1. Introduction
One of the most intriguing questions in the field of metal homeostasis concerns how biological systems distinguish and discriminate between different metal ions. This is important for understanding not only the metabolic pathways of essential metal ions, but also mechanisms of tolerance and detoxification including those for xenobiotic ions. As far as essential metal ions are concerned, it has been recognised that the cytosolic concentrations of essential metal ions are, in healthy conditions, regulated according to their relative position within the Irving-Williams series, and the proteins involved in their homeostasis typically have metal affinities to match these concentrations [
1]. This ensures, for example, that Cu(I), the most competitive essential metal ion, is kept out of the binding sites of all other metal ions—even when, and this is frequently the case, the affinity of the respective protein is higher for copper than for the “correct” metal ion. In the case of a non-essential, toxic metal ion such as cadmium, it may be inferred that at least one protein with sufficiently high affinity is required, with the added proviso that this protein, once expressed, should ideally not significantly interfere with the metabolism of other metal ions. This becomes particularly important when the toxic and essential metal ions have relatively similar coordination chemistry, such as in the case of essential Zn(II) and toxic Cd(II). The current study will highlight that certain metallothioneins (MTs), small proteins with an extraordinarily high proportion of cysteine thiols that endows them with high affinity towards both Zn(II) and Cd(II), may display distinctly different behaviour towards these two closely related ions.
MTs were one of the first protein families to be associated with metal metabolism in animals [
2,
3,
4]. Initially discovered in horse kidney cortex as major cadmium-binding proteins [
5], their physiological functions in mammals are now thought by most workers in the field to be predominantly if not exclusively concerned with the metabolism of the essential zinc and copper metal ions. In addition, because of their high thiol content, they can also function as antioxidants, and may also link cellular redox status to zinc dynamics [
6].
Unusually, and in contrast to most other protein families, MTs are a polyphyletic group of proteins held together not by significant similarities in protein sequence, but rather by a number of descriptors that refer to their overall composition (high proportion of sulfur and metals), and biophysical characteristics such as peculiar spectroscopic features indicative of metal–sulfur clusters. This fact should be reason enough to refrain from extrapolating the biological functions, structural features, or chemical reactivity of an MT from one phylum to another, but sadly, this is a frequent occurrence in the literature.
It has been pointed out that even though mammalian MTs undoubtedly bind cadmium
in vivo and, hence, play a role in cadmium metabolism [
7], it is unlikely that this action is a true, evolutionarily constrained function [
8,
9]. This may however be distinctly different for organisms in close contact with soils,
i.e., plants [
10] and terrestrial invertebrates [
11,
12,
13]. Typically, due to their relatively similar chemistries, toxic cadmium occurs in soils at concentrations that are only two to three orders of magnitude smaller than those of essential zinc. Moreover, in topsoils treated with rock phosphate fertilisers, cadmium levels can be significantly higher and reach up to 14 ppm [
14]. With 2015 being the international year of soil [
15], it seems appropriate to devote some attention to a group of very important soil organisms supremely adept at coping with cadmium: the earthworm.
Earthworms, master “soil engineers”, directly ingest and process soil. This does not only mean that they play a major role in the dynamics of nutrients and essential elements present in soil [
16], but also that they are exposed to any compound present, both through their digestive tract as well as through their skin. Earthworms are therefore also used for biomonitoring purposes in ecotoxicology, and have also been dubbed “soil sentinels” [
17].
At least some species, for example the common red earthworm
Lumbricus rubellus, can survive in the presence of 600 µg cadmium per g dry weight of soil [
18]. Intriguingly,
L. rubellus also specifically bio-accumulates cadmium to a staggering ratio of up to 1 mg per gram dry body weight, whilst lead, zinc and copper are not bio-accumulated [
18]. This difference clearly indicates that there must be distinct metabolic pathways for the chemically closely related Zn(II) and Cd(II). A similar conclusion has been drawn for other soil organisms; for example, the nematode
Caenorhabditis elegans uses a system that includes phytochelatins, cystathionine, and its two metallothioneins MTL-1 and MTL-2 to discriminate between zinc and cadmium [
19,
20], with subsequent distinct pathways for utilisation and detoxification [
21]. Given the differences in zinc and cadmium accumulation, it is conceivable that similar mechanisms may exist in earthworms, with a potential function for MTs in the discrimination between essential zinc and toxic cadmium.
Many eukaryotic species, including invertebrates, express several different MT homologues (In MT literature, it is common to call these different forms “isoforms”, although strictly speaking this is a term that should refer to different forms derived from a single gene. However, in most if not all cases, different forms of MTs in one particular species are derived from different genes; therefore we prefer to refer to these as “homologues” provided that an evolutionary relationship between them is likely.), sometimes, but not always in a tissue-specific manner. Evidence has been accumulating that different homologues in the same species can exhibit different metal selectivities; prime examples are the Cu- and Cd-MTs from snails [
22,
23,
24] as well as the
C. elegans MTs mentioned above [
19,
20]. MTs are being studied in several annelids, mainly in terms of gene expression [
25,
26,
27,
28]. Perhaps the best-studied system in terms of MTs is
L. rubellus; for this species, at least three MT genes are known [
29]. The corresponding wMT-1 and wMT-2 proteins have been isolated from adult earthworms [
18], whilst the protein sequence of a third homologue, wMT-3, has been derived from an EST library generated from developing cocoons [
29] (
Figure 1).
Figure 1.
Sequence alignment of selected annelid MTs, showing conservation of two blocks of Cys-rich regions with 11–12 and 8–9 Cys residues (highlighted by black boxes). All three sequences from L. rubellus are shown; the numbering refers to wMT-2. Non-conserved Cys residues are highlighted in grey.
Figure 1.
Sequence alignment of selected annelid MTs, showing conservation of two blocks of Cys-rich regions with 11–12 and 8–9 Cys residues (highlighted by black boxes). All three sequences from L. rubellus are shown; the numbering refers to wMT-2. Non-conserved Cys residues are highlighted in grey.
The protein sequences of wMT-1 and wMT-2 are 74.7% identical and 91.1% similar, whilst that of wMT-3 is only 56% identical and 67% similar to wMT-1 or wMT-2. The expression patterns of these three homologues also differ considerably; the
wMT-3 gene has been suggested to be highly expressed during embryonic development [
29], whereas
wMT-1 and
wMT-2 are both responsive to metal exposure, but to different extents.
wMT-2 is the homologue with the most pronounced responsiveness to cadmium—its expression may be upregulated several hundred- to thousand-fold in response to high cadmium levels in soil [
30]. The non-transience of this increased expression suggested that this constitutes the primary response to cadmium [
31]. Accordingly,
wMT-2 was also one of the most upregulated genes in response to chronic cadmium exposure, as identified in an extensive transcriptomic study [
32]. The mechanism for metallo-regulation of
wMT-2 genes—and indeed many other invertebrate MT genes—has puzzled researchers for some while, because even though three recognisable metal-response elements (MREs) are present in the pertinent upstream regions in all three identified
wMT-2 loci [
29], a protein corresponding to MTF-1, the transcription factor that recognises MREs in vertebrates and
Drosophila melanogaster [
33], could not be identified. A recent study involving EMSA and DNAse I footprinting revealed that cytosolic, but not nuclear extracts from
L. rubellus cells contain proteins capable of binding to the
wMT-2 promoter region in a zinc-dependent manner. The DNA footprinting experiments identified cAMP-responsive elements (CRE) as putative candidates for MT gene regulation in invertebrates [
34].
The
wMT-2 gene is not only supremely responsive to cadmium exposure, its product was also the major Cd-binding protein isolated from earthworms from a contaminated site [
18]. The highest levels of wMT-2 protein were found in Cd-exposed worms in the thyphlosole and gut epithelium (both alimentary surfaces), chloragogenous tissues (these have been likened to vertebrate livers), coelomocytes (a type of invertebrate immune cells) and nephridia (analogues of vertebrate kidneys) [
35]. Thus, even though wMT-2, like any other MT, is also capable of binding other metal ions such as Zn(II) and Cu(I) [
36], and even though gene expression may also be induced by metals other than cadmium [
37], there are multiple lines of evidence, that as far as biological function is concerned, wMT-2 is a “cadmium-MT”. We have used a combination of mass spectrometry and NMR spectroscopy to study whether and how biological function may be reflected in the biophysical properties, in particular metal affinities and protein folding, of this invertebrate MT.
3. Experimental Section
3.1. Materials
Unless stated otherwise, all chemicals and reagents were obtained from Fisher Scientific, Loughborough, UK. All solutions were prepared with purified water (MilliQ, Millipore, Nottingham, UK) and other reagents used were of analytical grade or better.
3.2. Protein Expression and Purification
Lumbricus rubellus MT-2 was recombinantly expressed in
E. coli Rosetta 2(DE3)pLysS (Novagen, Nottingham, UK) cells as a fusion protein with an N-terminal S-peptide tag, using a pET29a-derived plasmid, with the
mt2 gene cloned between
SalI and
NcoI restriction sites [
31]. Cells were grown in standard Luria-Bertani medium at 37 °C (180 rpm), and Kanamycin (50 µg/mL) and Chloramphenicol (34 µg/mL) were used as selective antibiotics. Once an optical density at 600 nm of 0.6–0.8 was reached, protein over-expression was induced by 1 mM IPTG (Isopropyl-
d-thiogalactopyranoside), and 0.5 mM ZnSO
4 or 0.2 mM CdSO
4 (both Sigma Aldrich, Dorset, UK) were added at the same time. Cells were harvested after 5–6 h of induction by centrifugation (5000×
g, 10 min, 4 °C). Cell pellets were resuspended in 4–8 mL sonication buffer (50 mM Tris–HCl; 0.1 M KCl; 3 mM β-mercaptoethanol, 1% TWEEN-20, pH 8.5) per g wet cell weight. To prevent metal loss during sonication, 1 mM ZnSO
4 or CdCl
2 was added. The lysate was separated from cell debris by centrifugation for 30 min at 30,000×
g, 4 °C. S-tagged wMT-2 was separated from the bulk of other contents of the cell lysate by gel filtration (GE Healthcare HiLoad 16/60, Superdex G75). After concentration to 1 mg/mL, the S-peptide tag was cleaved using bovine thrombin (50 units per mg protein; Sigma Aldrich, Dorset, UK) following manufacturer’s protocols, and the mixture was subjected to a second round of gel filtration. Protein concentrations were determined via measuring sulfur concentration by Inductively-Coupled Plasma Optical Emission spectroscopy (ICP-OES, Perkin Elmer, Seer Green, UK), or by determining thiol content via Ellman’s method [
60], after removal of bound metal ions by incubation with EDTA (2,2′,2′′,2′′′-(Ethane-1,2-diyldinitrilo)tetraacetic acid, Sigma-Aldrich, Dorset, UK).
3.3. Determination of Protein Concentration and Metal-Protein Stoichiometries by ICP-OES
Samples of approximately 1 ppm [S] were prepared in 0.1 M ultrapure HNO3 (prepared in-house by sub-boiling point distillation; DuoPUR, Milestone S.R.L, Sorisole, Italy). Each sample was simultaneously assessed for Zn, Cd, Cu and S against mixed-element standards of concentrations between 0 and 2 ppm, which were prepared gravimetrically from high grade commercial stocks (1000 ppm, Fluka, Buchs, Switzerland). ICP-OES measurements were performed on an Optima 5300 DV instrument (Perkin Elmer, Seer Green, UK).
3.4. Mass Spectrometry
Samples were concentrated to 30–50 µM protein concentration using either Amicon Ultra centrifugal filtration units, with 3 kDa molecular weight cut-off (Fisher Scientific, Loughborough, UK), or Vivaspin centrifugal concentrators, with 5 kDa molecular weight cut-off (Sigma-Aldrich, Dorset, UK), using 20 mM ammonium bicarbonate (pH 8.35) buffer. Prior to mass spectrometric analysis, 10% (v/v) HPLC grade methanol was added to the samples, to obtain mass spectra of metallated species. The pH was gradually deceased using either acetic acid or formic acid. All samples were analysed on an HCTultra ion-trap mass spectrometer (Bruker Daltonics, Coventry, UK) equipped with an ESI source. Samples were directly infused using a syringe pump at 240 µL/h. Typically, data were acquired for 0.5–1.0 min in the positive mode over an m/z range of 500–3000. Resulting mass spectra were averaged, smoothed using the Savitzky-Golay algorithm, and where required deconvoluted onto a true mass scale using the Bruker Data Analysis Suite (Bruker Daltonics, Coventry, UK).
3.5. Affinity Measurements by 19F NMR Spectroscopy
Zn- and Cd-loaded forms of recombinant wMT-2 were buffer-exchanged into 10 mM Tris–HCl and 10% D
2O, pH 8.1, using Amicon ultrafiltration devices (3 kDa molecular weight cut-off). Samples, approximately 450 µM in metal ion concentration, were incubated with 5-fluoro-1,2-bis(2-aminophenoxy)-ethane-
N,
N,
N′,
N′-tetraacetic acid (5F-BAPTA; 3 mM; Molecular Probes™, Invitrogen) overnight at 25 °C. Direct observe proton-decoupled 1D
19F NMR spectroscopy was carried out on a DRX400 spectrometer (Bruker) fitted with a QNP probe-head operating at 375.91 MHz for
19F. Datasets of 12,288 scans were acquired with a spectral width of 200 ppm, an acquisition time of 0.87 s and relaxation delay of 1.0 s. All spectra were acquired at 25 °C. FIDs were apodized with squared sine-bell functions, Fourier transformed with 64k data points and baseline corrected with TOPSPIN v. 2.1 software (Bruker Biospin, Coventry, UK). The apparent stability constants for metal-MT complexes were calculated using the method published by Hasler
et al. [
56]. The value for
KCd(BAPTA) at 30 °C and
I = 138 mM was corrected for temperature (25 °C) and ionic strength (
I = 4 mM) used in our experiments to give a log
K value of 11.75. The value for
KZn(BAPTA) was also recalculated and a log
K value of 9.91 was used. Metal and protein concentrations used in the calculations were determined accurately by ICP-OES.
3.6. 1H NMR Spectroscopy
Samples for 1H NMR spectroscopy were prepared in 20 mM NH4HCO3 buffer, pH 6.9, 10% D2O. All data were acquired for 0.5–1.0 mM samples on an AV II 700 (Bruker Biospin, Coventry, UK) equipped with a TCI cryoprobe.
Spectral conditions for TOCSY and NOESY experiments were: 25 °C; 48 scans; 4096 data points in F2 and 512 increments in F1, 90° pulse width ≈ 8.00 µs; spectral width 16.0 ppm in both F1 and F2. A mixing time of 60 ms was used for the TOCSY experiments; for NOESY experiments, several experiments with different mixing times were acquired (60–120 ms). Data were processed with TOPSPIN v. 2.1 (Bruker Biospin, Coventry, UK). Raw data were apodized using squared sine-bell functions and Fourier transformed with 2048 × 2048 data points, and visualised using SPARKY v.3.114 software [
61].
4. Conclusions
We have studied the composition, metal affinity, and folding behaviour of recombinant
L. rubellus wMT-2 in its Zn(II)- and Cd(II)-bound forms. As previously found for the S-tagged protein [
39], wMT-2 binds seven Cd(II) ions optimally, with a high likelihood for the formation of two separate domains—an N-terminal domain harbouring a Cd(II)
4Cys
11 cluster and a C-terminal domain with a Cd(II)
3Cys
9 cluster. Two-domain MTs are common in animals: vertebrate, crustacean, sea urchin, and mussel MTs all have two domains, despite the absence of significant sequence similarity between these MTs [
3]. At present, there is no clear opinion as to why such a two-domain arrangement may be advantageous—especially not in the case of MTs with a predominant role in detoxification. In MTs dealing with essential Zn(II) or Cu(I), two domains might serve in supporting interactions with different partners, and in some cases, the two domains might even handle different metal ions—a prime example is mammalian brain-specific MT-3 [
62,
63].
We have found significant differences in protein yields, metal stoichiometry, and folding behaviour between Zn-wMT-2 and Cd-wMT-2, which are congruent with the major biological function of wMT-2, which entails protection against Cd(II) toxicity [
29]. The concept that MTs have evolved to optimally bind their cognate metal ion is a still emerging and hence debated concept. It is clear that metal selectivity and discrimination cannot be based on overall affinity [
1], as all true MTs studied thus far show a similar order of binding affinity as small-molecule thiolate chelators,
i.e., Cu(I) > Cd(II) > Zn(II). This general trend, together with the knowledge that the protein backbones of MTs tend to be very flexible, may tempt one to regard MTs as not much more than larger versions of such small-molecule chelators, and to dismiss the relevance of protein folding. However, several recent studies on MTs from plants [
44,
55,
59,
64], snails [
22,
23,
24,
65,
66,
67], and nematodes [
19,
20,
68] have demonstrated a correlation between the biologically “correct” metal ion and the “foldedness” of the respective MT. We consider that a correlation between cognate metal and protein folding also makes sense in the case of wMT-2, both in terms of fundamental (bio-)physical principles, as well as biological function: (i) even in cases where Zn(II) and Cd(II) are bound “isostructurally”, the difference in ionic radii (0.74
vs. 0.92 Å), and hence bond lengths, will require small but potentially significant adaptations in side-chain conformation and backbone fold, and therefore, differences in the energetics for fold stability depending on which metal is bound can be expected; (ii) In recognition of the fact that the “foldedness” of proteins impacts on their persistence
in vivo [
69], we expect that MTs synthesised in the presence of the “wrong” metal ion (in the present case Zn(II)) will have shorter
in vivo lifetimes. This may help to remobilise the mis-incorporated metal ion, and prevent it from going down the wrong pathway.
These ideas are principally compatible with the finding that cadmium, but not other metal ions, are bio-accumulated in sulfur-rich vesicles by L. rubellus. As a next step, it would be desirable to test their validity by carrying out further metalloproteomic experiments in earthworms.