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1 Institute of Molecular Biology, University of Zurich, CH-8057, Zurich, Switzerland
2 Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, 08028-Barcelona, Spain
3 Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain
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Abstract |
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Introduction |
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The Drosophila genome contains four MT genes (MtnA, MtnB, MtnC and MtnD). The latter three are located in the same gene cluster and encode very similar peptides (67% amino acid identity) (Fig. 1) (Egli et al. 2003). In cultured cells, as in flies, MT gene duplications correlate with increased resistance to copper and cadmium, and cell lines selected for cadmium resistance always synthesize high levels of the MT proteins (Debec et al. 1985; Durnam & Palmiter 1987; Maroni et al. 1987; Czaja et al. 1991). Based on their metal binding properties, the Drosophila MTs MtnA and MtnB, also referred to as Mtn and Mto, respectively, were classified as copper-type thioneins (Valls et al. 2000; Domenech et al. 2003) with overall features closer to yeast CUP1 than to the well-known mammalian MTs, which are a paradigm of zinc-thioneins (Valls et al. 2001). At the gene level, the expression of MTs is transcriptionally regulated by metal-responsive transcription factor 1 (MTF-1), homolog to the mammalian MTF-1. Upon metal load, MTF-1 binds to the short DNA motifs termed metal-response elements (MREs) in the MT promoter, which are necessary and sufficient to mediate the transcriptional response to heavy metals (Stuart et al. 1984). Both in mammals and in Drosophila, basal and induced levels of transcription depend on MTF-1 activity, and consequently in both organisms a mutation of MTF-1 dramatically increases the sensitivity to heavy metals (Egli et al. 2003; Wang et al. 2004). Recently we reported the generation of a "MT family knockout" where all four Drosophila MT coding regions were inactivated by gene targeting (Egli et al. 2006). These flies are viable and fertile, but extremely sensitive to elevated concentrations of copper, cadmium and, to a lesser extent, zinc.
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Results |
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Null mutants for all four MTs, hereafter referred to as quadruple Mtn (qMtn) mutant flies, are viable and fertile, but highly sensitive to copper or cadmium load in the food. As shown in Fig. 2, survival of wild-type flies is only slightly affected by either 1 mM copper or 100 µM cadmium, qMtn flies do not survive when raised on this food. Interestingly, the sensitivity of qMtn flies to metals is similar to that observed in MTF-1 null mutants (Egli et al. 2003, 2006).
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Analysis of the Drosophila MT metal binding abilities and protein sequences
The zinc, cadmium and copper binding abilities of the four Drosophila MTs were determined by analyzing the metalated species recovered after recombinant synthesis (Table 1). Inductively coupled plasma–atomic emission spectroscopy (ICP-AES), flame photometric detection-gas chromatography (FPD-GC) and electrospray ionization mass spectroscopy (ESI-MS) were used to determine the metal-MT composition and stoichiometry, and additionally, their spectrometric (UV-vis) and chirooptical circular dichroism (CD) properties (Fig. 3A) yielded further information concerning the features of the metal sites of the different metal-MT clusters.
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Synthesis of the four MTs in Cd-enriched media yielded more substantial differences. MtnA and MtnB exhibit a similar cadmium binding capacity. In contrast, MtnC and MtnD yield preparations with a much lower cadmium content (nearly half that of MtnB, Table 1A). Surprisingly, the major species detected by ESI-MS are invariably Cd4- and Cd5- for the three MtnB-like forms, and therefore the diminished cadmium content of the MtnC and MtnD samples can possibly be attributed to the presence of undermetalated forms in the preparations. This may occur due to a partial instability of the metal–MtnC, MtnD complexes. This assumption is in accordance with Cd-MtnB exhibiting a higher chirality than Cd-MtnC and Cd-MtnD (Fig. 3A), which would indicate a less compact structure of the latter Cd-clusters. The GC-FPD data showed the presence of sulfide in all the Cd-preparations, and notably, in most of the cases the S2–-containing species could also be identified by ESI-MS analysis (Capdevila et al. 2005). CD absorbances in the 270–280-nm range in the four cases (Fig. 3A) are in concordance with sulfide anions acting as additional non-proteic Cd(II) ligands (Capdevila et al. 2005).
The four MTs yielded only homometallic copper-complexes when synthesized in copper-supplemented medium, thus all of them share a copper-thionein character (Valls et al. 2000; Domenech et al. 2003). Also, copper binding gives rise to important differences between MtnA and MtnB, and also MtnC and MtnD. First, as shown in Table 1A, the global copper content of the MtnB sample is clearly higher than that of MtnA, this again is in agreement with the higher number of cysteines in the MtnB peptide. On the contrary, MtnC and MtnD exhibit a reduced capacity to coordinate copper, yielding preparations with nearly half the metal content than those of MtnB (Table 1A). These differences are also noticeable in the respective CD spectra: those of Cu-MtnC and Cu-MtnD are quite similar, and less chiral than that of Cu-MtnB. The CD spectra of Cu-MtnA and Cu-MtnB (Fig. 3A) suggest similar but not equal numbers of Cu-SCys chromophores in both complexes, and the differences observed could be indicative of substantial differences in the Cu-clusters conformed by both main MT forms, MtnA and MtnB. These are also apparent when comparing their respective UV-visible spectra (Fig. 3B), which, in contrast to the almost perfect match of the Zn-MtnA and -MtnB complexes, show significant differences.
Additionally, to determine the composition of the MT complexes conformed under zinc and copper-binding competition, we recombinantly synthesized MtnA and MtnB in a mixed Zn,Cu-medium, at concentrations of 50 µM copper vs. 200 µM zinc, which approximately reproduced the internal Drosophila metal composition (Egli et al. 2006). Under these conditions, heterometallic Zn,Cu-MT preparations were obtained (Table 1B). For MtnA, ESI-MS identified a species containing four metal ions, with an average content of 2.6 Zn and 1.0 Cu. For MtnB, the two major species contained six and seven metals, with an average content of 3.3 Zn and 2.2 Cu. Most interestingly, the CD spectra of these samples (Fig. 3A) closely resemble those corresponding to intermediate stages of the in vitro Zn-to-Cu replacement reactions, previously reported for MtnA and MtnB. Specifically, they reproduce those obtained after the addition of 1–2 Cu(I) equivalents to Zn-MtnA (Valls et al. 2000) and 2–3 Cu(I) equivalents to Zn-MtnB (Domenech et al. 2003). These results are in accordance with the hypothesis that MTs are primarily synthesized in cells as Zn-complexes, and then the presence of other metal ions triggers the corresponding metal exchange. As the copper and zinc concentrations used in this experiment correspond to those observed in flies as a whole, it is likely that the basal, non-induced synthesis of MT in these organisms render heterometallic Zn,Cu-complexes.
Alignment and protein distance analyses (Fig. 1) readily highlighted the cluster formed by the so called MtnB-like forms (MtnB, MtnC and MtnD), with MtnD being the most similar to MtnB. Due to the overall sequence similarity, it might be assumed that an early duplication event of the primordial Drosophila MT generated the MtnA and MtnB forms, and that later on two additional duplications generated the MtnB-like subfamily, whereby MtnC and MtnD play only functionally minor roles, at least concerning metal detoxification.
Response of the four MT promoters to different metal induction
Basal and induced levels of MT expression depend on the transcription factor MTF-1 (Egli et al. 2003). The differential relevance of the individual MTs in the protection against copper and cadmium toxicity, deduced from the mutant survival experiments, prompted us to test the transcriptional induction rates of the corresponding genes in response to different metals. Promoter strength was examined using fluorescent reporter constructs (Fig. 4) as well as mRNA quantification assays (Fig. 5). Overall, the concentration of metals required to induce similar levels of MT differ vastly, e.g. about 100-fold between Cd and Zn. These differences often reflect differences in metal toxicity (Figs 2 and 5). Cadmium efficiently induces MT genes at lower concentrations than the other metals and also it is more toxic than either copper or especially zinc. Since the lethal dose equivalent to 50% fly survival (LD50) is at least tenfold lower for cadmium than for copper (Egli et al. 2006), a tenfold difference in Cu vs. Cd concentration was also used in the mRNA quantification assays (Figs 4 and 5). Interestingly, not all promoters respond equally to the same metal. The transcriptional induction ratio of copper/cadmium evaluated by fluorescent reporters is considerably higher for the MtnA promoter than for the MtnB-like forms (MtnB, MtnC and MtnD) (Fig. 4B). This difference is most obvious for MtnB and MtnD (threefold), whereas it is about twofold for MtnC. A similarly low ratio of copper/cadmium-mediated induction can also be seen with the synthetic promoters composed exclusively by four tandem copies of MREs, either derived from MtnB or from the copper importer Ctr1B. The endogenous Ctr1B gene is regulated by MTF-1, but unlike the MT genes, it is induced by copper scarcity and repressed by copper load (Zhou et al. 2003). However, when MREs from Ctr1B are isolated from their context and assembled in a mini-promoter, they respond to metal by activating gene expression, just as those from MTs (Selvaraj et al. 2005).
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When the levels of MT transcripts were evaluated (Fig. 5) results were clearly consistent with the previous ones. First, it is worth noting that in the absence of metal load, MT gene transcription is almost undetectable, with MtnA showing the major constitutive expression, perhaps related to some housekeeping function of this isoform. All assayed metals are good inducers of the four MT genes, but the response of MtnA to copper induction stands out among all. In addition, the induction capacity of cadmium vs. copper is strongest for the three MtnB-like forms, independently of the absolute levels of induction reached in each individual case.
Taken together, the results on the regulation of transcriptional induction by copper and cadmium correlate well with the relative importance of MtnA and MtnB to prevent copper and cadmium toxicity, respectively. MtnD, and to a lesser extent MtnC, show a similarly efficient induction by cadmium as MtnB. They probably play a minor role in the defense against cadmium, and/or protect against other adverse conditions not tested here (Fig. 2).
Phenotype rescue of copper deleterious effects in an ectopic expression system
To test if the lesser importance of MtnC and MtnD in metal detoxification was due to lower promoter strength, we directly compared the ability of all four MT coding sequences to prevent deleterious effects of copper. Copper toxicity in this system relies on ectopic over-expression of the copper importer Ctr1B in the Drosophila eye. This leads to tissue-specific copper accumulation, which results in a distorted development and a characteristic "rough eye" phenotype. Co-over-expression of MtnA or MtnB under the control of the eye-specific GMR-Gal4 promoter resulted in an almost complete rescue to the wild-type phenotype, while either MtnC or MtnD expression was unable to produce any improvement of the mutant eye appearance (Fig. 6). Since all MTs were expressed from the same promoter in this experiment, it is unlikely that dose effects are the basis of MtnC and MtnD's secondary role in metal detoxification in Drosophila.
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Discussion |
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Drosophila MT mutants are in many aspects similar to MT-null mice in that they are viable and fertile, but sensitive to heavy metal load (Masters et al. 1994). In Drosophila, even if all four MTs are involved in the defense against heavy metal stress, MtnA is the most important under copper intoxication, while MtnB is pivotal under cadmium load. Even though the MtnC and MtnD peptides share 67% amino acid identity with MtnB, their role in protection against these two metals is minor, at least as long as MtnA and MtnB are present. It remains to be seen if MtnC and MtnD play other particular roles not related to metal metabolism in Drosophila. In agreement with the phenotypes of the MT mutants, the metal-binding ability of the four MTs, as well as the transcriptional regulation of the individual MT genes, correlate well with one another.
It remains to be seen whether a preferential handling of copper and cadmium by specialized metallothionein isoforms is widespread among invertebrates. Other evidence for such a scenario comes from studies with the Roman snail (Helix pomatia) where separate metallothioneins in the mantle and in the midgut were found to be almost exclusively loaded with copper and cadmium, respectively (Dallinger et al., 1997). It would be interesting to determine for these snail MT isoforms, as was done in the present paper for an insect, the contribution to heavy metal resistance, the metal response of promoter constructs, and the metal affinities of purified recombinant proteins.
Unlike in mammals, Drosophila MTs play a minor role in the defense against zinc toxicity. This is supported by the poor ability of Drosophila MTs to bind zinc, as well as with the poor transcriptional induction observed in response to zinc load. This is consistent with an earlier classification of all four Drosophila MTs as Cu-thioneins, rather than as Zn-thioneins, according to their capacity to yield homometallic copper complexes. A similar situation is found in the yeast S. cerevisiae, whose genome encodes only copper-thioneins. In yeast, zinc fails to induce the synthesis of CUP1, the copper-MT paradigm, and zinc toxicity defense is exerted by metabolic pathways not related to MTs. Stoichiometric and spectroscopic analysis of metal complexes showed that MtnA and MtnB are more suitable for Cd(II) binding than MtnC and MtnD, due to the higher metal-content and chirality of the Cd-MtnA and Cd-MtnB complexes. Furthermore, MtnB has a slight advantage in cadmium binding over MtnA. MtnA and MtnB are also both suitable for Cu(I) binding, followed by the suboptimal MtnD and MtnC forms. These analytical data are completely supported by the results of in vivo experiments where all four different MTs were expressed under a heterologous promoter in the Drosophila eye, revealing a high ability of both MtnA and MtnB, but not of MtnC or MtnD, to protect against copper toxicity. In this system, the same promoter controls the expression of different MTs, indicating that the low metal-binding ability of MtnC and MtnD, rather than inefficient transcriptional induction, accounts for the minor role of MtnC and MtnD in the defense against metal toxicity.
Finally, the phenotype of MtnA and MtnB mutants also correlates well with the metal-inducibility of the respective gene promoters: MtnA and MtnB are preferentially induced by copper and cadmium, respectively. Since activation of all MT promoters depends on MTF-1, one might have expected a quantitatively equal induction of all MT genes by either cadmium or copper. The preferential response to cadmium of two synthetic minipromoters composed only of four tandem copies of MREs, derived from two unrelated genes, MtnB and Ctr1B, indicates that copper is, a priori, a less efficient inducer of the MTF-1/MRE response. However, a preferential response of MtnA to copper was observed, which may be due to an additional copper-specific factor binding to the MtnA promoter. Not only the MT promoters, but also other MTF-1-dependent promoters show a differential response to heavy metals: the copper importer Ctr1B is repressed by copper, but not affected by zinc, while the zinc transporter CG3994 shows induction by zinc and cadmium, but not by copper (Selvaraj et al. 2005; H. Yepiskoposyan and W.S., unpublished observation).
Overall, the exhaustive analysis of the four members of the Drosophila MT family reveals that MtnA and MtnB are of major importance in the heavy metal defense of Drosophila, with a leading role of MtnA and MtnB for copper and cadmium load, respectively. Individual activities of MTs are achieved by the corresponding specificity in induction and, probably some metal-cluster features that confer a more optimal character to the Cu-MtnA and Cd-MtnB complexes, respectively. This has been already reported for MtnB under high cadmium load (Domenech et al. 2003). In conclusion, the two founding members of the Drosophila MT family, MtnA and MtnB, have acquired some degree of metal-response specificity both at the gene level (specificity of transcriptional induction) and at the protein level (specificity of protein function).
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Experimental procedures |
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MT and MTF-1 Drosophila mutant strains were used as described in (Egli et al. 2003, 2006). The single mutant alleles, MtnA
ATG, MtnBATG, MtnCATG and MtnDATG had their ATG start codon removed by gene targeting, which led to the production of non-translatable mRNAs. These mutants can be assumed to be MT-null mutants, and are thereafter termed MtnA–, MtnB–, MtnC– and MtnD– in this work. MtnD* is a truncation allele with a premature stop codon that arose spontaneously in the OregonR stock (Egli et al. 2003). The construction of a fly strain mutant for several or all four MT genes was achieved using multiple rounds of gene targeting and recombination, as explained in detail in Egli et al. (2006).
Toxicity experiments
Flies were allowed to deposit eggs on food and eclosing adults were counted. From these eggs, approximately 50–70% develop to adulthood on normal food. Survival on metal-supplemented food was normalized to survival of the corresponding strain on normal food. Experiments were repeated several times and at different concentrations of the same metal. Fly food was composed of 55 g corn, 100 g yeast, 75 g sugar (glucose), 8 g Agar, 15 mL nipalgin and 10 g wheat per litre.
Fluorescent protein reporters
The reporter construct for MtnC was generated by inserting the coding region of EYFP or of dsRed into the NotI site of the construct used for gene targeting (Egli et al. 2006). The generation of the MtnA-EYFP and MtnB-EYFP reporter transgenic flies and of the MtnDdsRed knock-in allele were reported previously (Balamurugan et al. 2004; Egli et al. 2006). The constructs containing the synthetic minipromoter 4xMRE-EYFP, with MRE motifs derived either from the MtnB or the copper importer Ctr1B promoters, are described in Egli et al. (2006). Transgenics were produced by P-element mediated transgenesis. For analysis of EYFP expression by microscopy, flies were allowed to deposit eggs in the food which were allowed to develop until third instar larvae.
Metallothionein rescue experiments
MTF-1, Ctr1B and the MT genes were cloned into the widely used vector pUAST, containing binding sites for Gal4. The Gal4 driver line GMR-Gal4 was used for the eye-specific expression of Ctr1B, with or without co-expression of MTF-1 or any of the four MTs. At least two different transgene insertions were tested.
Imaging and microscopy
Images were taken with a Leica MZ FLIII fluorescence stereomicroscope and a Nikon COOLPIX950 digital camera for whole larvae.
Quantification of metallothionein transcripts
Larvae were either continuously raised on the indicated type of food, or transferred for 6 h to normal food or to metal-supplemented food. Only third instar feeding larvae were used for analysis. Total RNA was extracted using the TRIzol reagent (Life Technologies). Nuclease S1 mapping of transcripts with 50 µg of total RNA was performed as previously described (Weaver & Weissmann 1979). The gels were developed using PhosphorImager (Molecular Dynamics) and bands were quantified. The signal from the endogenous actin5c gene was used for normalization of metallothionein transcript levels. Sequence of the probe for EYFP (or EGFP): 5'-GGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTGGCCGccagaa-3'.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay (EMSA) was performed as previously described (Radtke et al. 1993; Zhang et al. 2001; Selvaraj et al. 2005). A 200-fold excess of cold MREd was used for competition analysis. The MRE oligonucleotides used for EMSA are as follows: consensus MRE-s as described. MREe (MtnA): 5'- AAAGCTTCTGCACACGTCTCCACTC-3' and 5'-TCGAGAGTGGAGACGTGTGCAGAAGCTTTAGCT-3' MREa (MtnB): 5'-TGCAATTTTGCACTCGTTCGAGTTC-3' and 5'-TCGAGAACTCGAACGAGTGCAAAATTGCAAGCT-3'.
Recombinant MT synthesis and characterization of their metal-binding abilities
MtnC and MtnD cDNAs were cloned in the expression vector pGEX, and expressed in E. coli cells growing in metal-supplemented media (300 µM ZnCl2, 300 µM CdCl2 or 500 µM CuSO4, final concentrations) to produce the corresponding recombinant metal-protein complexes, as previously reported for MtnA (Valls et al. 2000) and MtnB (Domenech et al. 2003). In a parallel experiment and in order to observe MtnA and MtnB competition for Zn and Cu binding, cultures were supplemented with a mixture of 200 µM ZnCl2 and 50 µM CuSO4, concentrations similar to the total zinc or copper concentration in the fly. The four Cd-MT species were obtained as described in Tio et al. (2004). Analytical and chemical characterization of the in vivo conformed metal-MT complexes was carried out by acid inductively coupled plasma–atomic emission spectroscopy (acid ICP-AES) (Capdevila et al. 2005) electrospray ionization mass spectroscopy (ESI-MS), UV-visible electronic absorption spectroscopy and circular dichroism (CD) spectropolarimetry, as previously done for the Zn- and Cu-complexes of MtnA (Valls et al. 2000) and -MtnB (Domenech et al. 2003). The presence of S2– ligands was evaluated as described in (Capdevila et al. 2005).
Protein sequence analyses
Protein sequences were aligned by ClustalW, v1.75, using Blosum62 as distance matrix (Thompson et al. 1994). The ClustalW alignments were the input for calculating protein distances through Protdist, which uses the Dayhoff-Pam matrix distance. The corresponding protein distance trees were constructed using Fitch, which uses the Fitch-Margoliash tree-building algorithm (Fitch & Margoliash 1967). Protdist and Fitch are included in the Phylip software package (Felsenstein 1989).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: satrian{at}ub.edu
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References |
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indicates the chiral intensity of the sample measured as the differential absorption of the left and right circularly polarized light. (B) UV-vis spectra of the Zn- and Cu-complexes of the two main MT forms, MtnA and MtnB.
