Various scientific events held on the metallothionein.
Metallothionein’s (MTs) are the lower molecular weight (6-7 kDa) proteins that are found to be present in almost all organism types ranging from prokaryotes to eukaryotes species. MT are the metal detecting proteins that can mitigate the effect caused by the excess metal ions. They are also found to be involved in cellular process such as cell growth regulation, ROS (Reactive Oxygen Species) and DNA repair. The protein was termed as Metallothionein due to the unusual higher metal (metallo) and the sulfur (thiol) content. They are further grouped into 3 classes viz., class I, II and III. The Class I and II MTs are polypeptides that were obtained from direct gene products, the class III MTs are from the cysteine-rich non-translational molecules that are termed as phytochelatins. The metal ions are been sequestered through the MTs with Cys rich motifs. All the cysteines are present in the reduced form and are been co-ordinated through the mercaptide bonds. The cysteines present in the MTs are preserved across the species, it is supposed that, cysteines are essential for the function and the MTs are required for the life. Metallothionins structure, conservation in evolution, their ubiquitous nature of occurrence, the genes redundancy and the programmed MTs synthesis in development, regeneration and reproduction of living organisms are some of the weighty arguments in suspecting MTs to also serve others and perhaps the high particular metal-related cellular roles. In this chapter, there is a detailed discussion about Metallothionein its structure, occurrence and function.
- heavy metals
Many of the elements are play a significant role in all kind of organisms and pose some functional outcomes in living organisms. Based on this outcome, elements are classified as essential and non-essential elements. Recent trace element research showed many of the metals also play a major significant role in metabolisms but they required very trace quantities. The metal ions of iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), cobalt (Co) molybdenum (Mo) and the non-metals selenium (Se) and iodine (I) bromine (Br), rubidium (Rb), aluminum (Al), nickel (Ni), titanium (Ti) and barium (Ba) etc. exceeded certain limits, it become toxic to organisms. Predicting the role of metals in living organisms is difficult since its intrinsic characteristics, redox properties, charge, and, above all, size. Toxicity of the metals mainly through altering the cell metabolism and thereby blocking synthesis of fundamental enzymes. To mitigate this, organisms have developed different detoxifying mechanisms such as blocking of toxic metals through oligopeptides or proteins and chelating metals encoding by genes, which are called metallothioneins (MTs) which possess residues of cysteine (Cys) having thiol or sulfhydryl groups (R-SH) and thereby immobilize the metallic ions. The prime function of MTs is the storage, transportation and binding of metals. Margoshes and Vallee’s discovery of Cd-binding, cystein-rich protein in the horse kidney marked a birth field for the research that focused to study a novel polypeptide super family, the metallothioneins (MTs). The MTs are family consisting of low molecular weight (6-7 kDa), cysteine (Cys)-rich, non-enzymatic proteins that are found ubiquitously in animals, in higher plants, in eukaryotic microorganisms, and many prokaryotes [1, 2]. The metal ions is sequestered through MTs by binding with Cys rich metal binding – motifs Cys-Cys, Cys-X-Cys, or Cys-X-X-Cys) that [3, 4]. Based on the Cys residues arrangement, MTs grouped into different classes [5, 6]. All the cysteines present in the reduced form are been coordinated to the metal ions that gives rise to the spectroscopic appearance of the metal-thiolate groups. Cysteines in the MTs are preserved across the species, it is suspected that, cysteines are essential for the function and the MTs is required for the life. MTs have some composition of unusual amino acid as they do not contain any aromatic amino acids and the most important is that, one third of its residues contacysteines. Moreover, MTs show spectroscopic features of metal thoilates (mercaptides) . MTs can bind Cd, Ni, Zn, Cu, and Pb with the affinities that depend up on the bioavailability and concentration of heavy metals. Three major scientist groups have contributed in the field of MTs include:
Toxicologists and physiologists for analyzing MTs role in the heavy metal metabolism and the de-toxification.
Protein chemists and spectroscopies intrigued by its usual structural features.
Molecular biologist interested in the gene regulation and use of MTs (promoter) sequences for the genetic engineering experiments.
MTs play a critical role in protecting the cell from harmful factors i.e. metals, free radicals, etc. through controlling cell growth, differentiation, proliferation, explicating their nuclear vs.cytoplasmic localization . MTs is cytoplasmic protein, accumulated in lysosomes, trandpoted to the nucleus and intermediated space in mitochondria [9, 10]. MTs transported the metals from one cell to other cell by receptor- mediated mechanism and metal is transported to the cytosol but protein remains in an endocytotic compartment [11, 12]. According to Wang et al. , MTs have exhibits four main functions and it is illustrated in the Figure 1.
Nevertheless, although MTs were discovered over four decades back, their physiological functions are not fully understood still . Based on the this prelude this review is being taken up to refresh the MTs structure, classification and role in metal detoxification.
2. Various scientific developments in metallothionein research
The scientific research history of MTs begins in 1950s almost seven decades back. The developments in MTs research includes the contributions from scientists in fields of environmental hygiene/medicine, biochemistry, medicine, pathology, nutrition and toxicology and are best illustrated in number of articles that are been published. During this time, in protein research methods, there is a considerable development in isolation and analytical methods for identification. In beginning the interest in MTs was focused on two main topics. The major interest were in the protein chemistry and in the toxicological,
|1964||Induction by cadmium|
|1971||Modification of Cd-toxicity|
|1972||Amino acid composition|
|1979||1st international meeting - Consesus nomenclature|
|1983||International meeting - Metallothionein and nephrotoxicity|
|1985||2nd International meeting on metallothionein|
|1992||3rd International meeting on metallothionein|
|1996||Workshop on metallothionein|
In 1957, the data related to cadmium binding protein in the equine tissue were published. However, in the beginning this study was first initiated through a report that was in a form of abstract that deals with cadmium in the human organs. In most of the animal species, cadmium is present in small amounts in tissues and the body fluids. For explaining this unexpected finding some of the hypotheses were postulated. Either cadmium can be present as bound to macromolecule or so had some natural function in the biological system or the cadmium also could be just a contaminant. The first report in 1960 was published on metallothionein . For the first time that a cadmium containing protein that are isolated from the equine renal tissue, was termed as ‘metallothionein’ based on the higher content of sulfur (4.1%) followed by Cadmium (2.9%) and Zinc (0.6%). Later researchers estimated that the molecular weight was found as 10000 ± 260 Da.
The absorption at specific wavelength of 250 nm revealed the cadmium mercaptide bonds. The MTs lack an aromatic amino acid due to the non absorption at precise wavelength of 280 nm. These were further examined through amino acid analyses. Higher sulfur concentration of about 4.1% was explained through the amino acid analyses that revealed high cysteine content. The eactive protein mercapto groups at this time were examined through titration with the p-chloro mercuri benzoate (CMB), N-ethylmaleimide (NEM) and silver ions. The Amino acids were determined using both ion exchange chromatography and two-dimensional paper chromatography. The cysteine residues were found as cysteic acid after metallothionein oxidation with the performic acid and as N-ethylmaleide derivatives. The Sedimentation constant were determined by Schlieren diagram with the use of sedimentation through ultracentrifugation to 1.75 (S020w). Partial specific volume, diffusion constant, and friction ratio were obtained. The estimated protein molecular weight was still varying between 9790 and 10500 Da. These were partly explained by several artifacts during the preparation. Metal analyses for Cd and Zn reported 5.9% of MTs weight and 5.2 g atoms mol−1 for cadmium and for zinc as 2.2% by weight and 3.3 g atoms mol−1. Some exchange between Cd and Zn was taking place obviously. It was suggested that the bonding with 3 SH-groups together with an atom of either Cd and Zn was formed.
In the years between 1970s and early 1980s, there were only few research groups performed researches that are related to MTs. A workshop was arranged with nearly 25 invited participants who had submitted their background manuscripts and the tentative report  was prepared and were distributed in advance to each of the participant. A consensus report was agreed during the meeting held in the year of 1978.
During this Ist international meeting about MTs, held in the Zurich consensus was reached about, e.g. nomenclature and methods for protein preparing. This first meeting had been followed by another meeting held at Zurich in 1985 that were more open but still with a workshop consensus. In between, a meeting was arranged at Aberdeen in1981.
3. Structure and occurrence of metallothionein
The structure of MTs was first reported by Kagi and Schafer . MTs have 2 domains consists of a cluster with three and other with 4 metal atoms. The gene are located on the chromosome 16. This protein contains a number of isoforms that are coded with several alleles. The ratio of the mRNA for MT-I and MT-II genes remains constant during the induction with metals, e.g. Cd, Cu and Zn. The MTs amino acid sequences from mammalian sources reveals that all of them contain nearly 61 amino acids that are more similar in their composition. All contain 20 cysteine residues which remain invariant to the amino acid sequence. Every cysteines do participate in the coordination of the seven moles of Zinc or cadmium per mole of the MT . The MTs are the cytoplasmic proteins; but, their occurrence during fetal development in nucleus are given by immuno histochemistry. The Plant MTs were first recognized in roots of the copper-resistant strain of
Metallothioneins from mammals contain 20 cysteins residues and bind seven cadmium or zinc ions in two discrete clusters. The thiolate side chains of these proteins act as bridging and terminal ligands to form Cd3SCy9 and Cd4 SCy11 clusters.
4. Classes of metallothionein
Based upon their similarities in structure, the MTs are divided into 3 classes:
Class I: This type present in mammalians, oysters, some fishes, crabs and mussels as Cys
Class II: Occurs in fungi, plants, yeasts, drosophila, cyanobacteria and nematodes in the form of Cys – x
Class III: Presence noticed in phytochelatins that present in all plants, some fungi and also algae as Cys – x – x - Cys.
The MTs belongs to the class I & II are been produced through direct mRNA translation. Whereas, the class III MTs belongs to the metal-binding polypeptides and have peptide bonds which are biologically synthesized as some of the enzymatic products. All vertebrates that are examined contain 2 or more distinct isoforms of MT, that are grouped into 2 classes, designated as MT-I and MT-II, depending upon elution position from the DEAE cellulose. In many cases each of the class actually consists of various different proteins that were designated as MT-IA, MT-IB, MT-IC, etc.
General characteristics of Metallothioneins are,
MT I and MT II’s molecular weight is 6-7 kDa, 61 amino acids, 20 Cysteine (30%), C-alanine, N-acetylmethionine, no histidine, no aromatics.
The have an unique amino acid sequence, metal clusters/tertiary structure
Metal content; Cu, Cd, Hg, Zn; 5-10% W/w
Light absorption are at 250 nm (Cd), 275 nm (Cu), 225 nm (Zn), 300 nm (Hg)
Induced synthesis through Zn and Cd.
There are no disulfide bonds, heat stability
Localization on chromosome and cytoplasm
MT expression in the crops serve as the key index in selecting for the heavy metal tolerance. In attempting for shedding light on their function, the researchers relied on the RNA blot hybridization for studying genes expression of MT during the development and also in response to several other environmental factors. Detailed localization of the MT mRNAs or gene promoter activity is obtained through reporter gene expression studies and
In general, affinity of the metal ions for binding sites follows the typical order of thiolate model complexes,
5. Metallothionein redox cycle and function
The MT redox cycle play an important role in the MT biologic function. It might link to homeostasis of the essential metals, detoxification of the toxic metals and protection against the oxidative stress. Advancement in MT research is shown by demonstrating redox regulation of the Zn-S interaction and zinc coupling. The Zn-MT cluster structure provides some chemical basis through which the cysteine ligand induces the oxido reductive properties. These structure permits for the thermo-dynamic stability of Zn in the MT. The decrease of oxidized MT can restore Zn binding ability, thereby replicates endogenous reservoir of zinc . These process constitutes the redox cycle of MT, as depicted in the Figure 2, that provides some new perspective upon MT biologic function.
Under the physiologic conditions, Zn that bounds to the MT are been released from the thiolate group oxidation. The MT disulfide and/or thionin formation follows a Zn release. The MT disulfide and/or the thionin undergoes some degradation, however, when the oxidized environment gets reduced. This reduction progress is more in occurrence of a catalyst (selenium). These processes constitute the MT redox cycle, which plays a critical role in biologic function of MT.
6. Proposed mechanism for metallothionein induction by metal cations
Cellular effects of exposure to the divalent metal cations and roles of particular resistance mechanism of the metallothionein expression, and expression of stress protein GroEL is shown in the Figure 3. GroEL forms essential component which reduces heavy metal stress . The MTs is a cysteine rich protein which binds with the metals and thereby detoxified through reducing its presense . The heavy metals enter the cells by the transport of the proteins which binds the metals. This metals could further binds SH groups with repressor protein of smtB. These attached metals further alter conforms the repressor protein and then released it. These process permits RNA polymerase to initiate the transcription from smtA, resulted the metallothionein expression.
7. Plant metallothioneins
After the Phytochelatin discovery, as a key metal ligand essential for the plant tolerance to Cadmium, a MT protein was identified in the wheat, and numerous MT genes was isolated from the plants. The plant Class II MT proteins are further classified based upon the amino acid sequence. In the year 1993, Robinson and his co-workers identified the two plant based MT which was based on cysteine residue positions in the predicted proteins. Since then, number of characterized plant MT genes has been increased drastically, as many do not conform to these 2 groups, some additional categories are added [24, 25].
The majority of the plant MT genes are identified in angiosperms. Numerous species, like
8. Expression pattern under influence of various factors
8.1 Metal ions
As homologs of animal MTs, it would be reasonable to assume that the MTs in plants are related to metal metabolism and detoxification of excess metal ions. Indeed, the expression of both Ec proteins and several class I MT genes vary under metal treatment. The wali l (type 2) in wheat was originally isolated as an aluminum-induced gene. Its expression is also increased by treatment with other toxic metal ions like Cd, Fe, Zn, Cu, Ga, In and La.
In rice, the transcript level of RgMT (type 1) was elevated by 250 μM CuCl2, in suspension-cultured cells [27, 28], while 100 μM CuSO4 increased ricMT (type 4) transcripts in both shoots and roots of seedlings. This expression-enhancing effect of Cu2+ are been reported in the type 2 MT genes from
Similarly, the type 3 MT LSC54 in rape (
8.3 Other stresses
Considerable effort are made to clarify the relationships between plant MTs and environmental factors other than metal ions. The expression of type 2 MT seems to be easily affected by stresses- For example; the MT in tobacco was strongly induced by tobacco mosaic virus and wounding  and expression of MT in
Regarding type I MT, the mRNA level of rgMT in rice increased under treatment with heat shock, a toxicant (DMSO) and starvation, and decreased in presence of abscisic acid.
9. Fungal metallothioneins
Fungal MTs, study are more sporadic and are described usually as copper binding proteins. Still, few are characterized functionally in the positions of metal organization in chemical properties or complementation tests in mutants of metal sensitive yeasts. Reputed MTs are mostly designated within collections of ESTs (Expressed Sequence Tag) based on sequence resemblance. These are occasion for the MT like arrangements that are present in the mycorrhizea, specifically in
Heterologous expression systems for demonstrating unambiguously that
There are more evidence for MTs role or MT-like proteins in the mutualistic and the pathogenic interactions in between fungi and plants. The genes of fungal MT are mostly regulated transcriptionally in the life stages mainly, during the colonization of plant. The first report was in 1995, when the 2 genes similar to MTs were expressed uniquely during appressorium formation by of conidia in
10. Cyanobacterial metallothionein
Numerous mechanisms of metal resistance exist in cyanobacteria. The first mechanism involves extracellular binding. Cells might synthesize and release some organic materials that could chelate the metals and reduce their bioavailability or metal ions might bound to outer cell surface . These complex forms are not readily transported into cell due to its complexity and structure. Secondly, cells could increase excretion rate of some metal ions using the energy-driven efflux pumps . The sequestration of internal metal, a 3rd resistance mechanism, is a key mechanisms through which the bacteria combats the exposure of the heavy metal and their subsequent accumulation. In cyanobacteria, metal ion sequestration inside the cell are performed by the Class II MTs.
The Class II MTs are a cysteine-rich, thiol-containing and are metal-binding proteins that can sequester the metals, thereby stopping the buildup of potentially some deadly free metals in cell. Metal binding happens by interactions of the metals with thiol clusters of the cysteine remainders. MTs gene was organized as operon termed as smt locus, that contains together the metallothionein protein (smtA) and the regulatory protein, repressor (smtB) genes. The SmtBs, are the managing repressor of the countenance from smtA promoter- operator province. The MTs countenance, from gene to the efficient protein, was encouraged through metals and regulation of the transcript to mRNA is based on the interface among the metals and repressor protein that regulates transcript, over by the interface by a thiol clusters that are contemporary on the repressor type of protein [38, 39].
11. Mammalian metallothionein
The MT genes were readily induced by several toxicologic and physiologic stimuli. Since the MT cysteines are conserved across the species, it is suspected that the cysteines are essential for the function and the MTs are required for the life. In attempts for determining the MT function(s), researches were carried out with 4 different experimental paradigms: (i) the animals were injected with chemicals that induce MT; (ii) the cells adapted to grow and survive in higher MT concentrations - that induces toxicants; (iii) the cells are transfected with the MT gene; and (iv) the MT-null mice and the MT transgenic. Very often, the reports from the researches with the first 3 approaches had indicated several purposes of MT in the cell biology is as follows: the MT (a) is “warehouse” for the “Zn” (b) the free radical hunter (c) protect from cadmium harmfulness. However, researches with MT transgenic with the null rats did not toughly reinforced initial 2 proposed purposes then they sturdily sustenance its purpose in the protection in contradiction of the cadmium harmfulness. Recurrent cadmium management to the MT null rats resulted in the nephron harmfulness at the 1/10 of the quantity that products the toxicity in the control rats. Researches in human being showed as 7% of overall populations had some renal malfunction by cadmium exposures. Consequently, if the human being did not consume the MT, “standard” exposure of cadmium will be toxic to human beings . Thus, it appears that during evolution, the MT ability to protect against the Cd toxicity would had taken a chief role for the life processes maintenance, as compared with its other proposed functions (
12. Studies conducted at Tamil Nadu agricultural university
Expression of metallothionein 3-like protein mRNA in the sorghum cultivars under the chromium (VI) stress was estimated . The
Study on the MT structure of plant and their function are impaired by difficulties that are encountered in the purification of this proteins since the innate bases, mostly because of intrinsic variability of the cysteine rich polypeptides with the occurrence of oxygen. Very few MTs are recuperated from the plant cells, and are uniform some as integral, noncleaved collections of metal . The heterologous countenance approaches stunned this complications, but the early results exhibited as the pea MTs produced in
MTs are stress related protein and might be involved in essential metal homeostasis and detoxification of excess metals during organism’s developmental process. The MT genes are readily induced by several toxicologic and physiologic stimuli. Due to the cysteines in MT are absolutely conserved across the species, it is suspected that cysteines are essential for the function and MT is required for the life. A clear role for MTs is yet to be established, although they are certainly thought to play a key role in the metal metabolism. MTs may function as the antioxidants and another possibility is the role in repair of the plasma membrane. Although MTs are expressed ubiquitously and conserved in living organisms, determining their function remains a future challenge. So further research work is necessary to better understand MT-metal interaction, the mechanism of MT expression and molecular transformation of novel MT genes to plant and microbial system for increasing heavy metal detoxification in the environment.