A de novo evolved domain improves the cadmium detoxification capacity of limpet metallothioneins.

Metallothioneins (MTs) constitute an important family of metal binding proteins. Mollusk MTs, in particular, have been used as model systems to better understand the evolution of their metal binding features and functional adaptation. In the present study two recombinantly produced MTs, LgiMT1 and LgiMT2, and their de novo evolved γ domain, of the marine limpet Lottia gigantea, were analyzed by electronic spectroscopy and mass spectrometry. Both MT proteins, as well as their γ domains, exhibit a strong binding specificity for Cd(II), but not for Zn(II) or Cu(I). The LgiMTs' γ domain renders an MII4(SCys)10 cluster with an increased Cd stoichiometry (binding 4 instead of 3 Cd2+ ions), representing a novel structural element in the world of MTs, probably featuring an adamantane 3D structure. This cluster significantly improves the Cd(II)-binding performance of the full length proteins and thus contributes to the particularly high Cd coping capacity observed in free-living limpets.

Sublethal cadmium exposure in the freshwater snail Lymnaea stagnalis meets a deficient, poorly responsive metallothionein system while evoking oxidative and cellular stress.

The Great Pond snail Lymnaea stagnalis (Gastropoda, Hygrophila) is a wide-spread freshwater gastropod, being considered as a model organism for research in many fields of biology, including ecotoxicology. The aim of the present study was to explore the Cd sensitivity of L. stagnalis through the measurement of a biomarker battery for oxidative, toxic and cellular stress. The interpretation of biomarker parameters occurred against the background of a truncated metallothionein protein with a limited Cd-binding capacity. Individuals of L. stagnalis were exposed through 14 days to uncontaminated water (controls) or to low (30 µg · L-1) or high (50 µg · L-1) Cd concentrations. The digestive gland of control and low-Cd exposed snails was processed for transcriptional analysis of the Metallothionein (MT) gene expression, and for determination of biomarkers for oxidative stress, toxicity and cellular stress. Digestive gland supernatants of high-Cd exposed snails were subjected to chromatography and subsequent analysis by spectrophotometry. It was shown that the MT system of L. stagnalis is functionally deficient, with a poor Cd responsiveness at both, the transcriptional and the protein expression levels. Instead, L. stagnalis appears to rely on alternative detoxification mechanisms such as Cd binding by phytochelatins and metal inactivation by compartmentalization within the lysosomal system. In spite of this, however, traces of Cd apparently leak out of the pre-determined detoxification pathways, leading to adverse effects, which is clearly indicated by biomarkers of oxidative and cellular stress.

Metal-Specificity Divergence between Metallothioneins of Nerita peloronta (Neritimorpha, Gastropoda) Sets the Starting Point for a Novel Chemical MT Classification Proposal.

Metallothioneins' (MTs) biological function has been a matter of debate since their discovery. The importance to categorize these cysteine-rich proteins with high coordinating capacity into a specific group led to numerous classification proposals. We proposed a classification based on their metal-binding abilities, gradually sorting them from those with high selectivity towards Zn/Cd to those that are Cu-specific. However, the study of the NpeMT1 and NpeMT2isoforms of Nerita peloronta, has put a new perspective on this classification. N. peloronta has been chosen as a representative mollusk to elucidate the metal-binding abilities of Neritimorpha MTs, an order without any MTs characterized recently. Both isoforms have been recombinantly synthesized in cultures supplemented with ZnII, CdII, or CuII, and the purified metal-MT complexes have been thoroughly characterized by spectroscopic and spectrometric methods, leading to results that confirmed that Neritimorpha share Cd-selective MTs with Caenogastropoda and Heterobranchia, solving a so far unresolved question. NpeMTs show high coordinating preferences towards divalent metal ions, although one of them (NpeMT1) shares features with the so-called genuine Zn-thioneins, while the other (NpeMT2) exhibits a higher preference for Cd. The dissimilarities between the two isoforms let a window open to a new proposal of chemical MT classification.

Responsiveness of metallothionein and hemocyanin genes to cadmium and copper exposure in the garden snail Cornu aspersum.

Terrestrial gastropods express metal-selective metallothioneins (MTs) by which they handle metal ions such as Zn2+ , Cd2+ , and Cu+ /Cu2+ through separate metabolic pathways. At the same time, they depend on the availability of sufficient amounts of Cu as an essential constituent of their respiratory protein, hemocyanin (Hc). It was, therefore, suggested that in snails Cu-dependent MT and Hc pathways might be metabolically connected. In fact, the Cu-specific snail MT (CuMT) is exclusively expressed in rhogocytes, a particular molluscan cell type present in the hemocoel and connective tissues. Snail rhogocytes are also the sites of Hc synthesis. In the present study, possible interactions between the metal-regulatory and detoxifying activity of MTs and the Cu demand of Hc isoforms was explored in the edible snail Cornu aspersum, one of the most common European helicid land snails. This species possesses CdMT and CuMT isoforms involved in metal-selective physiological tasks. In addition, C. aspersum expresses three different Hc isoforms (CaH ɑD, CaH ɑN, CaH ß). We have examined the effect of Cd2+ and Cu2+ exposure on metal accumulation in the midgut gland and mantle of C. aspersum, testing the impact of these metals on transcriptional upregulation of CdMT, CuMT, and the three Hc genes in the two organs. We found that the CuMT and CaH ɑD genes exhibit an organ-specific transcriptional upregulation in the midgut gland of Cu-exposed snails. These results are discussed in view of possible interrelationships between the metal-selective activity of snail MT isoforms and the synthesis and metabolism of Hc isoforms.

Modularity in Protein Evolution: Modular Organization and De Novo Domain Evolution in Mollusk Metallothioneins.

Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxification, and therefore, MTs have been crucial for the adaptation of the living beings to variable situations of metal bioavailability. The evolution of MTs is, however, not yet fully understood, and to provide new insights into it, we have investigated the MTs in the diverse classes of Mollusks. We have shown that most molluskan MTs are bimodular proteins that combine six domains-α, ß1, ß2, ß3, γ, and δ-in a lineage-specific manner. We have functionally characterized the Neritimorpha ß3ß1 and the Patellogastropoda γß1 MTs, demonstrating the metal-binding capacity of the new γ domain. Our results have revealed a modular organization of mollusk MT, whose evolution has been impacted by duplication, loss, and de novo emergence of domains. MTs represent a paradigmatic example of modular evolution probably driven by the structural and functional requirements of metal binding.

Preliminary Study of Multiple Stress Response Reactions in the Pond Snail Lymnaea stagnalis Exposed to Trace Metals and a Thiocarbamate Fungicide at Environmentally Relevant Concentrations.

Gastropod mollusks have achieved an eminent importance as biological indicators of environmental quality. In the present study, we applied a multibiomarker approach to evaluate its applicability for the pond snail Lymnaea stagnalis, exposed to common industrial and agricultural pollutants at environmentally relevant concentrations. The snails were exposed to copper (Cu2+, 10 µg L-1), zinc (Zn2+, 130 µg L-1), cadmium (Cd2+, 15 µg L-1), or the thiocarbamate fungicide "Tattoo" (91 µg L-1) during 14 days. Metal treatment and exposure to "Tattoo" caused variable patterns of increase or decrease of metal levels in the digestive gland, with a clear accumulation of only Cd and Zn after respective metal exposure. Treatment with Cu and "Tattoo" caused an increase of cytochrome P450-related EROD activity. Glutathione S-transferase was inhibited by exposure to Cu, Zn, and "Tattoo." Treatment with the "Tattoo" led to an inhibition of cholinesterase activity, whereas Cu and Cd increased its activity. Caspase-3 activity was enhanced by up to 3.3 times in all treatments. A nearly uniform inhibitory effect for oxidative stress response parameters was observed in all kinds of exposure, revealing an inhibition of superoxide dismutase (Mn-SOD) activity, a depression of glutathione (GSH and GSSG) and of protein carbonyl levels. Pollutant-specific effects were observed for the catalase activity, superoxide anion production, and lipid peroxidation levels. Due to the high response sensitivity of Lymnaea stagnalis to chemical impacts, we suggest our study as a contribution for biomarker studies with this species under field conditions.

Metallomics reveals a persisting impact of cadmium on the evolution of metal-selective snail metallothioneins.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic techniques, we show that Cd selectivity of snail MTs has resulted from convergent evolution of metal-binding domains that significantly differ in their primary structure. We also demonstrate how their Cd selectivity and specificity has been optimized by the persistent impact of Cd through 430 million years of MT evolution, modifying them upon lineage-specific adaptation of snails to different habitats. Overall, our results support the role of Cd for MT evolution in snails, and provide an interesting example of a vestigial abiotic factor directly driving gene evolution. Finally, we discuss the potential implications of our findings for studies devoted to the understanding of mechanisms leading to metal specificity in proteins, which is important when designing metal-selective peptides.

Cadmium Uptake, MT Gene Activation and Structure of Large-Sized Multi-Domain Metallothioneins in the Terrestrial Door Snail Alinda biplicata (Gastropoda, Clausiliidae).

Terrestrial snails (Gastropoda) possess Cd-selective metallothioneins (CdMTs) that inactivate Cd2+ with high affinity. Most of these MTs are small Cysteine-rich proteins that bind 6 Cd2+ equivalents within two distinct metal-binding domains, with a binding stoichiometry of 3 Cd2+ ions per domain. Recently, unusually large, so-called multi-domain MTs (md-MTs) were discovered in the terrestrial door snail Alinda biplicata (A.b.). The aim of this study is to evaluate the ability of A.b. to cope with Cd stress and the potential involvement of md-MTs in its detoxification. Snails were exposed to increasing Cd concentrations, and Cd-tissue concentrations were quantified. The gene structure of two md-MTs (9md-MT and 10md-MT) was characterized, and the impact of Cd exposure on MT gene transcription was quantified via qRT PCR. A.b. efficiently accumulates Cd at moderately elevated concentrations in the feed, but avoids food uptake at excessively high Cd levels. The structure and expression of the long md-MT genes of A.b. were characterized. Although both genes are intronless, they are still transcribed, being significantly upregulated upon Cd exposure. Overall, our results contribute new knowledge regarding the metal handling of Alinda biplicata in particular, and the potential role of md-MTs in Cd detoxification of terrestrial snails, in general.

Cadmium Pathways in Snails Follow a Complementary Strategy between Metallothionein Detoxification and Auxiliary Inactivation by Phytochelatins.

Metal detoxification is crucial for animals to cope with environmental exposure. In snails, a pivotal role in protection against cadmium (Cd) is attributed to metallothioneins (MTs). Some gastropod species express, in a lineage-specific manner, Cd-selective MTs devoted exclusively to the binding and detoxification of this single metal, whereas other species of snails possess non-selective MTs, but still show a high tolerance against Cd. An explanation for this may be that invertebrates and in particular snails may also synthetize phytochelatins (PCs), originally known to be produced by plants, to provide protection against metal or metalloid toxicity. Here we demonstrate that despite the fact that similar mechanisms for Cd inactivation exist in snail species through binding of the metal to MTs, the actual detoxification pathways for this metal may follow different traits in a species-specific manner. In particular, this depends on the detoxification capacity of MTs due to their Cd-selective or non-specific binding features. In the terrestrial slug Arion vulgaris, for example, Cd is solely detoxified by a Cd-selective MT isoform (AvMT1). In contrast, the freshwater snail Biomphalaria glabrata activates an additional pathway for metal inactivation by synthesizing phytochelatins, which compensate for the insufficient capacity of its non-selective MT system to detoxify Cd. We hypothesize that in other snails and invertebrate species, too, an alternative inactivation of the metal by PCs may occur, if their MT system is not Cd-selective enough, or its Cd loading capacity is exhausted.

The Solution Structure and Dynamics of Cd-Metallothionein from Helix pomatia Reveal Optimization for Binding Cd over Zn.

Metallothioneins (MTs) are cysteine-rich polypeptides that are naturally found coordinated to monovalent and/or divalent transition metal ions. Three metallothionein isoforms from the Roman snail Helix pomatia are known. They differ in their physiological metal load and in their specificity for transition metal ions such as Cd2+ (HpCdMT isoform) and Cu+ (HpCuMT isoform) or in the absence of a defined metal specificity (HpCd/CuMT isoform). We have determined the solution structure of the Cd-specific isoform (HpCdMT) by nuclear magnetic resonance spectroscopy using recombinant isotopically labeled protein loaded with Zn2+ or Cd2+. Both structures display two-domain architectures, where each domain comprises a characteristic three-metal cluster similar to that observed in the ß-domains of vertebrate MTs. The polypeptide backbone is well-structured over the entire sequence, including the interdomain linker. Interestingly, the two domains display mutual contacts, as observed before for the metallothionein of the snail Littorina littorea, to which both N- and C-terminal domains are highly similar. Increasing the length of the linker motionally decouples both domains and removes mutual contacts between them without having a strong effect on the stability of the individual domains. The structures of Cd6- and Zn6-HpCdMT are nearly identical. However, 15N relaxation, in particular 15N R2 rates, is accelerated for many residues of Zn6-HpCdMT but not for Cd6-HpCdMT, revealing the presence of conformational exchange effects. We suggest that this snail MT isoform is evolutionarily optimized for binding Cd rather than Zn.

Differential response to Cadmium exposure by expression of a two and a three-domain metallothionein isoform in the land winkle Pomatias elegans: Valuating the marine heritage of a land snail.

Through evolution, marine snails have adapted several times independently to terrestrial life. A prime example for such transitions is the adaptation to terrestrial conditions in members of the gastropod clade of Littorinoidea (Caenogastropoda). Some species of this lineage like the periwinkle (Littorina littorea), live in intertidal habitats, where they are intermittently exposed to semi-terrestrial conditions. Pomatias elegans is a close relative of Littorina littorea that has successfully colonized terrestrial habitats. Evolutionary transitions from marine to terrestrial conditions have often been fostered in marine ancestors by acquisition of physiological pre-adaptations to terrestrial life. Such pre-adaptations are based, among others, on the optimization of a wide repertoire of stress resistance mechanisms, such as the expression of metal inactivating metallothioneins (MTs). The objective of our study was to explore the Cd handling strategy in the terrestrial snail Pomatias elegans in comparison to that observed previously in Littorina littorea. After Cd exposure, the metal is accumulated mainly in the midgut gland of Pomatias elegans, in a similar way as in its marine relative. Upon Cd exposure, Pomatias elegans expresses Cd-specific MTs, as also described from Littorina littorea. In contrast to the latter species, however, the detoxification of Cd in Pomatias elegans is mediated by two different MT isoforms, one two-domain and one three-domain MT. Although the MT proteins of both species are homologous and clearly originate from one common ancestor, the three-domain MT isoform of Pomatias elegans has evolved independently from the three-domain MT of its marine counterpart, probably by addition of a third domain to the pre-existing two-domain MT. Obviously, the occurrence of homologous MT structures in both species is a hereditary character, whereas the differentiation into two distinct MT isoforms with different upregulation capacities in Pomatias elegans is an adaptive feature that probably emerged upon transition to life on land.

Successive Onset of Molecular, Cellular and Tissue-Specific Responses in Midgut Gland of Littorina littorea Exposed to Sub-Lethal Cadmium Concentrations.

Cadmium (Cd) is one of the most harmful metals, being toxic to most animal species, including marine invertebrates. Among marine gastropods, the periwinkle (Littorina littorea) in particular can accumulate high amounts of Cd in its midgut gland. In this organ, the metal can elicit extensive cytological and tissue-specific alterations that may reach, depending on the intensity of Cd exposure, from reversible lesions to pathological cellular disruptions. At the same time, Littorina littorea expresses a Cd-specific metallothionein (MT) that, due to its molecular features, expectedly exerts a protective function against the adverse intracellular effects of this metal. The aim of the present study was, therefore, to assess the time course of MT induction in the periwinkle's midgut gland on the one hand, and cellular and tissue-specific alterations in the digestive organ complex (midgut gland and digestive tract) on the other, upon exposure to sub-lethal Cd concentrations (0.25 and 1 mg Cd/L) over 21 days. Depending on the Cd concentrations applied, the beginning of alterations of the assessed parameters followed distinct concentration-dependent and time-dependent patterns, where the timeframe for the onset of the different response reactions became narrower at higher Cd concentrations compared to lower exposure concentrations.

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress.

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata, one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails.

The physiological role and toxicological significance of the non-metal-selective cadmium/copper-metallothionein isoform differ between embryonic and adult helicid snails.

Metal regulation is essential for terrestrial gastropods to survive. In helicid snails, two metal-selective metallothionein (MT) isoforms with different functions are expressed. A cadmium-selective isoform (CdMT) plays a major role in Cd2+ detoxification and stress response, whereas a copper-selective MT (CuMT) is involved in Cu homeostasis and hemocyanin synthesis. A third, non-metal-selective isoform, called Cd/CuMT, was first characterized in Cantareus aspersus. The aim of this study was to quantify the transcriptional activity of all three MT genes in unexposed and metal-exposed (Cd, Cu) embryonic Roman snails. In addition, the complete Cd/CuMT mRNA of the Roman snail (Helix pomatia) was characterized, and its expression quantified in unexposed and Cd-treated adult individuals. In embryos of Helix pomatia, the Cd/CuMT gene was induced upon Cu exposure. Its transcription levels were many times higher than that of the other two MT genes, and also exceeded by far the Cd/CuMT mRNA concentrations of adult snails. In the hepatopancreas of adult Roman snails, no Cd/CuMT could be detected at the protein level, irrespective of whether the snails had been exposed to Cd or not. This contrasts with the situation in the near relative, Cantareus aspersus. It appeared that the 3'-UTR of the Cd/CuMT mRNA differed largely between Cantareus aspersus and Helix pomatia, being larger in the latter species, with a number of putative binding sites for proteins and miRNAs known to inhibit mRNA translation. We suggest this as a possible mechanism responsible for the lack of Cd/CuMT protein expression in adult Roman snails.

Physiological, Diurnal and Stress-Related Variability of Cadmium-Metallothionein Gene Expression in Land Snails.

The terrestrial Roman snail Helix pomatia has successfully adapted to strongly fluctuating conditions in its natural soil habitat. Part of the snail's stress defense strategy is its ability to express Metallothioneins (MTs). These are multifunctional, cysteine-rich proteins that bind and inactivate transition metal ions (Cd(2+), Zn(2+), Cu(+)) with high affinity. In Helix pomatia a Cadmium (Cd)-selective, inducible Metallothionein Isoform (CdMT) is mainly involved in detoxification of this harmful metal. In addition, the snail CdMT has been shown to also respond to certain physiological stressors. The aim of the present study was to investigate the physiological and diurnal variability of CdMT gene expression in snails exposed to Cd and non-metallic stressors such as desiccation and oxygen depletion. CdMT gene expression was upregulated by Cd exposure and desiccation, whereas no significant impact on the expression of CdMT was measured due to oxygen depletion. Overall, Cd was clearly more effective as an inducer of the CdMT gene expression compared to the applied non-metallic stressors. In unexposed snails, diurnal rhythmicity of CdMT gene expression was observed with higher mRNA concentrations at night compared to daytime. This rhythmicity was severely disrupted in Cd-exposed snails which exhibited highest CdMT gene transcription rates in the morning. Apart from diurnal rhythmicity, feeding activity also had a strong impact on CdMT gene expression. Although underlying mechanisms are not completely understood, it is clear that factors increasing MT expression variability have to be considered when using MT mRNA quantification as a biomarker for environmental stressors.

Metallothionein gene expression in embryos of the terrestrial snail (Cantareus aspersus) exposed to cadmium and copper in the Bordeaux mixture.

The response specificity of three metallothionein (MT) genes (CdMT, CuMT and Cd/CuMT) was assessed after long-term exposure (20 days) of Cantareus aspersus eggs to cadmium (Cd) (2 to 6 mg/L) or to the fungicide Bordeaux mixture (BM) (2.5 and 7.5 g/L). MT gene expression measured by quantitative real-time PCR (qRT-PCR) revealed that in the unexposed embryos, the transcript levels of the three MT genes decreased significantly through embryonic development. However, the CdMT gene was strongly upregulated with increasing Cd exposure concentration, whereas the transcript levels of the other two genes increased less pronouncedly, but significantly above an exposure concentration of 4 mg Cd/L. Upon exposure to BM, all three MT genes were significantly upregulated above a BM concentration of 2.5 g/L. It is concluded that long-term Cd exposure in hatched snails induced patterns of MT gene expression that differed from those obtained after short-term exposure (24 h).

Differential sensitivity of snail embryos to cadmium: relation to age and metallothionein gene expression.

The aim of this study was to determine whether cadmium (Cd) sensitivity of Cantareus aspersus embryos is age-dependent and influenced by metallothionein (MT) gene expression. Hatching success and the expression of three MT isoform genes (Ca-CdMT, Ca-CuMT and Ca-Cd/CuMT) were measured in embryos exposed to increasing Cd concentrations for 24 h starting on the sixth day of development. Isoform gene expression was quantified on days 7 and 12 after exposure. Results were compared to those of embryos exposed to the same conditions as above, but from the beginning of embryogenesis (day 0). Transcription of the Cd-specific MT gene (Ca-CdMT) was observed from the first day of development, whereas the two other genes did not respond to Cd at all. Overall, Cd sensitivity of embryos decreased with increasing age of development, as assessed by age-dependent increase of EC50 values for hatching rate, and increasing Cd threshold concentrations for Ca-CdMT expression.

Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion.

Cognate and noncognate metal ion coordination in metal-specific metallothioneins: the Helix pomatia system as a model.

The Helix pomatia metallothionein (MT) system, namely, its two highly specific forms, HpCdMT and HpCuMT, has offered once again an optimum model to study metal-protein specificity. The present work investigates the most unexplored aspect of the coordination behavior of MT polypeptides with respect to either cognate or noncognate metal ions, as opposed to the standard studies of cognate metal ion coordination. To this end, we analyzed the in vivo synthesis of the corresponding complexes with their noncognate metals, and we performed a detailed spectroscopic and spectrometric study of the Zn(2+)/Cd(2+) and Zn(2+)/Cu(+) in vitro replacement reactions on the initial Zn-HpMT species. An HpCuMTAla site-directed mutant, exhibiting differential Cu(+)-binding abilities in vivo, was also included in this study. We demonstrate that when an MT binds its cognate metal, it yields well-folded complexes of limited stoichiometry, representative of minimal-energy conformations. In contrast, the incorporation of noncognate metal ions is better attributed to an unspecific reaction of cysteinic thiolate groups with metal ions, which is dependent on their concentration in the surrounding milieu, where no minimal-energy structure is reached, and otherwise, the MT peptide acts as a multidentate ligand that will bind metal ions until its capacity has been saturated. Additionally, we suggest that previous binding of an MT polypeptide with its noncognate metal ion (e.g., binding of Zn(2+) to the HpCuMT isoform) may preclude the correct folding of the complex with its cognate metal ion.

Evolutionary concepts in ecotoxicology: tracing the genetic background of differential cadmium sensitivities in invertebrate lineages.

In many toxicological and ecotoxicological studies and experimental setups, the investigator is mainly interested in traditional parameters such as toxicity data and effects of toxicants on molecular, cellular or physiological functions of individuals, species or statistical populations. It is clear, however, that such approaches focus on the phenotype level of animal species, whilst the genetic and evolutionary background of reactions to environmental toxicants may remain untold. In ecotoxicological risk assessment, moreover, species sensitivities towards pollutants are often regarded as random variables in a statistical approach. Beyond statistics, however, toxicant sensitivity of every species assumes a biological significance, especially if we consider that sensitivity traits have developed in lineages of species with common evolutionary roots. In this article, the genetic and evolutionary background of differential Cd sensitivities among invertebrate populations and species and their potential of adaptation to environmental Cd exposure will be highlighted. Important evolutionary and population genetic concepts such as genome structure and their importance for evolutionary adaptation, population structure of affected individuals, as well as micro and macroevolutionary mechanisms of Cd resistance in invertebrate lineages will be stressed by discussing examples of work from our own laboratory along with a review of relevant literature data and a brief discussion of open questions along with some perspectives for further research. Both, differences and similarities in Cd sensitivity traits of related invertebrate species can only be understood if we consider the underlying evolutionary processes and genetic (or epigenetic) mechanisms. Keeping in mind this perception can help us to better understand and interpret more precisely why the sensitivity of some species or species groups towards a certain toxicant (or metal) may be ranked in the lower or higher range of species sensitivity distributions. Hence, such a perspective will transcend a purely statistical view of the sensitivity distributions concept, and will enhance ecotoxicology in many respects.