Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80-90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30-50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

Gastropods are able to withstand fluctuating availabilities of nonessential trace elements such as cadmium by induction of Cd-specific metallothionein isoform (Cd-MT) expression. As in other species, the induction mechanism involves the binding of metal-regulatory transcription factors (MTF-1 or MTF-2) to metal responsive elements (MREs) in the MT promoter regions. Cd-dependent transcription of Cd-MT genes was assessed by quantitative real time PCR in two helicid gastropods, Helix pomatia and Cantareus aspersus, over a period of eight days. The promoter regions of the Cd-MT genes of the two species were sequenced and compared regarding the position of MREs and other relevant potential transcription factor binding sites (TFBs). Cd-MT gene transcription is induced after Cd exposure in Helix pomatia and Cantareus aspersus, showing a transient peak in Helix pomatia, contrasting with a persistent induction rate in Cantareus aspersus. Since the existence of MTF-2 was verified in both species, differing transcription patterns of Cd-MT genes must be due to functional differences in their metal-responsive promoter regions. Both promoters contain a proximal cluster of three MREs overlapping with TFBs for the transcriptional regulator Sp1. In contrast to Cantareus aspersus, however, the Cd-MT gene of Helix pomatia hosts an additional distal MRE overlapping with a Sp1 binding site and a CACCC box. Inhibitory effects of MRE overlapping Sp1 binding sites were observed in other MT genes. We therefore suggest that transient Cd-MT transcription upon Cd(2+) exposure in Helix pomatia may be the result of an inhibitory action of the distal MRE cluster.

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