Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance

Metallothioneins (MTs) are small cysteine-rich proteins found in various eukaryotes. Plant MTs are classified into four types based on the arrangement of cysteine residues. To determine whether all four types of plant MTs function as metal chelators, six Arabidopsis (Arabidopsis thaliana) MTs (MT1a, MT2a, MT2b, MT3, MT4a, and MT4b) were expressed in the copper (Cu)- and zinc (Zn)-sensitive yeast mutants, Deltacup1 and Deltazrc1 Deltacot1, respectively. All four types of Arabidopsis MTs provided similar levels of Cu tolerance and accumulation to the Deltacup1 mutant. The type-4 MTs (MT4a and MT4b) conferred greater Zn tolerance and higher accumulation of Zn than other MTs to the Deltazrc1 Deltacot1 mutant. To examine the functions of MTs in plants, we studied Arabidopsis plants that lack MT1a and MT2b, two MTs that are expressed in phloem. The lack of MT1a, but not MT2b, led to a 30% decrease in Cu accumulation in roots of plants exposed to 30 mum CuSO(4). Ectopic expression of MT1a RNA in the mt1a-2 mt2b-1 mutant restored Cu accumulation in roots. The mt1a-2 mt2b-1 mutant had normal metal tolerance. However, when MT deficiency was combined with phytochelatin deficiency, growth of the mt1a-2 mt2b-1 cad1-3 triple mutant was more sensitive to Cu and cadmium compared to the cad1-3 mutant. Together these results provide direct evidence for functional contributions of MTs to plant metal homeostasis. MT1a, in particular, plays a role in Cu homeostasis in the roots under elevated Cu. Moreover, MTs and phytochelatins function cooperatively to protect plants from Cu and cadmium toxicity.     

Age- and tissue-dependent metallothionein and cytosolic metal distribution in a native Mediterranean fish, Mullus barbatus, from the Eastern Adriatic Sea

The levels of metallothionein (MT), a biomarker of metal exposure, and of cytosolic metals (Zn, Cu, Cd), known as MT inducers, were investigated as variables of age (1 to 8 years) and tissue mass (liver, kidney, brain) of red mullet (Mullus barbatus). Within the age from 1 to 8 years the most significant increase is evident for cytosolic Cd in liver (43-fold) and in kidney (5-fold). MT and essential metals are constant with age or slightly increased. Over the growth period, statistically significant MT and metal increase is evident only between 1 and 6-8 years old specimens, while for Cd in liver and kidney cytosol significant increase already exists at 4 years old specimens. Metal distribution in all tissues follows the order: Zn>Cu>Cd, with even 500-800 times lower Cd levels than essential metal levels. Consequently, MTs follow the levels of essential metals, Zn and Cu, indicating MT involvement in homeostasis of essential metals. In contrast to kidney and brain, hepatic MT levels are not age-dependent. Inclusion of hepatic MT measurements and the associated cytosolic metals will be useful in the assessment of long-term metal effects in demersal fish M. barbatus.     

Chemistry and biology of mammalian metallothioneins

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular mass, cysteine- and metal-rich proteins containing sulfur-based metal clusters formed with Zn(II), Cd(II), and Cu(I) ions. In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are noninducible proteins, with their expression primarily confined to the central nervous system and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space. Recent reports established that these isoforms play an important protective role in brain injury and metal-linked neurodegenerative diseases. In the postgenomic era, it is becoming increasingly clear that MTs fulfill multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity, and oxidative damage. All mammalian MTs are monomeric proteins, containing two metal–thiolate clusters. In this review, after a brief summary of the historical milestones of the MT-1/MT-2 research, the recent advances in the structure, chemistry, and biological function of MT-3 and MT-4 are discussed.     

Localization effect on the metal biosorption capability of recombinant mammalian and fish metallothioneins in Escherichia coli

In this study, we examined the expression of mammalian and fish metallothioneins (MTs) in Escherichia coli as a strategy to enhance metal biosorption efficiency of bacterial biosorbents for lead (Pb), copper (Cu), cadmium (Cd), and zinc (Zn). In addition, MT proteins were expressed in either the cytoplasmic or periplasmic compartment of host cells to explore the localization effect on metal biosorption. The results showed that MT expression led to a significant increase (5-210%) in overall biosorption efficiency (eta(ads)), especially for biosorption of Cd. The MT-driven improvement in metal biosorption relied more on the increase in the biosorption rates (r(2), a kinetic property) than on the equilibrium biosorption capacities (q(max), a thermodynamic property), despite a 10-45% and 30-80% increase in q(max) of Cd and Zn, respectively. Periplasmic expression of MTs appeared to be more effective in facilitating the metal-binding ability than the cytoplasmlic MT expression. Notably, disparity of the impacts on biosorption ability was observed for the origin of MT proteins, as human MT (MT1A) was the most effective biosorption stimulator compared to MTs originating from mouse (MT1) and fish (OmMT). Moreover, the overall biosorption efficiency (eta(ads)) of the MT-expressing recombinant biosorbents was found to be adsorbate-dependent: the eta(ads) values decreased in the order of Cd > Cu > Zn > Pb.     

Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn

In this work we have studied the accumulation of heavy metals in two brown trout (Salmo trutta) populations in their natural environment and the participation of metal binding to metallothionein (MT) in this process. Cd, Cu and Zn concentrations, total MT (including Cu MT) and Cd/Zn MT were measured in the gills, liver and kidney of trout inhabiting two rivers, one Cu-contaminated and the other Cd/Zn-contaminated, located at Røros, Central Norway. In both populations, high levels of Cu were found in the liver, whereas Cd was accumulated in liver and particularly in the kidney. The proportions of Cd/Zn MT and Cu MT in liver and kidney, but not in gills, reflected the accumulated and the environmental concentrations of these metals. The total Cu MT concentrations in the investigated tissues, however, were highest in trout from the river with the lowest ambient Cu concentration. It is suggested that MTs are of less importance in Cu-acclimated trout. The data also suggest that acclimation to a Cu-rich environment involves reduced Cu accumulation or increased Cu elimination. In trout from the Cd-rich environment, this metal was mainly bound to MT, whereas in trout from the Cu-rich environment Cd was also associated with non-MT proteins. These findings emphasize the importance to determine both Cd/Zn MT and Cu MT levels, when the participation of this protein in metal handling in trout tissues is investigated.     

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.     

Metal binding properties and structure of a type III metallothionein from the metal hyperaccumulator plant Noccaea caerulescens

Metallothioneins (MT) are low molecular weight proteins with cysteine-rich sequences that bind heavy metals with remarkably high affinities. Plant MTs differ from animal ones by a peculiar amino acid sequence organization consisting of two short Cys-rich terminal domains (containing from 4 to 8 Cys each) linked by a Cys free region of about 30 residues. In contrast with the current knowledge on the 3D structure of animal MTs, there is a striking lack of structural data on plant MTs. We have expressed and purified a type III MT from Noccaea caerulescens (previously Thlaspi caerulescens). This protein is able to bind a variety of cations including Cd(2+), Cu(2+), Zn(2+) and Pb(2+), with different stoichiometries as shown by mass spectrometry. The protein displays a complete absence of periodic secondary structures as measured by far-UV circular dichroism, infrared spectroscopy and hydrogen/deuterium exchange kinetics. When attached onto a BIA-ATR biosensor, no significant structural change was observed upon removing the metal ions.     

His-containing plant metallothioneins: comparative study of divalent metal-ion binding by plant MT3 and MT4 isoforms

Metallothioneins (MTs) are a superfamily of Cys-rich, low-molecular weight metalloproteins that bind heavy metal ions. These cytosolic metallopeptides, which exist in most living organisms, are thought to be involved in metal homeostasis, metal detoxification, and oxidative stress protection. In this work, we characterise the Zn(II)- and Cd(II)-binding abilities of plant type 3 and type 4 MTs identified in soybean and sunflower, both of them being His-containing peptides. The recombinant metal-MT complexes synthesised in Zn(II) or Cd(II)-enriched Escherichia coli cultures have been analysed by ESI-MS, and CD, ICP-AES, and UV spectroscopies. His-to-Ala type 3 MT mutants have also been constructed and synthesised for the study of the role of His in divalent metal ion coordination. The results show comparable divalent metal-binding capacities for the MTs of type 3, and suggest, for the first time, the participation of their conserved C-term His residues in metal binding. Interesting features for the Zn(II)-binding abilities of type 4 MTs are also reported, as their variable His content may be considered crucial for their biological performance.     

Noncooperative metalation of metallothionein 1a and its isolated domains with zinc

Mammalian metallothioneins (MTs) are a family of small cysteine-rich proteins capable of binding 7 Zn(2+) or Cd(2+) ions into two distinct domains: an N-terminal β-domain that binds 3 Zn(2+) or Cd(2+) and a C-terminal α-domain that binds 4 Zn(2+) or Cd(2+). MT has been implicated in a number of physiological functions, including metal ion homeostasis, toxic metal detoxification, and as a protective agent against oxidative stress. Conventionally, MT has been understood to coordinate metal ions in a cooperative fashion. Under this mechanism of metalation, the only species of biological relevance would be the metal-free (apo-) form of the protein and the fully metalated (holo-) form of the protein. However, an increasing body of evidence suggests that metalation occurs in a noncooperative manner. If this latter mechanism is correct, then partially metalated forms of the protein will be stable and able to take part in cellular chemistry. We report in this paper conclusive evidence that shows that biologically essential zinc binds to MT in a noncooperative manner. In addition, we report for the first time the stability of a Zn(5)-MT species. The implications of these findings are discussed in terms of the mechanism of metalation.     

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.     

The potential use of metallothionein in the clam Ruditapes decussatus as a biomarker of in situ metal exposure

This work aimed to validate the relationship between metallothioneins (MTs) and metals (Cd, Cu and Zn) in field conditions. Specimens of the marine bivalve Ruditapes decussatus (Linné, 1758) from Gargour were transferred in two sites: Gargour and Sidi Mansour, both situated along the south-eastern coast of Tunisia. The bivalves were removed from pairs of cages at day 0 (date of transplantation), day 62 and day 132. Metals (Cd, Cu and Zn) and MTs were determined in the subcellular fractions of the digestive gland. In Gargour, metal and MT levels increased significantly after 62 days of transplantation. However, they showed modest and non-significant variations in Sidi Mansour. Zn was mainly associated with the insoluble fraction, whereas Cd and Cu percentages in the soluble and the insoluble fractions were equivalent. Simple correlation analysis showed a positive and significant relationship between MTs and each metal. If all metals were taken together, multiple correlations showed that MTs were significantly correlated with Cd and Zn, with an important coefficient for Cd, but no significant relationship was observed for Cu. Gel filtration chromatography showed that in the heat stable fraction, the only cytosolic SH rich compounds have an apparent low molecular mass (about 15 kDa), which could correspond to metallothioneins. In the digestive gland of R. decussatus MTs responded to moderate increases of metal contamination, without interference with other factors, and could be a promising biochemical indicator of metal exposure.     

Challenging the model for induction of metallothionein gene expression

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs bind essential and non-essential heavy metals. In mammalian cells MT genes are highly inducible by many heavy metals including Zn, Cd, Hg, and Cu. Aquatic systems are contaminated by different pollutants, including metals, as a result of man's activities. Bivalve molluscs are known to accumulate high concentrations of heavy metals in their tissue and are widely used as bioindicators for pollution in marine and freshwater environments, with MTs frequently used as a valuable marker of metal contamination. We here describe the MT isoform gene expression patterns of marine and freshwater molluscs and fish species after Cd or Zn contamination. Contamination was carried out at a river site polluted by a zinc ore extraction plant or in the laboratory at low, environmentally relevant metal concentrations. A comparison for each species based on the accumulated MT protein levels often shows discrepancies between gene expression and protein level. In addition, several differences observed in the pattern of MT gene expression between mollusc and mammalian species enable us to discuss and challenge a model for the induction of MT gene expression.     

Effects of dexamethasone on metallothionein induction by Zn, Cu, and Cd in Chang liver cells

Metallothioneins (MTs) were induced in Chang liver cells by the metals, Zn, Cu and Cd, and the glucocorticoid hormone, dexamethasone. When 116 microM Zn, 32 microM Cu and 18 microM Cd, and 10(-7) M dexamethasone, respectively, were administered for 9 h, MTs induced by each inducer in the cells reached maximum levels. The maximum accumulation of MT level induced by dexamethasone was the lowest of the four inducers investigated; the levels induced by Zn, Cu and Cd were 4.7, 1.2 and 1.5 times of that induced by dexamethasone. When dexamethasone was added to the cells together with the heavy metals (Zn, Cu and Cd), dexamethasone had an additive effect on the maximum MT accumulations induced by heavy metals as compared to when induction was conducted using one of heavy metals alone or by dexamethasone alone. However, dexamethasone did almost not effect the metal accumulations in the cells, although the maximum MT levels induced by heavy metal increased by dexamethasone. These results suggest that the process of MT induction by heavy metals and that by dexamethasone are independent of one another. When dexamethasone was added to the cells together with a high concentration of Cu (32 microM) induced the maximum MT accumulation, Cu transport into the cells decreased by 20-40% of that into non-treated cells, which was statistically significant.     

Role of metallothioneins in superoxide radical generation during copper redox cycling: defining the fundamental function of metallothioneins

In order to demonstrate the in vivo antioxidant properties of metallothioneins (MTs), the bacteria Escherichia coli was used as a cell reactor in which we compared the metal binding and antioxidative functions of MTs from different species, with different structures and polypeptide lengths. No protective effects of cytoplasmic MTs from cadmium (Cd) or zinc (Zn) contamination were observed in a wild-type E. coli strain, although these MTs can efficiently bind both Cd and Zn. To test their antioxidant properties, MTs were expressed within the cytoplasm of a sodA sodB deficient mutated strain (QC1726). However, a paradoxical MT toxicity was found when this strain was contaminated with Cd and Zn, suggesting that in a wild-type strain, superoxide dismutase counteracts MT toxicity. The most toxic MT was the one with the strongest Cd and Zn binding capacities. This toxic effect was linked to the generation of superoxide radicals, since a Cd-contaminated QC1726 strain expressing oyster MT isoforms produced 75-85% more O(2)*(-) than the control QC1726 strain. Conversely, under anaerobiosis or in the presence of a copper chelator, MTs protected QC1726 strain from Cd and Zn contamination. A model is proposed to explain the observed MT toxicity.