Metallothionein isoforms and their genes in Helix pomatia

The goal of the present project proposal is to test the hypothesis of metal-specific adaptation of the two metallothionein (MT) isoforms (Cd-MT and Cu-MT) and their genes from the terrestrial pulmonate mollusc, Helix pomatia at both, the protein expression and the gene regulatory levels. The background of this hypothesis is the fact that the two MT isoforms from this species apparently are differentially induced by and preferentially bind different metals (Cd2+ or Cu+ ) in vivo and perform metal-specific functions: while one isoform (Cd-MT) serves detoxification of Cd2+, the second isoform (Cu-MT) seems to be involved in Cu regulation and homaeostasis. We will achieve our goal by two different approaches: first, metal preference will be tested after recombinant expression of the two MT isoforms by metal replacement reactions of native Zn-thioneins with combined metal solutions (Cd2+ and Cu+ ). The resulting metal thionein complexes will then be analysed by UV / Vis and CD spectroscopy, as well as mass spectrometry. This part of the study will mainly be carried out in cooperation with the group of Prof. Dra. Silvia Atrian at the institute of Genetics of the University of Barcelona (Spain). Second, we will test the metal specificity of the two MT isoform genes on the basis of the gene sequence which we have elucidated during the last two years. Specifically, we would like to design reporter gene fusion constructs transfected in Biomphalaria glabrata embryonic cells (Bge), exposed to metal solutions (Cd2+, Zn2+ and Cu+ ) singly or in combination. Biomphalaria glabrata is a freshwater mollusc, the genomic DNA of which is currently being elucidated. For transfection, we propose to use an array of reporter gene constructs in which functional MT promoters of the two MT isoform genes from Helix pomatia will be fused to a reporter consisting of the mutually exchanged cDNA of the MT counterpart (i.e. Cd-MT promoter with Cu-MT cDNA and vice versa), tagged at its 3`- end by attachment of EGFP (Enhanced Green Fluorescence Protein) cDNA. The promoters will be modified by site-directed mutagenesis to obtain products with changed positions in MREs and in adjacent binding sites for enhancing or silencing nuclear transcription factors. This way, we will be able to study the metal-specific transcriptional activity of the two genes in dependence of the number and position of Metal Responsive Elements (MREs), and their interaction with other nuclear transcription factors (enhancers and silencers) which could possibly modulate metal-specific transcription. The transcriptional activity of the promoter elements will be detected and quantified by both, real-time detection PCR and laser scanninc microscopy, and the quantified transcriptional activity referred to the number of successfully transfected Bge cells. In addition to this, the cDNA of MT isoforms and of the Metal Transcription Factor (MTF) from Biomphalaria glabrata will be synthesized.

Pulmonate snails (lung snails) possess the ability to withstand stress impact under varying and fluctuating environmental conditions. In addition, they can accumulate and detoxify in their tissues increased concentrations of toxic trace elements. This exceptional capacity is, in a large part, owing to the expression in these animals of metallothioneins (MTs), a family of ubiquitous, metal binding, stress-responsive proteins. In contrast to MTs from most other animal taxa which normally form heterometallic metal complexes, this is not the case for some MT isoforms of pulmonate snails which seem to possess, with a Cd binding (CdMT) and a Cu binding (CuMT) isoform, strictly homometallic MT metal complexes, at least after isolation of these isoform from preparations in vivo. Within the frame of the present project, two questions had to be answered. First, it had to be clarified whether and by how much metal specificity of the Cd/Zn binding (CdMT) and the Cu binding (CuMT) isoform of Helix pomatia might be based on structural differences between the two isoform chains, as achieved during evolution. The second task was to identify the most important recognition motifs on the CdMT gene of Helix pomatia, which might serve as binding sites for transcription factors upon stress-dependent gene activation. One of the most significant findings is the fact that metal-specific MT isoforms apparently occur in all pulmonate snails, forming two MT subfamilies, one of them representing the CdMT isoforms, the second one comprising the CuMT isoforms. The CdMT isoforms evidently play an essential role for detoxification of Cd, whereas the CuMT isoforms are apparently involved in the homeostatic regulation of copper in connection with the cellular synthesis of haemocyanin. In addition, pulmonate snails possess a third isoform subfamily, comprising MT proteins that form heterometallic complexes with both, Cd2+ and Cu+ simultaneously, thus being not metal-specific and therefore called Cd/CuMTs. These unspecific isoforms seem to represent an ancestral variant of the metal-specific isoforms. They play an inferior physiological role in the metal metabolism of snails, being expressed constitutively at only very low concentrations. Intriguingly, the two metal-specific MT isoforms of pulmonates have arisen through repeated gene duplications from an unspecific, ancestral form. All three pulmonate isoforms share a highly homologous primary structure, differing among each other exclusively in their amino acid positions located between the conserved cysteine residues which provide the sulfur atoms for metal thiolate binding. This means that metal specificity of the respective isoforms must have achieved through evolution by modulation of amino acid positions not directly involved in metal binding. That metal specificity of the two CdMT and CuMT isoforms from Helix pomatia has indeed a structural basis, was shown experimentally through spectroscopic and mass spectrometric analyses of the recombinantly synthesized isoforms in metal-supplemented Escherichia coli cells. Thus it appeared that the two isoforms were able to form homometallic complexes only with their cognate metal ions. Moreover, upon transformation in yeast MT-knockout cells, the two Helix pomatia MT isoforms exhibited metal-specific functions according to their metal-specific binding behaviour. Interestingly, the metal-specific functionality of the two snail MT isoforms (CdMT and CuMT) is complemented by a metal-specific pattern of gene transcription, where the CdMT gene is exclusively induced by Cd2+ ions, whilst the CuMT gene does not respond to any kind of metal ions at all, which is in accordance with the homeostatic function of the respective expressed protein. There are, however, species-specific differences of transcriptional induction patterns, as shown by a comparison between the CdMT promoter regions of the two related pulmonate species, Helix pomatia and Cantareus aspersus. These differences in induction patterns are apparently due to the diverging number and position of binding sites for stress-involved transcription factors in the respective promoter regions. Precisely, the localization of MREs (Metal Responsive Elements) on the respective promoters is significant, since these elements may serve as binding sites for transcription factors upon transcriptional induction under metal and stress exposure. Whereas in vertebrates, the most important transcription factor under stress conditions is the so-called MTF-1 (Metal responsive Transcription Factor-1), this is apparently not the case in molluscs. Thus in these organisms, further research is needed to solve the question of about how MT genes are transcriptionally regulated under stress conditions.