Amide proton exchange in human metallothionein-2 measured by nuclear magnetic resonance spectroscopy

In human metallothionein-2, the exchange rate constants of ten amide protons were found to range from 1.7 x 10(-4) to 1 x 10(-1) min-1 at pH 6.3 and 8 degrees C. Most of these slowly exchanging protons could be associated with hydrogen bonds in secondary structure elements of the alpha-domain. Amide proton exchange rates thus present an additional criterion for the structural characterization of different metallothioneins, which could be particularly valuable for comparisons of different homologous protein preparations containing nuclear magnetic resonance-inactive metal ions, where the metal-polypeptide co-ordinative bonds cannot be identified directly.     

Comparison of the immunological properties of mammalian (rodent), bird,fish, amphibian (toad), and invertebrate (crab) metallothioneins

Summary Immunoassays such as rocket immunoelectrophoresis, western dot blot hybridization, and competitive ELISA analysis were used to estimate and compare homology among fishes (several species), crab, toad, chicken and rodent metallothioneins. The relative abundance of tissue-specific metallothionein in catfish was highest in liver followed by kidney and pancreas like mammalian systems. Immunologically, considerable homology exists among fish metallothioneins, although the extent of homology differs to some extent. No homology was found between fish or chicken metallothioneins. The amphibian (toad) and invertebrate (crab) metallothioneins showed only partial homology with fish metallothioneins in antigenic determinants.Abbreviations MT Metallothionein - PBS Phosphate Buffered Saline - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - ELISA Enzyme Linked Immunosorbent Assay - ME 2-Mercaptoethanol - BSA Bovine Serum Albumim     

Solution structure of MT_nc,a novel metallothionein from the Antarctic fish Notothenia coriiceps

The structure of [113Cd(7)]-metallothionein (MT_nc) of the Antarctic fish Notothenia coriiceps, the first three-dimensional structure of a fish metallothionein, was determined by homonuclear 1H NMR experiments and heteronuclear [1H, 113Cd]-correlation spectroscopy. MT_nc is composed of an N-terminal beta domain with 9 cysteines and 3 metal ions and a carboxy-terminal alpha-domain with 11 cysteines and 4 metal ions. The position of the ninth Cys of the alpha domain of MT_nc is different from the corresponding Cys of mammalian MTs. As a result, the last CXCC motif in the mammalian MT sequence becomes CXXXCC in the fish MT. This difference leads to a structural change of the alpha domain and, in turn, to a different charge distribution with respect to that observed in mammalian metallothioneins.     

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

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

Fish and mammalian metallothioneins: a comparative study

Structural studies show that fish and mammalian metallothioneins are endowed of distinctive features. In particular, the ninth cysteine residue present in the alpha domain of fish metallothionein is shifted of two positions with respect to the mammalian metallothionein, introducing a conformational modification in the protein structure. In addition, the fish metallothionein is less hydrophobic and more flexible than its mammalian counterpart. Our previous studies showed that the hydropathy of piscine and mammalian metallothioneins is significantly correlated with organismal temperature. In the present paper we have performed phylogenetic comparative analysis on metallothioneins of 24 species of fish and mammals. The results of such analysis failed to indicate that metallothionein hydropathy is an adaptive response to the thermal regime of the species. We concluded that metallothionein hydropathy is a trait that did not evolve in association with environmental changes.     

Purification and properties of plaice metallothionein, a cadmium-binding protein from the liver of the plaice (Pleuronectes platessa).

A low-molecular-weight protein induced in the liver of the plaice (Pleuronectes platessa) by exposure to cadmium was purified and characterized. It is closely similar to mammalian metallothioneins in all of its properties in that it is a single-chain cadmium-binding protein of approx. 7000 mol.wt. with a high cysteine content (31 mol%) and no aromatic amino acid residues. The thiol groups of the cysteine residues complex with the cadmium in a SH/Cd molar ratio of 3:1 and produce a characteristic absorption maximum at 250 nm. Unlike the mammalian metallothioneins, however, metal analyses reveal only traces of zinc and copper in addition to cadmium. The presence of carbohydrate previously assumed from a positive reaction with periodic acid/Schiff reagent has now been disproved, and the positive reaction attributed to interaction with the thiol groups in the protein.     

Toward a property/function relationship for metallothioneins: histidine coordination and unusual cluster composition in a zinc-metallothionein from plants

Early cysteine labeled (E(C)) proteins are plant metallothioneins, which were first identified in wheat embryos and are thought to be seed-specific. An exhaustive analysis of expressed sequence tag (EST) entries reveals that homologs are expressed in embryos of both classes of flowering plants (monocotyledons and dicotyledons), but also occur in conifers (gymnosperms) and seed-free spike moss (lycophyta). Mass spectrometric and elemental analysis results indicate that, contrary to the widely propagated number of five, E(C) binds predominantly six zinc ions in at least two zinc-thiolate clusters. 1H and 111Cd NMR experiments suggest that, in contrast to the majority of previously characterized metallothioneins, two conserved histidine residues participate in metal binding. The collected data is consistent with the presence of clusters unprecedented in metallothioneins so far. This novel cluster composition is accompanied by metal-binding properties that are substantially different from other metallothioneins; thus wheat E(C) binds zinc less strongly than either mammalian or cyanobacterial MTs. The unique biochemical properties of wheat E(C) render it ideally suited for a role in zinc donation to nascent proteins during seed development, a role that has been suggested based on the fact that E(C) is induced by the plant hormone abscisic acid, but not by heavy metals. Our results provide a step further toward developing a property/function relationship for metallothioneins.     

Coordination of three and four Cu(I) to the alpha- and beta-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes

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

Metal detoxification and homeostasis in Antarctic Notothenioids. A comparative survey on evolution, expression and functional properties of fish and mammal metallothioneins

Organisms from yeast to mammals contain cysteine-rich, heavy metal binding proteins termed metallothioneins. The putative roles of these proteins are trace metal homeostasis and detoxification of poisonous heavy metals. The highly conserved chemical composition and the structural constraints led to the conclusion that metallothioneins of different origin must display remarkably similar features. The present review aims at surveying the studies carried out on the metallothioneins of Antarctic Notothenioidei, a dominating fish group endowed of a number of striking adaptive characters, including reduced (or absent) hematocrit and presence of antifreeze glycoproteins. Given the unique peculiarities of the Antarctic environment, a comparative study of the features of notothenioid metallothioneins could provide new insights into the role of these proteins in physiology and toxicology. The results summarized here show that the metallothioneins of this fish group display a number of features at the level of evolution, expression pattern, structure and function remarkably different from those of mammal metallothioneins.     

Sequence-specific 1H-NMR assignments in rabbit-liver metallothionein-2

The complete sequence-specific assignment of the 1H nuclear magnetic resonance spectrum of a major subform of rabbit liver metallothionein-2 is presented. The sequential assignment procedures revealed a number of differences with regard to results obtained by earlier partial chemical sequencing of a preparation now known to be microheterogeneous. In particular, the present data indicate a polypeptide chain length of 62 amino acid residues as compared to the occurrence of 61 amino acids in all other known mammalian metallothioneins. In the new sequence, which was also fully confirmed by chemical means, the additional amino acid residue was identified as Ala8' inserted between Ala8 and Ala9 of the standard amino acid numeration. In addition to the predominant protein species all preparations contained a minor component, for which the two-dimensional 1H-nuclear magnetic resonance features are compatible with a chemically different, homologous metallothionein.     

Molecular cloning and sequencing of metallothionein in squamates: New insights into the evolution of the metallothionein genes in vertebrates

Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian metallothioneins, we have cloned and sequenced the cDNAs encoding for metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of metallothioneins in reptiles. Phylogenetic analysis shows that reptilian metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of metallothionein genes along the major vertebrate lineages.     

Expression of metallothionein in the liver and kidney of rats is influenced by excess dietary histidine

It is well known that excess dietary histidine induces the metabolic changes in copper and zinc. Therefore, this study was carried out to clarify whether excess dietary histidine alters the gene expressions of metallothionein-1 and metallothionein-2 in the liver and kidney. Male rats were fed the control (ad libitum and pair-fed) or histidine-excess (50 g of L-histidine per kg of diet) diet for 0, 1 and 3 days. The levels of liver metallothionein-1 and metallothionein-2 mRNA were markedly lower in the rats fed the histidine-excess diet as compared to those of the control (ad libitum and pair-fed) diet, when fed for 1 or 3 days. The levels of renal metallothionein-1 and metallothionein-2 mRNA in the rats fed the histidine-excess diet were higher or tended to be higher as compared with the rats fed the control (ad libitum and pair-fed) diet when fed for 1 or 3 days, respectively. At the same time, hepatic copper content was decreased and renal zinc content was increased by dietary histidine. It thus appears, that such a response on the level of liver metallothionein mRNA might be related to the contents of liver copper, but of kidney metallothionein mRNA might be due to the content of zinc.     

Nuclear import of metallothionein requires its mRNA to be associated with the perinuclear cytoskeleton

The influence of mRNA localization on metallothionein-1 protein distribution was studied by immunocytochemistry. We used Chinese hamster ovary cells that had been transfected with either a native metallothionein-1 gene construct or metallothionein-1 5'-untranslated region and coding sequences linked to the 3'-untranslated region from glutathione peroxidase. The change in the 3'-untranslated region caused the delocalization of the mRNA with a loss of the perinuclear localization and association with the cytoskeleton. Clones were selected which expressed similar levels of metallothionein-1 protein, as assessed by radioimmunoassay. The results showed that loss of metallothionein-1 mRNA localization was associated with a loss of metallothionein-1 protein localization, most notably with a lack of metallothionein-1 protein in the nucleus of synchronized cells which were beginning to synthesize DNA. This indicates that the association of metallothionein-1 mRNA with the cytoskeleton around the nucleus is essential for efficient shuttling of the protein into the nucleus during the G(1) to S phase transition. This is the first demonstration of a physiological role for perinuclear mRNA localization and we propose that such localization may be important for a wide range of nuclear proteins, including those that shuttle between nucleus and cytoplasm in a cell cycle dependent manner.     

Identification of mouse brain proteins associated with isoform 3 of metallothionein

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.     

Isolation and partial characterization of metallothionein in the liver of the red-eared turtle (Trachemys scripta) following intreperitoneal administration of cadmium

Cadmium was distributed predominantly in the liver (42% of the body burden) after intraperitoneal injection with 10 mg Cd/kg/day for 6 days, although the kidney, spleen, heart, gonads and shell also contained substantial amounts of the metal. The major cadmium-containing fraction of the liver cytosol eluted in the position of mammalian metallothionein on a Sephadex G-75 column and was further resolved into two isoforms by reverse-phase HPLC. The two isoforms had high cysteine contents (17–22 residues/molecule) and lacked aromatic amino acids, a composition similar to that of other vertebrate metallothioneins. The turtle metal-binding protein had other properties characteristic of vertebrate metallothioneins including heat stability (85°C for 10 min), a relatively high absorbance at 245 nm, a low absorbance at 280 nm and a high metal content (approximately 6 nmol cadmium/nmol protein).     

Isolation of a Gene for a Metallothionein-Like Protein from Soybean

Using a synthetic oligonucleotide that corresponded to the consensusnucleotide sequence of the N-terminal region of mammalian metallothioneinas probe, we isolated a cDNA clone from a soybean library. Theclone had an ORF that encode a protein of 79 amino acids whichshowed significant homology to both N- and C-terminal regionsof mammalian and Neurospora crassa metallothioneins ⁴Present address: Department of Biosciences, Teikyo University,Toyosatodai, Utsunomiya, Tochigi, 320 Japan (Received March 13, 1991; Accepted June 17, 1991)     

NMR structure of the sea urchin (Strongylocentrotus purpuratus) metallothionein MTA.

The three-dimensional structure of [(113)Cd7]-metallothionein-A (MTA) of the sea urchin Strongylocentrotus purpuratus was determined by homonuclear(1)H NMR experiments and heteronuclear [(1)H, (113)Cd]-correlation spectroscopy. MTA is composed of two globular domains, an N-terminal four-metal domain of the amino acid residues 1 to 36 and a Cd4Cys11cluster, and a C-terminal three-metal domain including the amino acid residues 37 to 65 and a Cd3Cys9cluster. The structure resembles the known mammalian and crustacean metallothioneins, but has a significantly different connectivity pattern of the Cys-metal co-ordination bonds and concomitantly contains novel local folds of some polypeptide backbone segments. These differences can be related to variations of the Cys sequence positions and thus emphasize the special role of the cysteine residues in defining the structure of metallothioneins, both on the level of the domain architecture and the topology of the metal-thiolate clusters.     

Mammalian metallothionein

Chemical, spectroscopic, and structural studies have established the metallothioneins (MTs) to be a widely occurring family of polypeptidic bioinorganic structures. They are distinguished by an extremely high metal (Zn, Cd, Cu) and Cys content and by the arrangement of these components in metal-thiolate clusters. By structural criteria the MTs have recently been subdivided into three classes (Fowler et al.,Experientia Suppl. 52, 19–22, 1987). Class I MTs include mammalian MTs and related forms. Class II MTs display no such relationships, and Class III MTs are atypical polypeptides made up of repetitive γ-glutamylcysteinyl units. Amino acid sequences of over 50 MTs are now known. In mammals, over 55% of the residues, including the 20 Cys, are conserved. Mammalian MTs are genetically polymorphous. Thus, in human tissues and cell lines closely related structures of ten functional isoMTs have been determined either by amino acid or nucleotide sequencing. A comparable degree of polymorphism also exists in the rabbit. Mammalian MTs have been inferred to bind a total of seven bivalent metal ions (Me) through thiolate coordination in two separate clusters, i.e., Me(II)₃(Cys)₉ and Me(II)₄(Cys)₁₁. This two-cluster model has now fully been confirmed by the spatial structures of rat MT-2 and rabbit MT-2a determined by 2D NMR spectroscopy in aqueous solution.     

Evidence for a dynamic structure of human neuronal growth inhibitory factor and for major rearrangements of its metal-thiolate clusters.

Human neuronal growth inhibitory factor (GIF), a metallothionein-like protein classified as metallothionein-3, impairs the survival and the neurite formation of cultured neurons. Despite its approximately 70% amino acid sequence identity with those of mammalian metallothioneins (MT-1 and MT-2 isoforms), only GIF exhibits growth inhibitory activity. In this study, structural features of the metal-thiolate clusters in recombinant Zn(7)- and Cd(7)-GIF, and in part also in synthetic GIF (68 amino acids), were investigated by using circular dichroism (CD) and (113)Cd NMR. The CD and (113)Cd NMR studies of recombinant Me(7)-GIF confirmed the existence of distinct Me(4)S(11)- and Me(3)S(9)-clusters located in the alpha- and beta-domains of the protein, respectively. Moreover, a mutual structural stabilization of both domains was demonstrated. The (113)Cd NMR studies of recombinant (113)Cd(7)-GIF were conducted at different magnetic fields (66.66 and 133.33 MHz) and temperatures (298 and 323 K). At 298 K the spectra revealed seven (113)Cd signals at 676, 664, 651, 644, 624, 622, and 595 ppm. A striking feature of all resonances is the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz), which account for the absence of cross-peaks in [(113)Cd, (113)Cd] COSY. On the basis of a close correspondence in chemical shift positions of the (113)Cd signals at 676, 624, 622, and 595 ppm with those obtained in our previous studies of (113)Cd(4)-GIF(32-68) [Hasler, D. W., Faller, P., and Vasák, M. (1998) Biochemistry 37, 14966], these resonances can be assigned to a Cd(4)S(11)-cluster in the alpha-domain of (113)Cd(7)-GIF. Consequently, the remaining three (113)Cd signals at 664, 651, and 644 ppm originate from a Me(3)S(9) cluster in the beta-domain. However, the latter resonances show a markedly reduced and temperature-independent intensity (approximately 20%) when compared with those of the alpha-domain, indicating that the majority of the signal intensity remained undetected. To account for the observed NMR features of (113)Cd(7)-GIF, we suggest that dynamic processes acting on two different NMR time scales are present: (i) fast exchange processes among conformational cluster substates giving rise to broad, weight-averaged resonances and (ii) additional very slow exchange processes within the beta-domain associated with the formation of configurational cluster substates. The implications of the structure fluctuation for the biological activity of GIF are discussed.