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

Abstract

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 Cd 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.

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

“…Seemingly higher basal md-MT gene transcription levels in unexposed individuals, and significantly higher gene transcription values in Cd-treated A. biplicata snails, compared to the respective CuMT gene, strongly suggest an elevated expression rate and Cd-dependent activation of the md-MT genes of this species and hence, their involvement in Cd inactivation (Figure 7). This is in accordance with the transcriptional expression patterns of the CdMT genes in the helicid snail species Helix pomatia and Cornu aspersum [22]. A minor different transcription pattern is observed for the CuMT gene of A. biplicata, which, contrary to CuMT genes of other snails [22,83], is slightly upregulated by Cd exposure, too (Figures 5 and 7).…”

“…In addition to all these primary structure features, the md-MT genes of A. biplicata exhibit a transcription pattern that is similar to other Cd-specific snail MTs [14,22]. Seemingly higher basal md-MT gene transcription levels in unexposed individuals, and significantly higher gene transcription values in Cd-treated A. biplicata snails, compared to the respective CuMT gene, strongly suggest an elevated expression rate and Cd-dependent activation of the md-MT genes of this species and hence, their involvement in Cd inactivation (Figure 7).…”

“…For example, under control and low-dose Cd exposure (Cd1) conditions, sufficient MT protein might already be available to bind the excess amounts of Cd and hence, no transcriptional upregulation of the respective MT genes would be necessary. In some non-exposed helicid snails, the CdMT gene is transcribed, but the respective encoded MT protein is not expressed [22]. Inhibition of mRNA translation and its activation upon stress is a phenomenon described for many different genes [69][70][71].…”

“…Metal-selective snail MTs were first discovered in the helicid snails Helix pomatia and Cornu aspersum. Typically, these MTs not only exhibit a metal-selective binding behavior but are also characterized by their distinct cellular expression patterns and physiological functions directly related to the selectively bound metal ion [6,[20][21][22]. For example, Cu-selective MT isoforms (CuMT) of snails preferentially bind monovalent Cu ions, being expressed in a particular cell type called rhogocyte, involved in Cu homeostasis and hemocyanin turnover [21,23].…”

“…A second, Cd-selective MT isoform (CdMT) binds Cd 2+ with high affinity and is devoted to Cd-detoxification and stress response [7,20]. In addition, a third, non-metal-selective MT isoform (Cd/CuMT) is able to bind both metal ions simultaneously [6,24], being involved in embryonic snail development [22]. Most snail MTs are structurally organized in two metal-binding domains (N-and C-terminal) connected through a short linker region, exhibiting a binding stoichiometry of six divalent (Cd 2+ , Zn 2+ ) or twelve monovalent (Cu + ) metal ions [23,25].…”