Metal-Specificity Divergence between Metallothioneins of Nerita peloronta (Neritimorpha, Gastropoda) Sets the Starting Point for a Novel Chemical MT Classification Proposal.

Metallothioneins' (MTs) biological function has been a matter of debate since their discovery. The importance to categorize these cysteine-rich proteins with high coordinating capacity into a specific group led to numerous classification proposals. We proposed a classification based on their metal-binding abilities, gradually sorting them from those with high selectivity towards Zn/Cd to those that are Cu-specific. However, the study of the NpeMT1 and NpeMT2isoforms of Nerita peloronta, has put a new perspective on this classification. N. peloronta has been chosen as a representative mollusk to elucidate the metal-binding abilities of Neritimorpha MTs, an order without any MTs characterized recently. Both isoforms have been recombinantly synthesized in cultures supplemented with ZnII, CdII, or CuII, and the purified metal-MT complexes have been thoroughly characterized by spectroscopic and spectrometric methods, leading to results that confirmed that Neritimorpha share Cd-selective MTs with Caenogastropoda and Heterobranchia, solving a so far unresolved question. NpeMTs show high coordinating preferences towards divalent metal ions, although one of them (NpeMT1) shares features with the so-called genuine Zn-thioneins, while the other (NpeMT2) exhibits a higher preference for Cd. The dissimilarities between the two isoforms let a window open to a new proposal of chemical MT classification.

Genome-Wide Identification and Analysis of the Metallothionein Genes in Oryza Genus.

Metallothionein (MT) proteins are low molecular mass, cysteine-rich, and metal-binding proteins that play an important role in maintaining metal homeostasis and stress response. However, the evolutionary relationships and functional differentiation of MT in the Oryza genus remain unclear. Here we identified 53 MT genes from six Oryza genera, including O. sativa ssp. japonica, O. rufipogon, O. sativa ssp. indica, O. nivara, O. glumaepatula, and O. barthii. The MT genes were clustered into four groups based on phylogenetic analysis. MT genes are unevenly distributed on chromosomes; almost half of the MT genes were clustered on chromosome 12, which may result from a fragment duplication containing the MT genes on chromosome 12. Five pairs of segmental duplication events and ten pairs of tandem duplication events were found in the rice MT family. The Ka/Ks values of the fifteen duplicated MT genes indicated that the duplicated MT genes were under a strong negative selection during evolution. Next, combining the promoter activity assay with gene expression analysis revealed different expression patterns of MT genes. In addition, the expression of OsMT genes was induced under different stresses, including NaCl, CdCl2, ABA, and MeJ treatments. Additionally, we found that OsMT genes were mainly located in chloroplasts. These results imply that OsMT genes play different roles in response to these stresses. All results provide important insights into the evolution of the MT gene family in the Oryza genus, and will be helpful to further study the function of MT genes.

Differential reactivity of closely related zinc(II)-binding metallothioneins from the plant Arabidopsis thaliana.

The dynamics of metal binding to and transfer from metalloproteins involved in metal homeostasis are important for understanding cellular distribution of metal ions. The dicotyledonous plant Arabidopsis thaliana has two type 4 seed-specific metallothionein homologues, MT4a and MT4b, with likely roles in zinc(II) homeostasis. These two metallothioneins are 84% identical, with full conservation of all metal-binding cysteine and histidine residues. Yet, differences in their spatial and temporal expression patterns suggested divergence in their biological roles. To investigate whether biological functions are reflected in molecular properties, we compare aspects of zinc(II)-binding dynamics of full-length MT4a and MT4b, namely the pH dependence of zinc(II) binding and protein folding, and zinc(II) transfer to the chelator EDTA. UV-Vis and NMR spectroscopies as well as native electrospray ionisation mass spectrometry consistently showed that transfer from Zn6MT4a is considerably faster than from Zn6MT4b, with pseudo-first-order rate constants for the fastest observed step of k obs = 2.8 × 10-4 s-1 (MT4b) and k obs = 7.5 × 10-4 s-1 (MT4a) (5 µM protein, 500 µM EDTA, 25 mM Tris buffer, pH 7.33, 298 K). 2D heteronuclear NMR experiments allowed locating the most labile zinc(II) ions in domain II for both proteins. 3D homology models suggest that reactivity of this domain is governed by the local environment around the mononuclear Cys2His2 site that is unique to type 4 MTs. Non-conservative amino acid substitutions in this region affect local electrostatics as well as whole-domain dynamics, with both effects rendering zinc(II) ions bound to MT4a more reactive in metal transfer reactions. Therefore, domain II of MT4a is well suited to rapidly release its bound zinc(II) ions, in broad agreement with a previously suggested role of MT4a in zinc(II) transport and delivery to other proteins.

Cd-metallothioneins in three additional tetrahymena species: intragenic repeat patterns and induction by metal ions.

Ciliate metallothioneins (MTs) possess many unique features compared to the "classic" MTs in other organisms, but they have only been studied in a small number of species. In this study, we investigated cDNAs encoding subfamily 7a metallothioneins (CdMTs) in three Tetrahymena species (T. hegewischi, T. malaccensis, and T. mobilis). Four CdMT genes (ThegMT1, ThegMT2, TmalMT1, and TmobMT1) were cloned and characterized. They share high sequence similarity to previously identified subfamily 7a MT members. Tetrahymena CdMTs exhibit a remarkably regular intragenic repeat homology. The CdMT sequences were divided into two main types of modules, which had been previously described, and which we name "A" and "B". ThegMT2 was identified as the first MT isoform solely composed of module "B". A phylogenetic analysis of individual modules of every characterized Tetrahymena CdMT rigorously documents the conclusion that modules are important units of CdMT evolution, which have undergone frequent and rapid gain/loss and shuffling. The transcriptional activity of the four newly identified genes was measured under different heavy metal exposure (Cd, Cu, Zn, Pb) using real-time quantitative PCR. The results showed that these genes were differentially induced after short (1 h) or long (24 h) metal exposure. The evolutionary diversity of Tetrahymena CdMTs is further discussed with regard to their induction by metal ions.

Metallothionein: an overview on its metal homeostatic regulation in mammals / Metalotioneina: una visión general de su regulación homeostática de metales en mamíferos

Metallothionein (MT) is a ubiquitous protein with a low molecular weight of 6-7 kDa weight and it was first identified in the kidney cortex of equines as a cadmium (Cd)-binding protein responsible for the natural accumulation of Cd in the tissue. The mammalian MT contains 61 to 68 amino acid residues, in which 18 to 23 cysteine residues are present. The expression of MT starts by binding of metal transcription factor-1 (MTF-1) to the regulative region of MT gene called metal responsive elements (MREs). The induction of MT through the MREs region can be initiated by several metal ions such as zinc (Zn), copper (Cu) and Cd. However, Zn is the only heavy metal which can reversibly and directly activate the DNA-binding activity of MTF-1. In mammals four types of MT are expressed and they are termed metallothionein-1 (MT1), metallothionein-2 (MT2), metallothionein-3 (MT3), and metallothionein-4 (MT4). MT1 and MT2 are expressed in almost all tissues while MT3 and MT4 are tissue-specific. MT is a key compound involved in the intracellular handling of a variety of essential and nonessential post-transitional metal ions. In order to the heavy metal binding ability of MT, it is suggested to play roles both in the intracellular fixation of essential trace elements Zn and Cu, in controlling the concentrations, and in neutralizing the harmful influences of exposure to toxic elements...

Metalotioneina (MT) es una proteína, con bajo peso molecular de kDa 6-7 y que fue primero identificada en la corteza renal de equinos como cadmio (Cd)-proteína responsable por la acumulación natural de Cd en los tejidos. La MT en mamíferos contiene 61 a 68 residuos de aminoácidos, de los cuales están presentes 18 a 23 residuos de cisteína. La expresión de MT se inicia por la unión del factor-1 de transcripción de metal (MTF-1) a la región reguladora del gen de la MT llamado elementos metálicos responsable (MREs). La inducción de MT a través de la región MREs puede ser iniciada por varios iones metálicos tales como zinc (Zn), cobre (Cu) y Cd. Sin embargo, el Zn es el único metal pesado que puede revertir y activar directamente la unión ADN de MTF-1. En los mamíferos se expresan cuatro tipos de MT y ellos se denominan metalotioneína-1 (MT1), metalotioneína-2 (MT2), metalotioneína-3 (MT3), y metalotioneína-4 (MT4). MT1 y MT2 se expresan en casi todos los tejidos mientras que MT3 y MT4 son tejido-específico. La MT es un compuesto clave implicado en la manipulación intracelular de una variedad de iones metálicos esenciales y no esenciales post-transicionales. Con el fin de evaluar la capacidad de unión de metales pesados de MT, se sugiere que éste desempeña ambos roles tanto en la fijación intracelular de trazas de elementos de Zn y Cu como en el control de las concentraciones, y neutralizando las influencias perjudiciales a la exposición de elementos tóxicos...

Metallothionein expression in colorectal cancer: relevance of different isoforms for tumor progression and patient survival.

Metallothioneins are a family of small, cysteine-rich proteins with many functions. Immunohistochemical evaluation of all metallothionein 1 + 2 isoforms in colorectal tumors has demonstrated an important down-regulation compared with normal tissue, although its prognostic significance is unclear. Moreover, the contribution of individual isoforms to overall metallothionein down-regulation is not known. To address these important issues, we analyzed the messenger RNA expression levels of all functional metallothionein 1 + 2 isoforms by quantitative reverse transcription polymerase chain reaction in 22 pairs of normal and tumor-microdissected epithelia and correlated these to the overall immunohistochemical protein expression. Our results showed that 5 isoforms (MT1G, 1E, 1F, 1H, and 1M) were lost during the transition from normal mucosa to tumor, whereas MT1X and MT2A were less down-regulated, and their expression was correlated with overall protein positivity. Second, we showed that MT1G hypermethylation occurred in cell lines and in 29% of tumor samples, whereas histone deacetylase inhibitors are able to induce most isoforms. Furthermore, we analyzed by immunohistochemistry 107 normal mucosae, 25 adenomas, 81 carcinomas, and 19 lymph node metastases to evaluate metallothionein expression during different stages of cancer development and to assess its relationship to patient survival. A lower immunohistochemical expression was associated with poorer survival, although it was not an independent predictor. Overall, this study identifies for the first time the relevant metallothionein isoforms for colorectal cancer progression, supports the concept that their loss is associated with worse prognosis, and suggests 2 mechanisms for epigenetic repression of metallothionein expression in colorectal tumors.

Zn- and Cu-thioneins: a functional classification for metallothioneins?

This report intends to provide the reader with a deeper insight in the chemical, and extensively biological, characteristics of the metallothionein (MT) system. We have devoted nearly 20 years to the study of MTs and this has allowed us to form what we believe is a more complete picture of this peculiar family of metalloproteins. At the beginning of the 1990s, the landscape of this field was quite different from the overall picture we have now. Many researchers have contributed to the readjustment of this part of scientific knowledge. In our case, we implemented a unified method for obtaining MTs, for characterizing their metal-binding features, and for applying a unified research rationale. All this has helped to enlarge the initial picture that was mainly dominated by mammalian MT1/MT2 and yeast Cup1, by introducing approximately 20 new MTs. It has also allowed some characteristics to be clarified and examined in more detail, such as the cooperativity or the coexistence of multiple species in the metal-substitution reactions, the availability of Ag(I) or Cd(II) for use as respective probes for the Cu(I) and Zn(II) binding sites, the participation of chloride or sulfide ligands in the metal coordination spheres, and the feasibility of using in vitro data as representative of in vivo scenarios. Overall, the results yield enough data to consider new criteria for a proposal of classification of MTs based on MT metal-binding features, which complements the previous classifications, and that can shed light on the still controversial physiological functions of this peculiar superfamily of metalloproteins.

Identification of two metallothionein genes and their roles in stress responses of Musca domestica toward hyperthermy and cadmium tolerance.

Stress proteins such as metallothioneins (MTs) play a key role in cellular protection against environmental stressors. In nature, insects such as houseflies (Musca domestica) are commonly exposed to multiple stressors including heavy metals (e.g. Cadmium, Cd) and high temperatures. In this paper, we identify two novel MT genes from the cDNAs of M. domestica, MdMT1 and MdMT2, which putatively encode 40 and 42 amino acid residues respectively. Expression of the two MTs' mRNAs, which are examined in the fat body, gut, hemocyte, and the epidermis. From our study, we saw that the expression of MdMT1 and MdMT2 are enhanced by Cd and thermal stress. Levels of expression are highest at 10 mM Cd(2+) within a 24-h period, and expressions increase significantly with exposure to 10 mM Cd for 12h. Levels of the mRNAs are up-regulated after heat shock and that of MdMT2 reaches its maximum peak faster than MdMT1. Both of the MT genes might be involved in a transient systemic tolerance response to stressors and they may play important roles in heavy metal and high temperature tolerance in the housefly. To detect whether or not the MTs bind heavy metals, the target genes are cloned into the prokaryotic expression vector pET-DsbA to obtain fusion protein expressed in Escherichia coli BL21 (DE3). Recombinant DsbA-MdMT1 significantly increases tolerance of the host bacteria to Cd(2+), but DsbA-MdMT2 is absent. These differential characteristics will facilitate future investigations into the physiological functions of MTs.

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.

HbMT2, an ethephon-induced metallothionein gene from Hevea brasiliensis responds to H(2)O(2) stress.

Metallothioneins (MTs) are the cysteine-rich proteins with low molecular weight, which play important roles in maintaining intracellular ion homeostasis, detoxification of heavy metal ions and protecting against intracellular oxidative damages. In this study a novel ethephon-induced metallothionein gene, designated as HbMT2, was isolated and characterized from Hevea brasiliensis. The HbMT2 cDNA contained a 237 bp open reading frame encoding 78 amino acids and the deduced protein showed high similarity to the type 2 MTs from other plant species. Expression analysis revealed more significant accumulation of HbMT2 transcripts in leaves and latex than in roots and barks. The transcription of HbMT2 in latex was strongly induced by ethephon and hydrogen peroxide (H(2)O(2)) stress. Overproduction of recombinant HbMT2 protein gave the Escherichia coli cells more tolerance on Cu(2+) and Zn(2+), and the recombinant HbMT2 could scavenge the reactive oxidant species (ROS) in vitro. All these results indicated that HbMT2 could respond to ethephon stimulation and H(2)O(2) stress as a ROS scavenger in H. brasiliensis. It is also suggested that HbMT2 function in improving the tolerance of rubber trees to heavy metal ions, and repressing the ethephon-induced senilism and tapping panel dryness (TPD) development by ROS scavenge system in H. brasiliensis.

The comparison of mouse full metallothionein-1 versus alpha and beta domains and metallothionein-1-to-3 mutation following traumatic brain injury reveals different biological motifs.

Traumatic injury to the brain is one of the leading causes of injury-related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock-out mice. We herewith report the results comparing full mouse MT-1 with the independent alpha and beta domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota-rod tests; all the proteins showed a modest effect in the former test, while only full MT-1 improved the performance of animals in the rota-rod, and the alpha domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host-response genes such as GFAP, Mac1 and ICAM, in some cases being the beta domain more effective than the alpha domain or even the full MT-1. A MT-1-to-MT-3 mutation blunted some but not all the effects caused by the normal MT-1, and in some cases increased its potency. Thus, splitting the two MT-1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions.

Two metallothionein genes from mud loach Misgurnus mizolepis (Teleostei; Cypriniformes): gene structure, genomic organization, and mRNA expression analysis.

Two metallothionein genes, MLMT-IA and MLMT-IB, were isolated and characterized from the mud loach Misgurnus mizolepis (Teleostei; Cypriniformes). For these MTs, we determined a tandem "tail-to-head" genomic organizational pattern, identified conserved genomic features, showed high sequence identities in the coding regions, and examined the closest phylogenetic affiliation, suggesting their divergence by a recent gene duplication event. However, the 5'-flanking upstream regions in MLMT-IA and MLMT-IB exposed large differences in the composition and distribution patterns of various transcription factor binding motifs, especially regarding the organization of the metal response element clusters. Real-time RT-PCR assays showed that mRNA levels of both MLMT-IA and MLMT-IB isoforms were variable among tissues and the ratios between them were also variable across tissues, although the MLMT-IA was always predominant in every adult tissue tested. We also found that the MLMT-IA and MLMT-IB mRNA expression levels were regulated dynamically during embryonic and larval development stages, in which the basal expression level of MLMT-IA was also consistently higher than that of MLMT-IB. Upon acute in vivo metal exposure to cadmium, chromium, copper, iron, manganese, nickel, or zinc at 5 microM for 48 h, the transcriptional modulations of MLMT-IA and MLMT-IB were quite different from each other and the type of response was affected significantly by the kind of metals and tissues.

The role of metallothionein in oncogenesis and cancer prognosis.

The antiapoptotic, antioxidant, proliferative, and angiogenic effects of metallothionein (MT)-I+II has resulted in increased focus on their role in oncogenesis, tumor progression, therapy response, and patient prognosis. Studies have reported increased expression of MT-I+II mRNA and protein in various human cancers; such as breast, kidney, lung, nasopharynx, ovary, prostate, salivary gland, testes, urinary bladder, cervical, endometrial, skin carcinoma, melanoma, acute lymphoblastic leukemia (ALL), and pancreatic cancers, where MT-I+II expression is sometimes correlated to higher tumor grade/stage, chemotherapy/radiation resistance, and poor prognosis. However, MT-I+II are downregulated in other types of tumors (e.g. hepatocellular, gastric, colorectal, central nervous system (CNS), and thyroid cancers) where MT-I+II is either inversely correlated or unrelated to mortality. Large discrepancies exist between different tumor types, and no distinct and reliable association exists between MT-I+II expression in tumor tissues and prognosis and therapy resistance. Furthermore, a parallel has been drawn between MT-I+II expression as a potential marker for prognosis, and MT-I+II's role as oncogenic factors, without any direct evidence supporting such a parallel. This review aims at discussing the role of MT-I+II both as a prognostic marker for survival and therapy response, as well as for the hypothesized role of MT-I+II as causal oncogenes.

A multiway approach for classification and characterization of rabbit liver apothioneins by CE-ESI-MS.

We applied a multiway approach to extract information from the analysis of protein isoforms by CE-ESI-MS. Metallothioneins (MT) are low-molecular-weight proteins (6-7 kDa) with a strong affinity for heavy-metal ions. Rabbit liver MT-I and MT-II fractions are purified from MT samples. At low pH, the bound metal ions were released from the amino acid structures, giving rise to apothioneins. MT-I, MT-II and MT apothioneins, which are complex mixtures of protein isoforms, were analyzed by CE-ESI-MS. After data pre-processing, parallel factor analysis (PARAFAC) and multivariate curve resolution-alternating least squares (MCR-ALS) were applied to the data sets. In both cases, the models enabled classification of the protein samples and identification of their characteristic sub-isoforms using a set of three components. MCR-ALS required an initial estimate of the pure mass spectra of the three components. Thus, PARAFAC loadings were used to initialize the MCR-ALS optimization. The classifications obtained with MCR-ALS were slightly better than those obtained with PARAFAC, probably because MCR-ALS was less affected by the small migration time shifts of the pre-processed electropherograms. However, no differences were found between the pure mass spectra of the three components in either model. Finally, MCR-ALS allowed us to obtain an individual electrophoretic profile of each of the three components for each of the samples analyzed, which proved valuable for characterization and quantification purposes.

Plant MTs-long neglected members of the metallothionein superfamily.

Occurrence of metallothioneins (MTs) was initially thought to be restricted to the animal kingdom, and the corresponding functions such as detoxification of heavy metal ions were assumed to be taken over in plants by the enzymatically synthesized phytochelatins. This perception was revised in the past years, and the existence of plant metallothioneins is generally accepted. Compared to the vertebrate forms, members of the plant MT family display a significantly larger sequence diversity, however, surprisingly little information is available concerning their possible functions, properties, and structures. Gene expression studies, and thus studies on the mRNA level, are the major source of data aiming at elucidating the function of plant MTs. However, so far it is not possible to unambiguously assign a specific function to a given metallothionein as proposed functions overlap, are complementary to each other, or even contradictory results are obtained. With respect to the structures and properties of plant metallothioneins even less scientific contributions are available illustrating the early stages, in which this research area resides. Existing data covers the metal ion content of the different plant metallothionein species and the pH stabilities of the resulting metal-thiolate clusters. Further, for a limited selection of proteins the number of clusters formed has been proposed and predictions towards the secondary structure of the protein backbone made. A recently determined three-dimensional structure of the larger domain of the wheat metallothionein E(c)-1 describes a metal ion coordination mode unprecedented for any metallothionein so far.

Induction of metallothionein synthesis by cilostazol in mice and in human cultured neuronal cell lines.

In this study, we examined the effect of Cilostazol to induce metallothionein (MT) in vivo and in vitro. Intraperitoneal injection of Cilostazol increased the expression of both MT-1 and MT-2 mRNA and total MT protein in the mouse liver. Cilostazol also augmented MT-1 mRNA levels in the murine brain. In vitro exposure to Cilostazol significantly augmented intracellular MT protein levels in cultured human brain microvascular endothelial cells (HBMEC) and in the neuroblastoma cell line IMR32. Taken together, these findings suggest that Cilostazol is an inducer of MT in the murine liver and brain, and that it has the potential to directly induce MT in cells. The contribution of the anti-oxidative effect of MT to the anti-stroke effect of Cilostazol was discussed.

Specificity and divergence in the neurobiologic effects of different metallothioneins after brain injury.

Brain injury and neuroinflammation are pathophysiologic contributors to acute and chronic neurologic disorders, which are progressive diseases not fully understood. Mammalian metallothioneins I and II (MT-I&II) have significant neuroprotective functions, but the precise mechanisms underlying these effects are still unknown. To gain insight in this regard, we have evaluated whether a distant, most likely single-domain MT (Drosophila MTN) functions similarly to mammalian MT-I&II (recombinant mouse MT-I and human MT-IIa and native rabbit MT-II) after cryogenic injury to the cortex in Mt1&2 KO mice. All the recombinant proteins showed similar neuroprotective properties to native MT-II, significantly reducing brain inflammation (macrophages, T cells, and pro-inflammatory cytokines), oxidative stress, neurodegeneration, and apoptosis. These results in principle do not support specific protein-protein interactions as the mechanism underlying the neuroprotective effects of these proteins because a non-homologous and structurally unrelated MT such as Drosophila MTN functions similarly to mammalian MTs. We have also evaluated for the first time the neurobiologic effects of exogenous MT-III, a major CNS MT isoform. Human rMT-III, in contrast to human nMT-IIa, did not affect inflammation, oxidative stress, and apoptosis, and showed opposite effects on several growth factors, neurotrophins, and markers of synaptic growth and plasticity. Our data thus highlight specific and divergent roles of exogenous MT-III vs. the MT-I&II isoforms that are consistent with those attributed to the endogenous proteins, and confirm the suitability of recombinant synthesis for future therapeutic use that may become relevant to clinical neurology.

The plant MT1 metallothioneins are stabilized by binding cadmiums and are required for cadmium tolerance and accumulation.

The small Arabidopsis genome contains nine metallothionein-like (MT) sequences with classic, cysteine-rich domains separated by spacer sequences, quite unlike the small conserved MT families found vertebrate genomes. Phylogenetic analysis revealed four ancient and divergent classes of plant MTs that predate the monocot-dicot divergence. A distinct cysteine spacing pattern suggested differential metal ion specificity for each class. The in vivo stability of representatives of the four classes of plant MT proteins and a mouse MT2 control expressed in E. coli were enhanced by cadmium (Cd). Particular MTs were also stabilized by arsenic (As), copper (Cu), and or zinc (Zn). To understand why plants have such a diversity of MT sequences, the Arabidopsis MT1 class, comprised of three genes, MT1a, MT1b, and MT1c, was characterized in more detail in plants. MT1 family transcripts were knocked down to less than 5-10% of wild-type levels in Arabidopsis by expression of a RNA interference (RNAi) construct. The MT1 knockdown plant lines were all hypersensitive to Cd and accumulated several fold lower levels of As, Cd, and Zn than wildtype, while Cu and Fe levels were unaffected. The ancient class of MT1 protein sequences may be preserved in plant genomes, because it has distinct metal-binding properties, confers tolerance to cadmium, and can assist with zinc homeostasis.

Variations in plant metallothioneins: the heavy metal hyperaccumulator Thlaspi caerulescens as a study case.

Plant metallothioneins (MTs) are extremely diverse and are thought to be involved in metal homeostasis or detoxification. Thlaspi caerulescens is a model Zn/Cd hyperaccumulator and thus constitutes an ideal system to study the variability of these MTs. Two T. caerulescens cDNAs (accession: 665511; accession: 665515), that are highly homologous to type 1 and type 2 Arabidopsis thaliana MTs, have been isolated using a functional screen for plant cDNAs that confer Cd tolerance to yeast. However, TcMT1 has a much shorter N-terminal domain than that of A. thaliana and so lacks Cys motifs conserved through all the plant MTs classified as type 1. A systematic search in plant databases allowed the detection of MT-related sequences. Sixty-four percent fulfil the criteria for MT classification described in Cobbett and Goldsbrough (2002) and further extend our knowledge about other conserved residues that might play an important role in plant MT structure. In addition, 34% of the total MT-related sequences cannot be classified strictly as they display modifications in the conserved residues according to the current plant MTs' classification. The significance of this variability in plant MT sequences is discussed. Functional complementation in yeast was used to assess whether these variations may alter the MTs' function in T. caerulescens. Regulation of the expression of MTs in T. caerulescens was also investigated. TcMT1 and TcMT2 display higher expression in T. caerulescens than in A. thaliana. Moreover, their differential expression patterns in organs and in response to metal exposure, suggest that the two types of MTs may have diverse roles and functions in T. caerulescens.

New metallothionein mRNAs in Gobio gobio reveal at least three gene duplication events in cyprinid metallothionein evolution.

This paper reports the identification and analysis of the primary structure of three novel metallothionein cDNA sequences in the gudgeon, Gobio gobio (Cyprinidae). Two different 180 bp coding regions were identified, resulting in two MT isoforms differing in one amino acid. The primary structure of the amino acid sequence was compared to other cyprinid MT sequences. Furthermore, two differently sized cDNAs were discovered in one of the two transcripts. We present a phylogenetic comparison of our sequences to other, previously published cyprinid MT gene sequences. Our analysis reveals an unexpected complexity in cyprinid MT evolution, with at least three gene duplication events. Differences and homologies between the evolution of cyprinid MT genes are compared to other teleost families. Finally, possible implications for metallothionein classification are discussed.