Background: The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu⁺ or Cd²⁺. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. Results: HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd²⁺, Zn²⁺ or Cu²⁺ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd₆-HpCdMT and Cu₁₂-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions. Conclusion: The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Earthworms have been shown to accumulate trace elements in general, and particularly high amounts of metal ions such as cadmium, copper and zinc. The earthworm's response to metal contamination has been linked to the induction and expression of metallothionein (MT) proteins, a detoxification strategy analogous to that found in other biological systems. The present study focuses on an inducible Cd‐MT isolated from the compost‐dwelling brandling worm Eisenia foetida (Savigny). A full characterization of the protein (including protein induction, MT cDNA, amino‐acid sequence and metal stoichiometry) revealed a new dimension of knowledge to the molecular genetic information available to date. Whereas the elucidated cDNA codes for a putative protein which possesses 80 amino‐acid residues, the characterized protein bears only 41 amino acids. The isolated product has evidently attained its size and shape by cleavage near the N‐terminal site and at the linker region between the two putative metal‐binding domains of the translated product, yielding a small MT moiety which contains 12 Cys residues (including one triple Cys‐motif) binding four cadmium ions. It can be shown that the isolated MT molecule represents a self‐sufficient one‐domain MT which is stable in vitro. The isolation of the single‐domain MT peptide raises the question about the method of formation and significance in vivo of such small MT moieties from tissues of E. foetida and possibly other terrestrial invertebrates. In this respect, two hypotheses are discussed: firstly, the possibility of formation of small MT peptides due to enzymatic cleavage of the intact protein during the process of preparation and isolation; and secondly, the possibility of deliberate post‐translational processing of the translated gene product to yield functional one‐domain MT moieties.

A sublethal dose of cadmium (Cd²⁺) administered via the diet during short‐term exposure over 10 d induced programmed cell death in the hepatopancreas of the terrestrial pulmonate snail Helix pomatia. Condensed cell residues were predominantly phagocytosed by calcium cells, suggesting a specific function of these epithelial cells in metal detoxification or in clearing the organ of cellular debris from cell death. The considerable cell loss recorded by histological analysis was accompanied by enhanced cell proliferation. Intoxication with Cd was further associated with the pronounced abundance of residual bodies, predominantly recorded in excretory cells, and with pathological changes in the endoplasmic reticulum. During long‐term Cd exposure, mortality increased with increasing Cd concentrations in the diet, as demonstrated by feeding experiments in the laboratory. Lethal effects of Cd appeared to be correlated with Cd overloading of the Cd‐specific metallothionein isoform (Cd‐MT), isolated and characterized previously from the animal's hepatopancreas. Stoichiometric analysis shows that the capacity of Cd‐MT to bind six molar equivalents of Cd corresponds to a tissue Cd concentration of approximately 4 μmol/g dry weight. At this tissue concentration, all high‐affinity metal‐binding sites of Cd‐MT are occupied by Cd²⁺. Cadmium exposure beyond this level gives rise to progressive destabilization of Cd‐MT cluster structure in vitro, resulting in increasing proportions of weakly bound, or even unbound, Cd²⁺ ions. Our results suggest that in vivo, the observed overburdening of Cd‐MT with Cd²⁺ reduces the viability of affected animals.

Like all other animal species, terrestrial pulmonate snails require Cu as an essential trace element. On the other hand, elevated amounts of Cu can exert toxic effects on snails. The homeostatic regulation of Cu must therefore be a pivotal goal of terrestrial pulmonates to survive. Upon administration of Cu, snails accumulate the metal nearly equally in most of their organs. Quantitative studies in connection with HPLC and electrospray ionization mass spectrometry reveal that a certain fraction of Cu in snails is bound to a Cu-metallothionein (Cu-MT) isoform that occurs in most organs at constant concentrations, irrespective of whether the animals had been exposed to physiological or elevated amounts of Cu. In situ hybridization demonstrates that at the cellular level, the Cu-binding MT isoform is exclusively expressed in the so-called pore cells (or rhogocytes), which can be found in all major snail organs. The number of pore cells with Cu-MT mRNA reaction products remains unaffected by Cu exposure. Rhogocytes also are major storage sites of Cu in a granular form, the metal quickly entering the snail tissues upon elevated exposure. The number of rhogocytes with granular Cu precipitations strongly increases upon Cu administration via food. Thus, whereas Cu-MT in the rhogocytes represents a stable pool of Cu that apparently serves physiological tasks, the granular Cu precipitations form a second, quickly inducible, and more easily available pool of the metal that serves Cu regulation by responding to superphysiological metal exposure.