Structure-activity relationships of metallothionein isoforms in terrestrial gastropods: Metal specificity versus multifunctionality

Research project P 14593 Metallothionein Isoforms in Terrestrial Gastropods Reinhard DALLINGER 09.10.2000 Metallothioneins (MTs) are hydrophilic, low molecular mass proteins with a high affinity towards certain trace metal ions such as Cd2+, Zn2+ and Cu+ . The main function of these proteins has been suggested to be linked to cellular trace clement metabolism, with metal detoxification and homeostatic regulation of essential metal ions being the most prominent tasks of these peptides. During the last years, novel aspects of structural and Ainctional relationships have been reported from terrestrial invertebrate MTs. In these animals, MTs apparently exhibit a large degree of structural diversity, with the perspective of discovering new models of MT functioning One example for such an intriguing model has been provided by terrestrial gastropods. There, two structurally divergent MT isoforms were discovered which apparently exhibit different metal-specific functions, with one of the two isoforms serving as a detoxification molecule against Cd2+, the second one performing metal-regulatory tasks in favour of Cu+ (see Dallinger et al. 1997, Nature 388, 237-238). However, the particular conditions under which the two MT isoforms act in a. metaldiscriminating manner are largely unknown.. The goal of the present proposal will be to answer these open questions by examining four central working hypothesis along which our project work and methodology will develop. First hyphothesis: Differences in the behaviour of the two isoforms towards Cd and Cu are based on differences in gene structure and molecular regulation. To test this hypothesis, it will be necessary to elucidate the gene structure and characterize the regulatory elements supposedly present in the promoter region of both isoform. genes. This will be achieved by using the already known cDNA sequences of both isoforms as starting points, with the possibility to obtain information on gene structure and regulatory elements by means of long-distance PCR or, alternatively, partial miverse PCR and modified tail-PCP- Moreover, MT. mRNA and isoform expression will be quantified by quantiative PCR and metal-specific saturation assays. Second Hypothesis: Differences in metal preference between the two isoforms are based on differences in their primary structures. This hypothesis will be tested by recombinant expression of both IVff isoforms with a bacterial expression system, and by analyzing the two isoforms with respect to some of their most important biophysical, biochemical and structural features by means of titration calorimetry, UV electronic absorption and circular dichroism (CD) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and 113Cd nuclear magnetic resonance (NMR). An intriguing perspective for the future would be to carry out metal binding studies with chimeric forms of the two isoforms, by stepwise introducing amino acid substitutions from one isoforin to another using,,Splicing by. Overlap Extensioe` (SOE) PCPL Third Hypothesis: The metal specificity of the two isoforms in. vivo is based on cellular and tissuespecific discrimination processes. A decisive clue in this concern is probably the differential expression of the two metal- specific isoforms in different tissues. We plan to test this hypothesis by two approaches allowing to trace differential expression patterns of the two MT isoforms in snad tissues based on specific visualization of the two isoforms by means of polyclonal antibodies and mRNA in-situ-hybridization. Fourth Hypothesis: The metal-specific functions of the two isoforms can only be understood by considering possible interferences with other metal-binding molecules. The validity of this hypothesis will be examined by adopting different biochemical methods to demonstrate: 1) the presence of other metal-binding IVIT peptides in mail tissues, apart from the Cd- and Cu-specific isoforms alreeady known; 2) the role of glutathione in Ze binding and its possible interaction with a Ze binding MT isoform; and 3) possible interferences of Cu-IVIT with hemocyamn, and in particular the Ce-donating function of Cu-MT. We are convinced that the dual MT isoform model in terrestrial gastropods will offer the opportunity to elucidate some of the most basic mechanisms of metal-specificity and multifunctionality in these proteins, and we believe that these aspects will confer to our studies some kind of general importance. To achieve our ambitious goals, many activities planned within the frame of the project will be done in cooperation with internationally reputed experts.

Terrestial snails of the helicid family (such as, for example, the Roman snail, Helix pomatia) are avid accumulators of certain non-essential trace elements (Cd), while at the same time, they are capable of maintaining in their tissues the homeostatic regulation of essential trace elements (Cu). This exceptional ability of snails is based on the presence and activity of two metal-specific Metallothionein (MT) isoforms, one of them being responsible for the detoxification of Cd, the second one serving homeostatic regulation of Cu. The goal of the present project work was to study and elucidate the molecular, structural, cellular and physiological background determining the metal-specific function of the two MT isoforms. This was achieved by following four working hypotheses which should be tested by means of different molecular, biochemical and cell-physiological approaches. In the first hypothesis it was suggested that the metal-spcicific function of the two MT isoforms may be based on the structure and regulatory activity of the two MT isoform genes. This hypothesis was, in part, verified by our experiments. The gene of the Cd-specific isoform, for example, can be induced by Cd. As a consequence, the protein encoded by this gene is synthesized at increasing rates upon Cd exposure. This induction is consistent with the gene structure of this isoform, which exhibits a series of metal-responsive elements (MREs) in the nucleotide sequence of the gene`s control region. Apart from MREs, the Cd-MT encoding gene contains a number of additional regulatory elements, which suggest that the gene may also be responsive to a range of stress factors such as heat, or physiological stgressors related to mineral homeostasis and reproduction activities. The second working hypothesis suggested that some of the metal-specific features of the two MT isoforms may be linked to the peptide primary structure. As one of the results of these studies we could show that the differential stoichiometry of the two MT isoforms for Cd and Cu is related to diverging peptide sequences. This may also hold for several other biochemical features of the two metal-specific isoforms. Our third hypothesis was that the metal-specific functions performed by the two MT isoforms may partially be determined and modified by cell-specific features and mechanisms. This hypothesis was verified for both isoforms. The Cd-specifid MT peptide, for example, is preferentially expressed in epithelial cells of the gut and the digestive gland, where the isoform apparently serves detoxification for those Cd fractions which enter the cells upon metal uptake via the digestive tract. In contrast to this, the Cu-specific MT isoform is exclusively found to be expressed in one single cell type called rhogocyte. The Cu-MT in the rhogocytes is not inducible, however, by any of the two metals (Cd or Cu), suggesting that its function might be related to physiological tasks rather than to metal detoxification. This latter function is merely linked to the depositikon of Cu within rhogocytes in granular vesicles, which are quickly formed in response to supraphysiological administration of Cu. The last hypothesis claimed that the function of the two metal-specific MT isoforms can only be completely understood in the context of other metal ligands and metal pools which may also interfere and compete for the metals with the two MT isoforms. In this concern, a Cu-transport protein could be identified the primary structure of which is actually being elucidated.