Low Molecular Weight Metal-binding Proteins Research Articles

Identification of cadmium-binding proteins from rice (Oryza sativa L.)

Metal-binding proteins play an important role in maintaining intracellular metal homeostasis and eliminating heavy metal toxification. Many metallothioneins (MTs) have been isolated from mammalian sources, which are a family of low molecular weight metal-binding proteins that are rich in cysteine. However, plants contain a different type of cadmium-binding protein that contain fewer cysteine residues. In this study, cadmium affinity chromatography coupled with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) has been used to separate and identify cadmium-binding proteins from different parts (root, stem, leaf and grain) of rice (Oryza sativa L.) cultivated under cadmium stress conditions. Seven cadmium-binding proteins with low isoelectric points containing relatively few cysteine residues were chosen for expression in Escherichia coli. The cadmium removal efficiency of protein A3AGZ4 (OsJ_10480) from Escherichia coli △zntA-BL21 was the highest (57.35%), which compares favorably with the cadmium removal efficiency of metallothionein MT (48.99%, mt from mouse,) and SMT (55.84%, smt from Sinopotamon honanense). In addition, for the strain A3AGZ4-△zntA-BL21, most of the bound cadmium was found to accumulate in the cytoplasm and not the cell wall. These results indicate that these plant proteins can bind cadmium to reduce heavy metal toxicity, thus contributing towards bioremediation of cadmium in the environment.

Metallothionein is elevated in liver and duodenum of Atp7b(\u2212/\u2212) mice

Different mutations in the copper transporter gene Atp7b are identified as the primary cause of Wilson’s disease. These changes result in high copper concentrations especially in the liver and brain, and consequently lead to a dysfunction of these organs. The Atp7(−/−) mouse is an established animal model for Wilson’s disease and characterized by an abnormal copper accumulation, a low serum oxidase activity and an increased copper excretion in urine. Metallothionein (MT) proteins are low molecular weight metal-binding proteins and essential for the zinc homeostasis but also play a role for the regulation of other metals, e.g. copper. However the molecular mechanisms of MT in regard to Atp7b remain still elusive. In this study we investigate the expression of MT in the liver and duodenum of Atp7b(−/−) mice and wildtype mice. Hepatic and duodenal expression of MT was measured by real-time reverse transcription-polymerase chain reaction and post-translational expression was analyzed by immunoblot and immunofluorescence. Expression of MT in liver und duodenum was significantly higher in Atp7b(−/−) mice than in controls. Hepatic and duodenal copper, iron and zinc content were also studied. Compared to control hepatic copper and iron content was significantly higher while hepatic zinc content was significantly lower in Atp7b(−/−) mice. In the duodenum copper and zinc content of Atp7b(−/−) mice was significantly lower than in controls. Duodenal iron content was also lower in Atp7b(−/−) mice, but did not reach statistical significance. The results of our study suggest that metallothionein is elevated in the liver and duodenum of Atp7b(−/−) mice.

Protective Role of Metallothionein in Chemical and Radiation Carcinogenesis

Metallothionein (MT) is a low molecular weight metal-binding protein induced by endogenous and exogenous stimuli such as cytokines and heavy metals. In 1993 and 1994, two research groups (Choo et al. and Palmiter et al., respectively) produced MT-I/II double-knockout mice (MT-I/II null mice) with null mutations of the MT-I and MT-II genes. Subsequently, MT-I/II null mice have been used to clarify the biological function, physiological role, and pathophysiological relevance of MT by many research groups. Recent studies using MT-I/II null mice to investigate the role of MT in metal toxicity and distribution, oxidative stress, and some disease were reviewed. In addition, several research groups including our laboratory have reported that MT-I/II null mice are highly susceptible to several carcinogenesis caused by 7,12-dimethylbenz[a]anthracene, X-ray, benzo[a]pyrene, N-butyl-N-(4-hydroxybutyl) nitrosamine, lead, and cisplatin. These results suggest that MT is an important protective factor against not only metal toxicity and oxidative stress but also chemical and radiation carcinogenesis. In this review, we present the findings of MT-I/II null mice with regard to the protective role of MT in carcinogenesis and mutagenesis caused by chemical agents and X-ray.

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.

Characterization of MtnE, the fifth metallothionein member in Drosophila

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight metal-binding proteins which occur in almost all forms of life. They bind physiological metals, such as zinc and copper, as well as nonessential, toxic heavy metals, such as cadmium, mercury, and silver. MT expression is regulated at the transcriptional level by metal-regulatory transcription factor1 (MTF-1), which binds to the metal-response elements (MREs) in the enhancer/promoter regions of MT genes. Drosophila was thought to have four MT genes, namely, MtnA, MtnB, MtnC, and MtnD. Here we characterize a new fifth member of Drosophila MT gene family, coding for metallothionein E (MtnE). The MtnE transcription unit is located head-to-head with the one of MtnD. The intervening sequence contains four MREs which bind, with different affinities, to MTF-1. Both of the divergently transcribed MT genes are completely dependent on MTF-1, whereby MtnE is consistently more strongly transcribed. MtnE expression is induced in response to heavy metals, notably copper, mercury, and silver, and is upregulated in a genetic background where the other four MTs are missing.

Human metallothioneins 2 and 3 differentially affect amyloid‐beta binding by transthyretin

Transthyretin (TTR), an amyloid-beta (Abeta) scavenger protein, and metallothioneins 2 and 3 (MT2 and MT3), low molecular weight metal-binding proteins, have recognized impacts in Abeta metabolism. Because TTR binds MT2, an ubiquitous isoform of the MTs, we investigated whether it also interacts with MT3, an isoform of the MTs predominantly expressed in the brain, and studied the role of MT2 and MT3 in human TTR-Abeta binding. The TTR-MT3 interaction was characterized by yeast two-hybrid assays, saturation-binding assays, co-immunolocalization and co-immunoprecipitation. The effect of MT2 and MT3 on TTR-Abeta binding was assessed by competition-binding assays. The results obtained clearly demonstrate that TTR interacts with MT3 with a K(d) of 373.7 +/- 60.2 nm. Competition-binding assays demonstrated that MT2 diminishes TTR-Abeta binding, whereas MT3 has the opposite effect. In addition to identifying a novel ligand for TTR that improves human TTR-Abeta binding, the present study highlights the need to clarify whether the effects of MT2 and MT3 in human TTR-Abeta binding observed in vitro have a relevant impact on Abeta deposition in animal models of Alzheimer's disease.

From animals to humans: evidence linking oxidative stress as a causative factor in muscle atrophy

From animals to humans: evidence linking oxidative stress as a causative factor in muscle atrophy

The Physiological Significance of Metallothionein in Oxidative Stress

Metallothionein (MT), a ubiquitous family of low-molecular weight metal-binding proteins, comprises 30% cysteine residues. Although all of the thiol residues in MT are bound to metals, it still remains active to reactive oxygen species. Each cysteine residue in MT is more effective at protecting DNA from hydroxyl radical attack than the glutathione cysteine in vitro. Prooxidative agents such as paraquat and carbon tetrachloride induce MT synthesis mediated by some responsive elements. MT demonstrates strong antioxidant properties, yet the physiological relevance of its antioxidant action is not clear. An injection of ferric nitrilotriacetate (Fe-NTA), which produces reactive oxygen species, caused transcriptional induction of MT synthesis in the liver and kidney. Pretreatment of mice with Zn attenuated nephrotoxicity induced by Fe-NTA. After a Fe-NTA injection, a loss of Cd-binding properties of preinduced MT was observed only in kidneys of Zn-pretreated mice but not in liver. MT-enriched hepatocytes are resistant to Fe-NTA toxicity, oxidative DNA, and cell damage during conditions of glutathione depletion. In glutathione-depleted cells, but not in non-treated cells, Cd-binding properties of cellular MT decreased with increasing concentration of Fe-NTA. Moreover, Cd released from MT after an injection of Fe-NTA induced new MT protein again. Thus MT may act as a secondary antioxidant in cellular protection system against oxidative stress.

A sensitive time-resolved fluorescent immunoassay for metallothionein protein

Metallothioneins (MTs) are a family of low molecular weight metal-binding proteins induced by a broad range of stress conditions, including exposure to transition metal ions. Biochemical and immunological methods to measure MT protein levels in tissues and cultured cells have been reported, but accuracy and sensitivity is impeded by high background levels, low specificity of currently available reagents, and relatively laborious and time-consuming multistep procedures. To address these difficulties, a protocol has been developed to measure MT protein levels using a competitive solid phase assay based on dissociation enhanced lanthanide fluoroimmuno (DELFIA) detection of anti-MT monoclonal antibody bound to solid phase MT. This assay allows time-resolved detection of antibody binding, based on binding and exchange of different lanthanide chelates followed by fluorescent detection, designed to reduce background fluorescence and increase sensitivity. The method allows measurement of low MT levels that are undetectable using current radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) protocols, and yields reproducible results with low background over a wide range of MT concentrations. Improved sensitivity of MT protein detection is of value in toxicological measurement of stress responses and assessment of MT expression and function.

Zinc accumulation and metallothionein gene expression in the proliferating epidermis during wound healing in mouse skin.

Metallothionein (MT), a low molecular weight metal-binding protein, has been related to zinc and copper metabolism, the acute-phase response, and cellular proliferation. In this study, we investigated changes in zinc metabolism and MT gene expression occurring in tissue damage and repair during wound healing in mouse skin. Northern blot analysis revealed that a significant increase of MT mRNA was observed in the liver for 18 h after wounding, and serum zinc downfall and hepatic zinc uptake were observed. In situ hybridization analysis showed that no significant expression of MT mRNA was detected within the first 9 h after wounding. However, it was expressed restrictively in the proliferating epidermis of the wound margin after 12 h. Zinc began to accumulate in wounded skin after MT gene expressed. Northern blotting and immunocytochemical staining revealed that MT has been synthesized actively during the growth phase compared with the stationary phase in normal human epidermal keratinocytes in vitro. Intracellular zinc accumulation was observed in the proliferating cells. We concluded that hepatic MT plays an important role as an acute phase protein against host damage, and epidermal MT contributes in the supply of zinc to wounded tissue and activates proliferation for the regeneration of epidermis.

Epidermal Proliferation of the Skin in Metallothionein-Null Mice

Metallothionein (MT) is a low-molecular weight metal-binding protein. Although the physiologic function of MT is not fully known, it is present in various species and various organs including the skin. MT is strongly stained in hyperplastic epidermal tissues in normal skin and in hyperplastic skin lesions, and increased expression of mRNA of the MT gene has been demonstrated in skin stimulated by proliferative agents, suggesting that MT is involved in the proliferation of epidermal keratinocytes. To improve our understanding of the role of MT in epidermal hyperplasia, mice with null mutations in their MT-1 and MT-2 genes were used in this study. We compared the epidermal hyperplasia in MT-null mice and in normal C57BL/6 J mice after treatments with cholera toxin, 12-0-tetradecanoylphorbol-13-acetate, and ultraviolet B irradiation, which stimulate epidermal proliferation. Immunostaining of MT was not detected in the skin of MT-null mice, and these mice developed significantly less epidermal hyperplasia than the normal mice after exposure to each stimulator. We determined the metal contents of skin samples by the proton-induced x-ray emission method. The zinc content of the skin of the MT-null mice was lower than that of the control mice before stimulation. After stimulation of epidermal hyperplasia, MT-null and normal mice showed significantly reduced levels of zinc. These findings indicate that cellular MT is involved in the proliferative process of the epidermis induced by cholera toxin, 12-0-tetradecanoylphorbol-13-acetate, and ultraviolet B light through its regulatory action on the metal metabolism required for cell growth.

Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity.

Metallothioneins (MT) are ubiquitous low molecular weight metal-binding proteins that may act as antioxidants. We examined the sensitivity of NIH 3T3 cells transfected with a plasmid containing mouse metallothionein-I gene (NIH3T3/MT) to the membrane permeant oxidant, tert-butyl hydroperoxide (tBH). NIH3T3/MT cells had a 4-fold increase in intracellular metallothionein as compared to cells transfected with a plasmid containing an inverted gene (NIH3T3/TM). Newly expressed metallothionein appeared to be localized to the cytoplasm as determined by immunofluorescence and confocal microscopy. NIH3T3/MT cells were 6 times more resistant than NIH3T3/TM cells to the cytotoxic effects of tBH. The antioxidant activity of NIH3T3/MT cells was greater than NIH3T3/TM cells, since exposure to tBH resulted in significantly less: (a) thiobarbituric acid-reactive substances and (b) fluorescence after loading cells with the oxidant-sensitive dye, 2'7'-dichlorodihydrofluorescein diacetate. Furthermore, homogenates of NIH3T3/MT cells were more capable of scavenging in vitro generated phenoxyl radicals as quantified by electron spin resonance detection. In contrast, overexpression of cytoplasmic MT did not protect against tBH-induced DNA damage, suggesting that subcellular location of MT is important for its function and that DNA damage is not a key determinant of cytotoxicity. These data provide direct support for an antioxidant role for MT, since physiologically relevant elevations in cytoplasmic MT interfere with tBH-induced cytotoxic peroxidation.

Preliminary purification and partial characterization studies of a low-molecular weight cytosolic lead-binding protein in liver of the channel catfish (Ictalurus punctatus)

The present study reports the isolation and partial characterization of a lead-binding protein (PbBP) in the liver of the channel catfish ( Ictalurus punctatus). The protein has a molecular weight of 10 kDa as determined by SDS polyacrylamide gel electrophoresis and contains relatively large amounts of glycine (18.3%), aspartic acid (10.2%) and serine (15.1%). Western blot studies conducted using polyclonal antibodies to the rat renal PbBP and metallothionein (MT) showed no cross-reactivity, suggesting that the fish hepatic protein is immunologically distinct from these low-molecular weight metal-binding proteins in mammals.

Copper-binding proteins and copper tolerance in Pisam sativum L.

The effect of high nutrient levels of copper on the low-molecular-weight copper-proteins of leaves from plants of two cultivars of Pisum sativum L., with different sensitivity to copper, was investigated. Gel-filtration chromatography of leaf extracts from Cu-tolerant and Cu-sensitive plants grown with 1 μM Cu(II), showed the presence of only two copper peaks (I and II), but growth of plants with 240 μM Cu(II) induced two additional copper fractions (III and IV). Fractions II and III were purified by solvent extraction, gel-filtration and ion-exchange chromatography, and their molecular weights, subunit sizes, absorption spectra, metalprotein stoichiometry and amino-acid contents were determined. Fraction II was a polypeptide of Mr 15000 composed of a single chain. The purification of fraction III produced a copper-containing fraction (III-1) of Mr 3700, and a copper-protein (III-2) with an Mr, by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, of 66000. The metal contents of fractions III-1 and III-2 were higher in Cu-tolerant than in Cu-sensitive plants. On the basis of amino-acid analyses, fraction III-1 appeared to be complexes of Cu(II)-poly-isoleucine and Cu(II)-poly-leucine. The results rule out the existence, in pea leaves, of any protein similar to either animal metallothioneins or to any of the low-molecularweight metal-binding proteins or peptides described in other plants and reported to be involved in metal tolerance. In the mechanism of copper tolerance at the leaf level, fractions III-1 (Mr 3700), III-2 (Mr 66000), and IV (Mr 2000) appear to have a role, fraction IV being specifically induced in the tolerant cultivar by Cu(II). Fractions III-1 and III-2 could participate in a different mechanism, adaptive in character, involving an enhanced capacity to bind copper in Cu-tolerant plants.

Cadmium accumulation and metallothionein-like proteins in the Sea star Asterias rubens

Sea stars Asterias rubens, were exposed to cadmium (50 μg Cd 2+/litre) for 4 months. Accumulation rates were highest in body wall and pyloric caeca, while in the gonads a relatively low accumulation rate was measured. In all tissues Cd accumulation showed saturation kinetics. In the cytosolic fraction of pyloric caeca from Cd-exposed sea stars, Cd-binding proteins with a molecular weight of approximately 10 800 Da could be demonstrated. In cytosolic fractions of testes or ovaries, low molecular weight metal-binding proteins were absent and the accumulated Cd was bound exclusively to high molecular weight proteins.

Evidence of presence of a low molecular weight, non-metallothionein-like metal-binding protein in the marine gastropod Nassarius reticulatus L.

1. 1. A low molecular weight metal-binding protein was found in the snail Nassarius reticulatus cytosol, which was induced in heavy metal contaminated environments. 2. 2. In our sodium dodecyl sulfate-mercaptoethanol polyacrylamide gel systems it behaved as a protein of 19 kDa mol. wt. 3. 3. Amino acid composition studies definitely established this protein not to be metallothionein (Mt) like, because it had a much lower level of cysteine and substantial amounts of aromatic amino acids and histidine. 4. 4. The metal-binding strength of this protein was concluded to be much weaker than that of Mt. 5. 5. In the crustacean Pagurus bernhardus L. such a protein could not be demonstrated. 6. 6. In both the snail and the crustacean Zn may inhibit the accumulation of Hg. The premise for studying the induction of the metal-binding Nassarius protein as a supplement to environmental metal monitoring purposes is briefly discussed.

A fish hepatocyte model for the investigation of the effects of environmental contaminants

An isolated fish hepatocyte culture system was developed as a models ystem to investigate the mechanisms of action of environmental contaminants. Hepatocytes were isolated from striped bass ( Morone saxatilis) by an adaptation of the two stage perfusion technique of Seglen. 1 The system was used to evaluate metal binding protein (MBP) induction in response to cadmium, a primary inducer of metallothionein (MT) in rat hepatocytes. Striped bass hepatocytes appeared to be refractory to the induction of MBP by cadmium, since there was no significant increase in the synthesis of MBP for any of the doses at any of the time points investigated. However, when a similar experiment was performed using rat hepatocytes there was induction of MBP that was related to both dose and time. These comparative experiments indicate that although there are similarities between the hepatocytes of the two species in regard to 35S incorporation into low molecular weight metal-binding proteins, there appear to be significant quantitative differences as well in regard to MBP kinetics. This in vitro model system could potentially be utilized to investigate the toxicological properties of other environmental contaminants.

Evidence of presence of heavy metal-binding proteins in polychaeta species

1. 1. Four groups of polychaete worms, differing in feeding habits, have been studied with respect to presence of metal-binding proteins. 2. 2. The presence of low molecular weight metal-binding proteins was clearly demonstrated in the species Lumbrineris fragilis as well as in Harmothoë sp. both being carnivorous animals. One of the proteins found in L. fragilis was similar to mammalian metallothionein (Mt).

Partitioning of metals among metal-binding proteins in the bay mussel, Mytilus edulis

Metabolism of metals was assessed in populations of Mytilus edulis exposed to metals in the laboratory and in the field. Chronic exposure to metals in both environments resulted in increased concentrations of metals in both the low molecular weight metal-binding protein fraction, which contains metallothioneins, and in the high molecular weight metal-binding protein fraction, which contains metalloenzymes. Our results from analysis of laboratory-exposed populations and from monitoring indigenous and transplanted populations indicate that the capacity for production of metallothioneins is limited and that the quantities present differ greatly with seasonal changes in the environment.

Isolation and Quantification of Cadmium and Copper-Species in Lettuce Plants

Cadmium and copper-species from native lettuce plants were isolated and quantified after Ultra Turrax homogenization in buffer and subsequent ltracentrifugation and gel filtration of the cytosol. From the cytoplasma, a high molecular weight metal binding-protein (>75000g/mol) containing 55% of the cytoplasmic cadmium and 20% of the cytoplasmic copper and a low molecular weight metal binding-protein (3200g/mol) containing 45% of the cytoplasmic cadmium and 70% of the cytoplasmic copper were isolated. Beside these there were two more copper binding-proteins of about 57000 and 25000g/mol, containing only small percentage of copper. The solid fragments of the cell, containing 50% of the total cadmium and 20% of the total copper respectively were not further examined. A broad variation in the metal balance from plant to plant was observed.