergence of the different MT types. We recovered 77 MT coding sequences from 20 representative vertebrates with annotated complete genomes. We found multiple MT genes, also in reptiles, which were thought to have only one MT type. Phylogenetic and synteny analyses indicate the existence of a eutherian MT1 and MT2, a tetrapod MT3, an amniote MT4, and fish MT. The optimal gene-tree/species-tree reconciliation analyses identified the best root in the fish clade. Functional analyses reveal variation in hydropathic index among protein domains, likely correlated with their distinct flexibility and metal affinity. Analyses of functional divergence identified amino acid sites correlated with functional divergence among MT types. Uncovering the number of genes and sites possibly correlated with functional divergence will help to design cost-effective MT functional and gene expression studies. This will permit further understanding of the distinct roles and specificity of these proteins and to properly target specific MT for different types of functional studies. Therefore, this work presents a critical background on the molecular evolution and functional divergence of vertebrate MTs to carry out further detailed studies on the relationship between heavy metal metabolism and tolerances among vertebrates.

Expression and function analysis of metallothionein in the testis of Portunus trituberculatus exposed to cadmium

Metallothioneins (MTs) possess a unique molecular structure that provides metal-binding and redox capabilities. These capabilities include the maintenance of metal equilibria that protect against heavy metals (especially cadmium) and oxidative damage. Past studies have focused on the function of MTs in vertebrates. However, the functions of MTs during spermiogenesis in invertebrates remain unclear. In order to investigate the function of MTs during spermiogenesis in Portunus trituberculatus, we used RT-PCR and RACE to identify two MT complete cDNA sequences in the total RNA from the P. trituberculatus testis. The 450bp MT-1 cDNA consists of a 77bp 5′ untranslated region, a 196bp 3′ untranslated region, and a 177bp open reading frame that encodes 58 amino acids including 19 cysteines. The 581bp MT-2 cDNA consists of 73bp 5′ untranslated region, a 328bp 3′ untranslated region, and a 180bp open reading frame that encodes 59 amino acids including 18 cysteines. MT-1 and MT-2 of P. trituberculatus more closely resemble invertebrate (especially crab) MT homologues than vertebrate MT homologues as indicated by protein alignment comparisons and phylogenetic tree analysis. MT-1 and MT-2 were detected in the heart, testis, muscle, hepatopancreas, and gill of P. trituberculatus by tissue expression analysis. In addition, MT-1 and MT-2 are present during the entire process of spermiogenesis in P. trituberculatus as indicated by H&E staining and in situ hybridization. MT-1 and MT-2 expression levels significantly increase after cadmium (Cd) exposure as measured by real-time quantitative PCR analysis. Therefore, we suggest that MT-1 and MT-2 perform important functions in spermiogenesis and testis detoxification in P. trituberculatus.

Metal Dealing at the Origin of the Chordata Phylum: The Metallothionein System and Metal Overload Response in Amphioxus

Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd-thioneins or Cu-thioneins, according to the ecophysiological needs of each kind of organisms.

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.

CiMT‐1, an unusual chordate metallothionein gene in Ciona intestinalis genome: structure and expression studies

The present article reports on the characterization of the urochordate metallothionein (MT) gene, CiMT-1, from the solitary ascidian Ciona intestinalis. The predicted protein is shorter than other known deuterostome MTs, having only 39 amino acids. The gene has the same tripartite structure as vertebrate MTs, with some features resembling those of echinoderm MTs. The promoter region shows the canonical cis-acting elements recognized by transcription factors that respond to metal, ROS, and cytokines. Unusual sequences, described in fish and echinoderms, are also present. In situ hybridization suggests that only a population of hemocytes involved in immune responses, i.e. granular amebocytes, express CiMT-1 mRNA. These observations support the idea that urochordates perform detoxification through hemocytes, and that MTs may play important roles in inflammatory humoral responses in tunicates. The reported data offer new clues for better understanding the evolution of these multivalent proteins from non-vertebrate to vertebrate chordates and reinforce their functions in detoxification and immunity.

The β E-Domain of Wheat E c-1 Metallothionein: A Metal-Binding Domain with a Distinctive Structure

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn II, Cu I, and Cd II that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the β E-domain of the early cysteine-labeled protein (E c-1) from wheat (β E-E c-1), which represents the first three-dimensional structure of a plant MT. The β E-domain comprises the 51 C-terminal residues of E c-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear Zn II binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn II ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys–metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by 15N relaxation experiments support the results of the structure determination in that β E-E c-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn II with Cd II ions and examined the effects of metal ion release on incubation with a metal ion chelator.

The plant metallothionein 2 from Cicer arietinum forms a single metal–thiolate cluster

The plant metallothionein 2 from Cicer arietinum (chickpea; cicMT2) is a typical member of this subfamily and features two cysteine-rich regions containing eight and six cysteine residues, respectively, separated by a linker region 41 amino acids in length. This metallothionein thus differs significantly from the well-studied vertebrate forms. A synthetic gene encoding cicMT2 was designed, cloned into a suitable vector, and the protein was over-expressed in Escherichia coli. For the first time, an in-depth spectroscopic characterization of cicMT2 in the presence of divalent metal ions is performed showing a binding capacity for five Zn(II), Cd(II), or Co(II) ions and the typical features of metal-thiolate clusters. Based on proteolytic digestion experiments, the cluster arrangement formed by the divalent metal ions and the cysteine thiolate groups connects the amino-terminal with the carboxy-terminal cysteine-rich region. The cluster formation process, put into effect with the addition of the fourth metal ion to the apo protein, was investigated using the characteristic shift of absorption bands observed in the UV/Vis spectra upon titration with Co(II). The pH-dependent Zn(II)- and Cd(II)-thiolate cluster stability is one of the highest observed for plant MTs so far, but lower than that usually found in vertebrate metallothioneins. The dependence of the pH stability on the ionic strength of the solution is more pronounced for the Cd(II)- than for the Zn(II)-form of the protein.

The two distinctive metal ion binding domains of the wheat metallothionein Ec-1

Metallothioneins are small cysteine-rich proteins believed to play a role, among others, in the homeostasis of essential metal ions such as Zn(II) and Cu(I). Recently, we could show that wheat E(c)-1 is coordinating its six Zn(II) ions in form of metal-thiolate clusters analogously to the vertebrate metallothioneins. Specifically, two Zn(II) ions are bound in the N-terminal and four in the C-terminal domain. In the following, we will present evidence for the relative independence of the two domains from each other with respect to their metal ion binding abilities, and uncover three intriguing peculiarities of the protein. Firstly, one Zn(II) ion of the N-terminal domain is relative resistant to complete replacement with Cd(II) indicating the presence of a Zn(II)-binding site with increased stability. Secondly, the C-terminal domain is able to coordinate an additional fifth metal ion, though with reduced affinity, which went undetected so far. Finally, reconstitution of apoE(c)-1 with an excess of Zn(II) shows a certain amount of sub-stoichiometrically metal-loaded species. The possible relevance of these finding for the proposed biological functions of wheat E(c)-1 will be discussed. In addition, extended X-ray absorption fine structure (EXAFS) measurements on both, the full-length and the truncated protein, provide final evidence for His participation in metal ion binding.

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.

Spectroscopic characterization of Cicer arietinum metallothionein 1

The plant metallothioneins differ distinctively from other metallothionein families with respect to the cysteine distribution patterns, the presence of aromatic amino acids in most and histidine in some forms, as well as long cysteine-free amino acid stretches between cysteine-rich regions. Although known for more than 25 years, research activity on plant metallothioneins has been low increasing only in the past few years. In the following, we will present the first characterization of Cicer arietinum (chickpea) MT1. In this root-specific protein two cysteine-rich regions with six cysteine residues each are separated by a 42 amino acids long linker region. A synthetic gene encoding MT1 was designed, cloned into a suitable vector, and the protein was over-expressed in Escherichia coli. We could show, that MT1 has the ability to coordinate up to five Zn 2+ or Cd 2+ ions and even higher amounts of Hg 2+. According to titration experiments pH-dependent zinc– and cadmium–thiolate cluster stability in MT1 is considerably lower than in vertebrate metallothioneins. The approximate contribution of secondary structural elements to the overall structure was assessed with circular dichroism and infrared spectroscopy. Hypothetical metal–thiolate cluster structures will be presented.

Coordination of three and four Cu(I) to the α− and β‐domain of vertebrate Zn‐metallothionein‐1, respectively, induces significant structural changes

Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d10-metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1H NMR using two synthetic fragments representing the alpha- and the beta-domain. The 1H NMR data and thus the structures of Zn4alpha metallothionein (MT)-1 and Zn3betaMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1. Cu(I) titration of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing alpha- and the four Cu(I)-containing beta-domains. The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addition of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the separately prepared aqueous three and four Cu(I) loaded alpha- and beta-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.

Comparative metal binding and genomic analysis of the avian (chicken) and mammalian metallothionein

Chicken metallothionein (ckMT) is the paradigm for the study of metallothioneins (MTs) in the Aves class of vertebrates. Available literature data depict ckMT as a one-copy gene, encoding an MT protein highly similar to mammalian MT1. In contrast, the MT system in mammals consists of a four-member family exhibiting functional differentiation. This scenario prompted us to analyse the apparently distinct evolutionary patterns followed by MTs in birds and mammals, at both the functional and structural levels. Thus, in this work, the ckMT metal binding abilities towards Zn(II), Cd(II) and Cu(I) have been thoroughly revisited and then compared with those of the mammalian MT1 and MT4 isoforms, identified as zinc- and copper-thioneins, respectively. Interestingly, a new mechanism of MT dimerization is reported, on the basis of the coordinating capacity of the ckMT C-terminal histidine. Furthermore, an evolutionary study has been performed by means of in silico analyses of avian MT genes and proteins. The joint consideration of the functional and genomic data obtained questions the two features until now defining the avian MT system. Overall, in vivo and in vitro metal-binding results reveal that the Zn(II), Cd(II) and Cu(I) binding abilities of ckMT lay between those of mammalian MT1 and MT4, being closer to those of MT1 for the divalent metal ions but more similar to those of MT4 for Cu(I). This is consistent with a strong functional constraint operating on low-copy number genes that must cope with differentiating functional limitation. Finally, a second MT gene has been identified in silico in the chicken genome, ckMT2, exhibiting all the features to be considered an active coding region. The results presented here allow a new insight into the metal binding abilities of warm blooded vertebrate MTs and their evolutionary relationships.

Fish and molluscan metallothioneins

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.

Structural and functional studies of vertebrate metallothioneins: cross-talk between domains in the absence of physical contact

In previous studies, we showed that the chemical and dynamic properties of fish and mouse MTs (metallothioneins) present a number of distinctive differences linked to their primary structures, and that phylogenetic relationships of mammal and fish MTs correlate with their three-dimensional structures. The different behaviours of MTs may also be linked to the interaction between their two domains. In the present study, we have compared the physicochemical properties of the isolated recombinant domains constituting Notothenia coriiceps and mouse MTs, and compared them with those of the corresponding whole MTs. NMR spectra of the separated domains of N. coriiceps are almost superimposable on those of the parent MT, suggesting an apparent lack of interaction between the two domains in the protein. However, certain dynamic and physicochemical features of the isolated domains are unlike those of the whole protein. In particular, the temperature-induced changes in the chiroptical properties, thiol reactivity of the Zn-MT domains and the Zn2+/Cd2+ rate of exchange are different for the two domains and with respect to the whole protein. Taken together, these results provide a strong argument in favour of the interaction of the two domains in the MT molecule, in spite of the elusive evidence provided by the structural analyses.

Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and expression analysis

Novel metallothionein (MT) complementary DNA and genomic sequences were isolated from a cartilaginous shark species, Scyliorhinus torazame. The full-length open reading frame (ORF) of shark MT cDNA encoded 68 amino acids with a high cysteine content (29%). The genomic ORF sequence (932 bp) of shark MT isolated by polymerase chain reaction (PCR) comprised 3 exons with 2 interventing introns. Shark MT sequence shared many conserved features with other vertebrate MTs: overall amino acid identities of shark MT ranged from 47% to 57% with fish MTs, and 41% to 62% with mammalian MTs. However, in addition to these conserved characteristics, shark MT sequence exhibited some unique characteristics. It contained 4 extra amino acids (Lys-Ala-Gly-Arg) at the end of the beta-domain, which have not been reported in any other vertebrate MTs. The last amino acid residue at the C-terminus was Ser, which also has not been reported in fish and mammalian MTs. The MT messenger RNA levels in shark liver and kidney, assessed by semiquantitative reverse transcriptase PCR and RNA blot hybridization, were significantly affected by experimental exposures to heavy metals (cadmium, copper, and zinc). Generally, the transcriptional activation of shark MT gene was dependent on the dose (0-10 mg/kg body weight for injection and 0-20 microM for immersion) and duration (1-10 days); zinc was a more potent inducer than copper and cadmium.

Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus

A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

Cloning of a complementary DNA encoding an Ambystoma mexicanum metallothionein, AmMT, and expression of the gene during early development.

We have used a polymerase chain reaction strategy to isolate a metallothionein (MT) cDNA from the amphibian Ambystoma mexicanum (axolotl). This cDNA is 875-bp long and encodes a 60 amino acid protein, AmMT, typical for family 1 MTs. It contains 20 cysteine (Cys) residues that can be aligned with those of other vertebrate MTs. The overall structure of the protein is unique among vertebrates in having only two amino acid residues before the first Cys at the amino-terminal end. Northern analyses showed that AmMT is expressed throughout embryogenesis, giving rise to three mRNA species of 650, 750, and 1,600 nucleotides (nt). The 750 and 1,600 nt transcripts appear to result from differential use of polyadenylation signals, whereas the 650 nt RNA could arise from deadenylation of the 750-nt transcript. Both the 750- and 1,600-nt RNAs were presented in embryos before the mid-blastula transition (MBT). After the MBT, the 750-nt RNA was replaced by the 650-nt RNA which was gradually degraded to undetectable levels in post-neurulation embryos. Levels of the 1,600-nt transcript increased at gastrulation and reach a maximum in Stage 30 embryos. In adult animals, levels of the 750-nt RNA were high in liver and testes, and very low in lung, gut, skin, and oviducts, whereas levels of the 1,600-nt transcript were similar and moderately elevated in all tissues examined. In contrast, in Xenopus laevis, Northern analysis did not detect XIMT-A mRNA in embryos before late neurulation (Stage 24). XIMT-A mRNA levels then increased sharply in Stage 36 hatched embryos at levels similar to those found in adult livers. These results show that AmMT presents a unique expression pattern among metazoans being transcribed as two transcripts differing in the length of their 3' untranslated regions, the levels of which vary during embryogenesis and in adult tissues.