Michael Niederwanger

Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene.

Structural Adaptation of a Protein to Increased Metal Stress: NMR Structure of a Marine Snail Metallothionein with an Additional Domain

In this study, we present an NMR structure of the metallothionein (MT) from the snail Littorina littorea (LlMT) in complex with Cd2+ . LlMT is capable of binding 9 Zn2+ or 9 Cd2+ ions. Sequence alignments with other snail MTs revealed that the protein is likely composed of three domains. The study revealed that the protein is divided into three individual domains, each of which folds into a single well-defined three-metal cluster. The central α2 and C-terminal β domains are positioned with a unique relative orientation. Two variants with longer and shorter linkers were investigated, which revealed that specific interdomain contacts only occurred with the wild-type linker. Moreover, a domain-swap mutant in which the highly similar α1 and α2 domains were exchanged was structurally almost identical. It is suggested that the expression of a three-domain MT confers an evolutionary advantage on Littorina littorea in terms of coping with Cd2+ stress and adverse environmental conditions.

Physiological, Diurnal and Stress-Related Variability of Cadmium-Metallothionein Gene Expression in Land Snails

The terrestrial Roman snail Helix pomatia has successfully adapted to strongly fluctuating conditions in its natural soil habitat. Part of the snail’s stress defense strategy is its ability to express Metallothioneins (MTs). These are multifunctional, cysteine-rich proteins that bind and inactivate transition metal ions (Cd2+, Zn2+, Cu+) with high affinity. In Helix pomatia a Cadmium (Cd)-selective, inducible Metallothionein Isoform (CdMT) is mainly involved in detoxification of this harmful metal. In addition, the snail CdMT has been shown to also respond to certain physiological stressors. The aim of the present study was to investigate the physiological and diurnal variability of CdMT gene expression in snails exposed to Cd and non-metallic stressors such as desiccation and oxygen depletion. CdMT gene expression was upregulated by Cd exposure and desiccation, whereas no significant impact on the expression of CdMT was measured due to oxygen depletion. Overall, Cd was clearly more effective as an inducer of the CdMT gene expression compared to the applied non-metallic stressors. In unexposed snails, diurnal rhythmicity of CdMT gene expression was observed with higher mRNA concentrations at night compared to daytime. This rhythmicity was severely disrupted in Cd-exposed snails which exhibited highest CdMT gene transcription rates in the morning. Apart from diurnal rhythmicity, feeding activity also had a strong impact on CdMT gene expression. Although underlying mechanisms are not completely understood, it is clear that factors increasing MT expression variability have to be considered when using MT mRNA quantification as a biomarker for environmental stressors.

Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character

After the resolution of the 3D structure of the Cd9-aggregate of the Littorina littorea metallothionein (MT), we report here a detailed analysis of the metal binding capabilities of the wild type MT, LlwtMT, and of two truncated mutants lacking either the N-terminal domain, Lltr2MT, or both the N-terminal domain, plus four extra flanking residues (SSVF), Lltr1MT. The recombinant synthesis and in vitro studies of these three proteins revealed that LlwtMT forms unique M9-LlwtMT complexes with Zn(II) and Cd(II), while yielding a complex mixture of heteronuclear Zn,Cu-LlwtMT species with Cu(I). As expected, the truncated mutants gave rise to unique M6-LltrMT complexes and Zn,Cu-LltrMT mixtures of lower stoichiometry with respect to LlwtMT, with the SSVF fragment having an influence on their metal binding performance. Our results also revealed a major specificity, and therefore a better metal-coordinating performance of the three proteins for Cd(II) than for Zn(II), although the analysis of the Zn(II)/Cd(II) displacement reaction clearly demonstrates a lack of any type of cooperativity in Cd(II) binding. Contrarily, the analysis of their Cu(I) binding abilities revealed that every LlMT domain is prone to build Cu4-aggregates, the whole MT working by modules analogously to, as previously described, certain fungal MTs, like those of C. neoformans and T. mesenterica. It is concluded that the Littorina littorea MT is a Cd-specific protein that (beyond its extended binding capacity through an additional Cd-binding domain) confers to Littorina littorea a particular adaptive advantage in its changeable marine habitat.

Successive Onset of Molecular, Cellular and Tissue-Specific Responses in Midgut Gland of Littorina littorea Exposed to Sub-Lethal Cadmium Concentrations

Cadmium (Cd) is one of the most harmful metals, being toxic to most animal species, including marine invertebrates. Among marine gastropods, the periwinkle (Littorina littorea) in particular can accumulate high amounts of Cd in its midgut gland. In this organ, the metal can elicit extensive cytological and tissue-specific alterations that may reach, depending on the intensity of Cd exposure, from reversible lesions to pathological cellular disruptions. At the same time, Littorina littorea expresses a Cd-specific metallothionein (MT) that, due to its molecular features, expectedly exerts a protective function against the adverse intracellular effects of this metal. The aim of the present study was, therefore, to assess the time course of MT induction in the periwinkle’s midgut gland on the one hand, and cellular and tissue-specific alterations in the digestive organ complex (midgut gland and digestive tract) on the other, upon exposure to sub-lethal Cd concentrations (0.25 and 1 mg Cd/L) over 21 days. Depending on the Cd concentrations applied, the beginning of alterations of the assessed parameters followed distinct concentration-dependent and time-dependent patterns, where the timeframe for the onset of the different response reactions became narrower at higher Cd concentrations compared to lower exposure concentrations.

Biomphalaria glabrata metallothionein: lacking metal specificity of the protein and missing gene upregulation suggest metal sequestration by exchange instead of through selective binding

The wild-type metallothionein (MT) of the freshwater snail Biomphalaria glabrata and a natural allelic mutant of it in which a lysine residue was replaced by an asparagine residue, were recombinantly expressed and analyzed for their metal-binding features with respect to Cd2+, Zn2+ and Cu+, applying spectroscopic and mass-spectrometric methods. In addition, the upregulation of the Biomphalaria glabrata MT gene was assessed by quantitative real-time detection PCR. The two recombinant proteins revealed to be very similar in most of their metal binding features. They lacked a clear metal-binding preference for any of the three metal ions assayed—which, to this degree, is clearly unprecedented in the world of Gastropoda MTs. There were, however, slight differences in copper-binding abilities between the two allelic variants. Overall, the missing metal specificity of the two recombinant MTs goes hand in hand with lacking upregulation of the respective MT gene. This suggests that in vivo, the Biomphalaria glabrata MT may be more important for metal replacement reactions through a constitutively abundant form, rather than for metal sequestration by high binding specificity. There are indications that the MT of Biomphalaria glabrata may share its unspecific features with MTs from other freshwater snails of the Hygrophila family.

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata, one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails.

Differential response to Cadmium exposure by expression of a two and a three-domain metallothionein isoform in the land winkle Pomatias elegans: Valuating the marine heritage of a land snail

Through evolution, marine snails have adapted several times independently to terrestrial life. A prime example for such transitions is the adaptation to terrestrial conditions in members of the gastropod clade of Littorinoidea (Caenogastropoda). Some species of this lineage like the periwinkle (Littorina littorea), live in intertidal habitats, where they are intermittently exposed to semi-terrestrial conditions. Pomatias elegans is a close relative of Littorina littorea that has successfully colonized terrestrial habitats. Evolutionary transitions from marine to terrestrial conditions have often been fostered in marine ancestors by acquisition of physiological pre-adaptations to terrestrial life. Such pre-adaptations are based, among others, on the optimization of a wide repertoire of stress resistance mechanisms, such as the expression of metal inactivating metallothioneins (MTs). The objective of our study was to explore the Cd handling strategy in the terrestrial snail Pomatias elegans in comparison to that observed previously in Littorina littorea. After Cd exposure, the metal is accumulated mainly in the midgut gland of Pomatias elegans, in a similar way as in its marine relative. Upon Cd exposure, Pomatias elegans expresses Cd-specific MTs, as also described from Littorina littorea. In contrast to the latter species, however, the detoxification of Cd in Pomatias elegans is mediated by two different MT isoforms, one two-domain and one three-domain MT. Although the MT proteins of both species are homologous and clearly originate from one common ancestor, the three-domain MT isoform of Pomatias elegans has evolved independently from the three-domain MT of its marine counterpart, probably by addition of a third domain to the pre-existing two-domain MT. Obviously, the occurrence of homologous MT structures in both species is a hereditary character, whereas the differentiation into two distinct MT isoforms with different upregulation capacities in Pomatias elegans is an adaptive feature that probably emerged upon transition to life on land.

Metal binding functions of metallothioneins in the slug Arion vulgaris differ from metal-specific isoforms of terrestrial snails

Arion vulgaris is a land-living European slug belonging to the gastropod clade of Stylommatophora. The species is known as an efficient pest organism in vegetable gardening and horticulture, which may in part be the consequence of its genetically based innate immunity, along with its high ability to withstand toxic metal stress by intracellular detoxification. Like many species of terrestrial snails, slugs possess a distinct capacity for Cd accumulation in their midgut gland, where the metal is stored and inactivated, conferring to these animals an increased metal tolerance. Although midgut gland Cd fractions in slugs have been shown to be variably allocated between different metal-binding protein pools, depending on the level of environmental metal contamination, a true metallothionein (MT) was so far never characterized from slugs. Instead, the Cd binding proteins identified so far were described as Metallothionein-like proteins (MTLPs). In the present study, the slug A. vulgaris was used as a model organism, in order to verify the presence of true MTs in experimentally metal-exposed slugs. We wanted to find out if these suggested slug MTs have similar metal binding properties and metal-selective features like those previously reported from helicid snails. To this aim, two MT isoform genes (AvMT1 and AvMT2) were characterized from midgut gland extracts and localized in the cells of this tissue. The AvMT1 and AvMT2 proteins were purified and partially sequenced, and their metal-binding features analysed after recombinant expression. Eventually, we wanted to understand if and by how much the metal binding features of the two MT isoforms of A. vulgaris may be related, owing to their reciprocal amino acid sequence similarities, to the binding properties of metal-specific MTs from terrestrial snails.