Purification and characterization of metallothionein-like cadmium binding protein from Asian periwinkle Littorina brevicula

Although mussels and oysters in the ocean are known to act as bioconcentrators for contaminants such as heavy metals, their ability to survive in heavily polluted water is relatively limited. The Asian periwinkle, Littorina brevicula, is one species that can accumulate a variety of environmental heavy metals, and the expression of its metal binding protein (MBP) is induced by cadmium. To better characterize this protein and its detoxification mechanism against cadmium, the present work examined the induction of a cadmium binding protein (Cd-BP) in Littorina brevicula exposed to 400 microg/l CdCl(2) for 30 days. The induced Cd-BP was purified by chromatography from the supernatants of homogenized organs (digestive gland, gonad, gill and kidney). This Cd-BP was found to consist of 103 amino acids, was rich in Cys (21 residues), and partial C-terminal sequence obtained by MALDI-TOF MS analysis revealed a Cys-XXX-Cys motif, which resembles a typical feature of mollusc metallothionein (MT). The Cd-BP molecular weight of 9.8 kDa is a little larger than that of other MTs.     

Heavy metals in rat liver cadmium binding protein.

The simultaneous determination of heavy metals in microsamples of chromatographically isolated cadmium-binding protein (Cd-BP) from rat liver was performed by neutron activation analysis. The results suggested that metals other than those already reported (Cd, Zn, Cu, and Hg) can bind the protein. These observations were confirmed by in vivo radiotracer experiments by injecting i.p. 21 labelled metal ions in cadmium-treated rats. Of the metals tested, 109Cd, 65Zn, 64Cu, 203Hg, 106Ag and 113Sn were found incorporated in the Cd-BP. The incorporation of 35S-cysteine, used as an indicator of Cd-BP biosynthesis, was increased in rats exposed to cadmium as compared to untreated animals. In order to establish the influence of other metal ions on the biosynthesis of Cd-BP and the incorporation of cadmium in the protein, in vivo experiments were carried out by i.p. injection of 109Cd and 35S-cysteine. In the presence of 42 metal ions no influence was observed on the incorporation of the two radioisotopes in the Cd-BP. These observations tend to support the hypothesis that cadmium can act as a highly specific inducer of Cd-BP and that this protein might be involved in the metabolism of several heavy metals.     

融和蛋白GST-SUMO-MT在大肠杆菌中的表达、纯化及其活性研究

将编码融合蛋白GST-SUMO-MT的DNA片段连接到大肠杆菌表达载体pET-28a中,构建重组表达质粒pET-GS-MT并转化到大肠杆菌Origami（DE3）中。20℃,1mM的IPTG诱导20h后,获得分子量约为43Kd的融合蛋白,表达量占菌体上清总蛋白的38.4％。利用谷胱甘肽交联琼脂糖（Glutathione Sepharose 4B）凝胶柱和Sephardex G-25分子筛联用可以得到纯度为95%以上的融合蛋白,得率约为70mg/L。该融合蛋白可与GST抗体产生阳性反应。融合蛋白GST-SUMO-MT可以显著提高宿主对Cd2+、Zn2+和Cu2+离子聚积的能力,其耐受能力比对照组分别提高4.2倍、4倍及1.6倍。此外,原子吸收光谱法测定,每分子GST-SUMO-MT可以结合2-3个Cd2+离子。     

McsA and the roles of metal-binding motif in Staphylococcus aureus

McsA is a key modulator of stress response in Staphylococcus aureus that contains four CXXC potential metal-binding motifs at the N-terminal. Staphylococcus aureus ctsR operon encodes ctsR, clpC, and putative mcsA and mcsB genes. The expression of the ctsR operon in S. aureus was shown to be induced in response to various types of heavy metals such as copper and cadmium. McsA was cloned and overexpressed, and purified product was tested for metal-binding activity. The protein bound to Cu(II), Zn(II), Co(II), and Cd(II). No binding with any heavy metal except copper was found when we performed site-directed mutagenesis of Cys residues of three CXXC motifs of McsA. These data suggest that two conserved cysteine ligands provided by one CXXC motif are required to bind copper ions. In addition, using a bacterial two-hybrid system, McsA was found to be able to bind to McsB and CtsR of S. aureus and the CXXC motif was needed for the binding. This indicates that the Cys residues in the CXXC motif are involved in metal binding and protein interaction.     

Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase

Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 microg g(-1) Zn and 10,000 microg g(-1) Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils. In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T. caerulescens. A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P1B subfamily of purported heavy metal-translocating ATPases. A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead [Pb], and copper [Cu]) out of the cell. However, in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance. Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens. Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding. When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation. The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.     

Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD

SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal binding capabilities, and previous work demonstrated that the protein can coordinate several types of first-row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To improve our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals [Mn(II), Fe(II), Co(II), Cu(I), and Zn(II)] were examined by using a combination of optical spectroscopy and mass spectrometry. Binding of SlyD to Mn(II) or Fe(II) ions was not detected, but the protein coordinates multiple ions of Co(II), Zn(II), and Cu(I) with appreciable affinity (K(D) values in or below the nanomolar range), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ? Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed.     

Concentrations of cadmium,zinc, copper,iron, and metallothionein in liver and kidney of nonhuman primates

To evaluate the species specificity of Cd accumulation and the relationship of Cd with other essential metals and metallothionein (MT), the concentrations of Cd, Zn, Cu, and Fe in the liver and kidney and the MT concentrations in the soluble fractions of the liver and kidney were determined in Cd-uncontaminated nonhuman primates (11 species, 26 individuals) kept in a zoo and two wild-caught Japanese macaques. The compositions of metal-binding proteins in the soluble fractions were also investigated by high-performance liquid chromatography (HPLC). The hepatic Cd concentration was 0.03–14.0 μg/g and the renal Cd concentration was 0.35–99.0 μg/g, both varying greatly and being higher in nonhuman primates, which were more closely related to man. The hepatic Zn concentration was 24.0–176 μg/g and the renal Zn concentration was 13.5–138 μg/g, showing 7- to 10-fold differences, and a correlation (r=0.558, p<0.01) was found between renal Zn and renal Cd concentrations. It was proved that in the liver, MT is more closely correlated with Zn (r=0.795, p<0.001) than with Cd (r=0.492, p<0.01) and that in the kidney MT is correlated with both Cd (r=0.784, p<0.001) and Zn (r=0.742, p<0.001). HPLC analysis of metals bound to MT-like protein in chimpanzees, de Brazza’s monkeys, and Bolivian squirrel monkeys showed that more than 90% of Cd in both the liver and kidney, approx 40% of Zn in liver and 28–69% of Zn in kidney were bound to MT-like protein. The higher percentage Zn was bound to high-molecular protein.     

Occurrence of acid-labile sulfide in cadmium-binding peptide 1 from fission yeast

Two kinds of Cd-binding peptides (Cd-BP1 and Cd-BP2) are induced in fission yeast upon addition of CdCl2 to the culture medium (l). It was also reported that Cd-BP1 and Cd-BP2 consisted of the same components, unit peptides (Cys3, Glu3, Gly1) and Cd atoms, though the respective amounts of components in each molecule were different (1, 2). Now, we have found that Cd-BP1 contains about 1 mol of acid-labile sulfide per mol, and Cd-BP2 contains no labile sulfide. The existence of the labile sulfide explains the unique physicochemical characteristics of Cd-BP1. Since acid-labile sulfide has not been found in metallothioneins or other metallothionein-like metal-binding proteins, the occurrence of labile sulfide in Cd-BP1 is the first instance in this field.     

Metallothionein-like proteins and zinc-copper interaction in the hindgut of Porcellio scaber (crustacea: Isopoda) exposed to zinc

Metallothioneins (MTs) are ubiquitous low-molecular-weight metalbinding proteins, with a variety of functions in metal metabolism ascribed to them. Among terrestrial invertebrates, MTs have been studied in nematodes, insects, snails, and earthworms. The aim of this study was the characterization of MT-like proteins in the terrestrial isopod crustacean Porcellio scaber in order to analyze their probable role in the metaboliss of copper (Cu) and zinc (Zn). Dietary Zn supplementation (793 μg Zn/g dry food, 6 d) was applied to stimulate MT synthesis. After separation of the hindgut postmicrosomic supernatant (cytosol) of Zn-exposed animals by gel filtration on a Sephadex G-75 column, a Cu- and Zn-containing peak was detected in the position of V _c/V_o≈2, where MTs are expected to elute. Rechromatography of these fractions by size-exclusion chromatography-high-performance liquid chromatography revealed that the 215-nm absorbance peak coincided with the absorbance peak of the rabbit MT II standard. These low-molecular-weight Cu- and Zn-binding compounds, detected in the cytosol of the hindgut cells in Zn-exposed P. scaber. are considered to be Cu, Zn-MT-like proteins. To our knowledge, this is the first report on the characterization of MT-like proteins in isopod crustaceans. These results also indicate that both Zn and Cu dynamics in P. scaber hindgut are affected at the given dietary Zn supplementation and that MT-like proteins are involved in this Zn-Cu interaction.     

Cu(II)-binding properties of a cytochrome c with a synthetic metal-binding site: His-X3-His in an alpha-helix.

A metal-binding site consisting of two histidines positioned His-X3-His in an alpha-helix has been engineered into the surface of Saccharomyces cerevisiae iso-1-cytochrome c. The synthetic metal-binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two-phase polymer systems containing a polymer-metal complex, Cu(II)IDA-PEG, and by metal-affinity chromatography. The stability constant for the complex formed between Cu(II)IDA-PEG and the cytochrome c His-X3-His site is 5.3 x 10(4) M-1, which corresponds to a chelate effect that contributes 1.5 kcal mol-1 to the binding energy. Incorporation of the His-X3-His site yields a synthetic metal-binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility.     

Characterization of jack-bean alpha-D-mannosidase as a zinc metalloenzyme.

1. Two methods were used to obtain alpha-mannosidase free from unbound Zn2+, (a) by removal of excess of metal ion from preparations purified in the presence of Zn2+ and (b) by purification under conditions that eliminate the need to add Zn2+. 2. The purified enzyme is homogeneous on ultracentrifugation, polyacrylamide-gel electrophoresis and gel chromatography. 3. The molecular weight is estimated to be 230 000. 4. The enzyme contains between 470 and 565 mug of zinc/g of protein, corresponding to between 1.7 and 2 atoms of zinc/enzyme molecule. The contents of other metals are much lower. 5. The enzyme is inactivated by chelating agents and activity is restored by Zn2+. 6. No other metal ion was found to replace Zn2+ with retention of activity. Some bivalent metal ions, e.g. Cu2+, rapidly inactivate the enzyme. 7. The results indicate that jack-bean alpha-mannosidase exists naturally as a zinc-protein complex and may be considered as a metalloenzyme.     

Relationship between metallothionein and metal contents in red-blooded and white-blooded Antarctic teleosts

Metal bioaccumulation and metallothionein were investigated in different organs of the red-blooded teleost, Trematomus bernacchii and the haemoglobinless Chionodraco hamatus. Specimens of the two Antarctic fish were sampled from Terra Nova Bay (Ross Sea), and their levels of Cd, Cu and Zn in homogenates and in soluble fractions of liver, muscle, gills, heart and plasma were determined. Dosages of metallothioneins (MTs) or MT-like proteins were assayed by the silver saturation method in soluble fractions of the same organs. In both T. bernacchii and C. hamatus the highest MT contents were found in liver. Hepatic MT and Cd, Cu and Zn concentrations correlated positively in T. bernacchii, whereas in C. hamatus hepatic MT showed a positive correlation only with Cd. Positive correlations were also found between Cd and MT in gills of the two species. A metal-binding protein containing a high percentage of cysteine from C. hamatus was purified and compared with the MT from T. bernacchii and mammals. Accepted: 27 November 1999     

Metallothionein induction and heavy metal accumulation in white shrimp Litopenaeus vannamei exposed to cadmium and zinc

Metallothioneins (MTs) have been widely considered for their potential use as specific biomarkers to reflect the existence of heavy metal pollution, because their induction has been observed to be obviously elevated after heavy metal exposure in a large number organism studied. However, relatively fewer efforts have been made in MT-related studies of prawn species, such as the white shrimp Litopenaeus vannamei, a globally important aquaculture species. With the results from gel filtration chromatography, we demonstrate the existence of MTs or MT-like proteins in L.vannamei. We further studied the relationship between MT induction and metals accumulation after long-term exposure to the heavy metals Cd and Zn. From our results, it is very clear that the response of L. vannamei to Cd differs from that to Zn, and this should be considered when using MTs in field applications to monitor metals contamination.     

Relative cadmium-binding capacity of metallothionein and other cytosolic fractions in various tissues of the rat.

The Cd-binding capacity of soluble proteins in 10 tissues of normal rats not excessively exposed to heavy metals was measured by saturation of freshly isolated cytosol with 109CdCl2 in vitro followed by Sephadex G-75 chromatography. The Cd-binding capacity of a 10,000 molecular weight Cd-binding peak (10,000 MW Cd-BP), which had a high affinity for Cd and was probably metallothionein, was the highest in kidney (78nmol Cd/g fresh tissue), followed by testis (63 nmol/g), liver (38 nmol/g) and then by brain (14 nmol/g). The amount of the Cd-BP in these tissues (assuming that it was metallothionein and bound 9 mol Cd/10,000g) was calculated to be 87, 70, 42 and 16 mg/kg fresh tissue in kidney, testis, liver and brain, respectively, or in the order of 10(-5) to 10(-6) mol/kg tissue. A significant amount of the 10,000 MW Cd-BP was also found in small intestine. It was present in rather small amounts in heart and lung, and possibly in spleen and skeletal muscle as well. In contrast, the protein was not detectable by this technique in plasma. The results suggest that metallothionein is a rather ubiquitous, intracellular protein in tissues of normal animals and may have other biological functions, besides its possible fortuitous role in heavy metal detoxification. A 30,000 molecular weight Cd-binding peak (30,000 MW Cd-BP) having a very high affinity Cd, apparently higher than that of the 10,000 MW Cd-BP, was found only in testes, among the 10 tissues examined. Its estimated Cd-binding capacity was 51 nmol Cd/g of testis, slightly less than that of metallothionein in testis. These findings support the hypothesis that the 30,000 MW Cd-BP is a plausible target of Cd in Cd-induced testicular injury, and suggest a basis for the peculiar sensitivity of the rat testis to Cd.     

金属结合蛋白(肽)与环境重金属生物修复

重金属污染是全球关注的重要环境问题。针对重金属的生物修复技术 ,因其特有的优势 ,越来越受到重视 ,其中一个重要的研究领域是利用金属离子和金属结合蛋白或结合肽之间存在的强亲和能力特性进行的生物修复研究。就金属结合蛋白 (肽 )的种类、结构特点、以及金属结合的作用机理进行了总结 ,同时综述了展示或表达有不同金属结合蛋白或结合肽的微生物和植物对重金属污染进行生物修复的最新研究进展 ,对基于金属结合蛋白 (肽 )的环境重金属生物修复的进一步研究 (如肽库的构建和筛选 ,金属与蛋白 (肽 )的相互作用 )进行了讨论。     

A new zinc-protein coordination site in intracellular metal trafficking: solution structure of the Apo and Zn(II) forms of ZntA(46-118)

Zinc, a metal ion that functions in a wide variety of catalytic and structural sites in metalloproteins, is shown here to adopt a novel coordination environment in the Escherichia coli transport protein ZntA. The ZntA protein is a P-type ATPase that pumps zinc out of the cytoplasm and into the periplasm. It is physiologically selective for Zn(II) and functions with metalloregulatory proteins in the cell to keep the zinc quota within strict limits. Yet, the N-terminal cytoplasmic domain contains a region that is highly homologous to the yeast Cu(I) metallochaperone Atx1. To investigate how the structure of this region may influence its function, this fragment, containing residues 46-118, has been cloned out of the gene and overexpressed. We report here the solution structure of this fragment as determined by NMR. Both the apo and Zn(II)-ZntA(46-118) structures have been determined. It contains a previously unknown protein coordination site for zinc that includes two cysteine residues, Cys59 and Cys62, and a carboxylate residue, Asp58. The solvent accessibility of this site is also remarkably high, a feature that increasingly appears to be a characteristic of domains of heavy metal ion transport proteins. The participation of Asp58 in this ZntA metal ion binding site may play an important role in modulating the relative affinities and metal exchange rates for Zn(II)/Pb(II)/Cd(II) as compared with other P-type ATPases, which are selective for Cu(I) or Ag(I).     

紫羊茅重金属抗性和敏感品种中类金属硫蛋白基因的鉴定及表达初探

重金属对生命机体的作用具有双重性。一方面,作为多数辅酶的辅助因子对细胞的正常代谢必不可少;另一方面,当重金属超过一定的浓度时对细胞有较大的毒性。在长期的进化过程中,生物可能形成一种调节细胞内重金属浓度的机制。这种机制在动物和真菌中被认为同金属硫蛋白(metallothionein,MT)的作用密切相关。植物中也存在类似的与重金属结合的低分子量蛋白(heavy metal binding pep-tide)。最近对拟南芥菜和水稻中类金属硫蛋白(MT-like)基因的研究证实其作用与动物MT相似。紫羊茅品种“Merlin”是一种从锌铅矿区的重金属污染地采集的单子叶草种,对镉和铜的抗性都较高,分别达到50mg/L和30mg/L,而5mg/L Cd~(2 )或2mg/L Cu~(2 )便可抑制敏感品种“S59”的正常生长。目前     

Expression of the pea gene PSMTA in E. coli. Metal-binding properties of the expressed protein

The pea (Pisum sativum L.) gene PSMTA has an ORF encoding a predicted protein with sequence similarity to class I metallothioneins (MTS). To examine the metal-binding properties of the PSMTA protein it has been expressed in E. coli as a carboxyterminal extension of glutathione-S-transferase (GST). Metal ions were associated with the expressed protein when purified from lysates of E. coli grown in metal supplemented media. The pH of half-dissociation of Zn, Cd and Cu ions from the recombinant fusion protein was determined to be 5.35, 3.95 and 1.45 respectively, compared with equivalent estimates of 4.50, 3.00 and 1.80 for equine renal MT.