Tài liệu Báo cáo khoa học: The two Caenorhabditis elegans metallothioneins (CeMT-1 and CeMT-2) discriminate between essential zinc and toxic cadmium docx

The two Caenorhabditis elegans metallothioneins (CeMT-1
and CeMT-2) discriminate between essential zinc and toxic
cadmium
Sukaina Zeitoun-Ghandour
1
, John M. Charnock
2
, Mark E. Hodson
3
, Oksana I. Leszczyszyn
4
,
Claudia A. Blindauer
4
and Stephen R. Stu
¨
rzenbaum
1
1 School of Biomedical & Health Sciences, King’s College London, UK
2 School of Earth, Atmospheric and Environmental Sciences, University of Manchester, UK
3 Department of Soil Science, University of Reading, UK
4 Department of Chemistry, University of Warwick, Coventry, UK
Introduction
Metal pollution in the environment is a matter of con-
cern. Many studies have focused on the use of terres-
trial biomonitors to determine how organisms, in
particular invertebrates, control and tolerate increased
exposure to elevated levels of metals [1–7]. Responses
may include avoidance, excretion, chelation or
Keywords
affinity; C. elegans; cadmium; metal

speciation; metallothionein; zinc
Correspondence
S. Stu
¨
rzenbaum, School of Biomedical &
Health Sciences, Pharmaceutical Science
Division, King’s College London, 150
Stamford Street, London SE1 9NH, UK
Fax: +44 2078484500
Tel.: +44 2078484406
E-mail:
(Received 22 January 2010, revised 23
March 2010, accepted 30 March 2010)
doi:10.1111/j.1742-4658.2010.07667.x
The nematode Caenorhabditis elegans expresses two metallothioneins (MTs),
CeMT-1 and CeMT-2, that are believed to be key players in the protection
against metal toxicity. In this study, both isoforms were expressed in vitro in
the presence of either Zn(II) or Cd(II). Metal binding stoichiometries and
affinities were determined by ESI-MS and NMR, respectively. Both isoforms
had equal zinc binding ability, but differed in their cadmium binding
behaviour, with higher affinity found for CeMT-2. In addition, wild-type
C. elegans, single MT knockouts and a double MT knockout allele were
exposed to zinc (340 lm) or cadmium (25 lm) to investigate effects in vivo.
Zinc levels were significantly increased in all knockout strains, but were most
pronounced in the CeMT-1 knockout, mtl-1 (tm1770), while cadmium
accumulation was highest in the CeMT-2 knockout, mtl-2 (gk125) and the
double knockout mtl-1;mtl-2 (zs1). In addition, metal speciation was
assessed by X-ray absorption fine-structure spectroscopy. This showed that
O-donating, probably phosphate-rich, ligands play a dominant role in
maintaining the physiological concentration of zinc, independently of

metallothionein status. In contrast, cadmium was shown to coordinate with
thiol groups, and the cadmium speciation of the wild-type and the CeMT-2
knockout strain was distinctly different to the CeMT-1 and double knock-
outs. Taken together, and supported by a simple model calculation, these
findings show for the first time that the two MT isoforms have differential
affinities towards Cd(II) and Zn(II) at a cellular level, and this is reflected at
the protein level. This suggests that the two MT isoforms have distinct in vivo
roles.
Abbreviations
EXAFS, extended X-ray absorption fine structure; ICP-OES, inductively coupled plasma optical emission spectrometry; XANES, X-ray
absorption near-edge structure.
FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2531
immobilization of metal ions, or activation of general
stress response mechanisms ⁄ proteins [8,9]. A promi-
nent response pathway involved in the chelation of
metal ions involves metallothioneins (MTs). These are
proteins of low molecular mass that are characterized
by a high cysteine content [15–30%], high heat stability
and lack of aromatic amino acids (including histidine)
[10,11]. Although the discovery of MTs dates back to
1957 [12], their precise physiological functions are still
debated. It has become evident that a single function
does not exist for this heterogeneous superfamily of
proteins, and that they are ‘multipurpose’ proteins
[13], with roles in protection against cadmium toxicity
[14], essential Cu(I) and Zn(II) homeostasis [15], and
response to oxidative stress [16].
There is growing evidence that the existence of mul-
tiple MT isoforms is associated with functional differ-
entiation, for example in snails [17], earthworms [18],

plants [19] and vertebrates [16]. So far, studies have
focused on the discrimination between monovalent
Cu(I) and divalent Zn(II) and Cd(II) [20]. As the coor-
dination geometries of mono- and divalent metal ions
are very distinct (digonal or trigonal planar versus tet-
rahedral), it is easily conceivable that the steric require-
ments imposed by binding of these metal ions will
differ, and this offers a straightforward mode of dis-
crimination.
In contrast, discrimination between the essential
Zn(II) and toxic Cd(II), which have relatively similar
coordination chemistry, presents a major challenge for
organisms that are exposed to both metal ions. The
soil nematode Caenorhabditis elegans is a case in point
[21,22], and offers a unique biological system for the
study of MT isoform specificity, because its fully
sequenced genome contains only two metallothioneins
CeMT-1 and CeMT-2 [23]. The encoded proteins bear
the hallmarks of metallothioneins, i.e. they are small
and cysteine-rich, and their expression is induced by
metals [24]. More recently, RNA interference (RNAi)
and chromosomal deletion of the C. elegans MT loci
have highlighted an increased sensitivity of mutant
strains to metal toxicity, reflected by reduced growth,
brood size and lifespan [23,25]. In addition, phytochel-
atins, which are small, non-ribosomally synthesized,
Cd-binding peptides, play a prominent role in protec-
tive responses to Cd exposure [26–28].
Significantly, the two MT isoforms show differential
expression profiles [24]. CeMT-2 is only induced in

intestinal cells in the presence of cadmium, but CeMT-1
is also constitutively active in three cells of the lower
pharyngeal bulb [24]. These studies provided the first
evidence that CeMT-1 and CeMT-2 may have distinct
in vivo functions, but although additive sensitivity
towards cadmium was observed in C. elegans metallo-
thionein knockout alleles, isoform-specific in vivo effects
have not been observed to date, even by detailed meta-
bolomic profiling analysis [28].
At the protein sequence level, CeMT-1 and CeMT-2
display intriguing differences, and are more different
from one another than vertebrate MT isoforms.
CeMT-1 contains a 15 amino acid insert with two
additional histidines and one cysteine [23,24,29], with a
further histidine at position 54 (see Fig. S1A for
sequence alignment). Recent in vitro characterization
of recombinantly expressed CeMT-1 and CeMT-2 by
ESI-MS and CD spectroscopy has begun to determine
the differences in metal binding properties of the two
isoforms [30]. A clear preference for divalent metal
ions was discovered, but, most significantly, this study
suggested that CeMT-1 and CeMT-2 show differential
metal preferences, with CeMT-1 biased towards Zn(II)
and CeMT-2 biased towards Cd(II).
In the present study, we explore whether these quali-
tative findings are reflected by overall in vivo metal
accumulation and speciation of metallothionein-
mutated C. elegans strains, as well as the in vitro metal
ion affinities of the two isoforms under metal-replete
and metal-excess conditions.

Results
Metal-binding properties of recombinant
metallothioneins
For characterization and quantification of the metal-
binding properties of CeMT-1 and CeMT-2, an expres-
sion strategy was adapted that avoids the use of fusion
tags, as we have previously observed that tags can influ-
ence the metal binding properties of recombinantly
expressed metallothioneins [31]. In contrast to most
expression tag systems, our protocol also allows the
expression of proteins with no additional residues at the
termini. Careful chemical precipitation followed by gel
filtration chromatography yielded pure proteins
(> 95%, as judged by ESI-MS analysis) with no addi-
tional species (see Fig. S1B,C for expression and purifi-
cation, respectively).
Both metallothioneins were expressed in the presence
of either Zn(II) or Cd(II) in the culture medium. The
metal ion stoichiometry of the purified proteins was
determined by inductively coupled plasma optical
emission spectrometry (ICP-OES) and ESI-MS (Fig. 1
and Table 1). Consistent with previous work [30,32],
CeMT-2 expressed in the presence of Cd(II) had six
Cd(II) ions bound. We found the same stoichiometry
for Zn(II), with no discernible peaks for metal-depleted
C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al.
2532 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS
species in the mass spectrum, and corresponding ICP-
OES results. Consistent with recent findings [30], our
analysis also confirmed that CeMT-1 binds seven metal

ions, with Zn
7
-CeMT-1 the only species observed in
mass spectra at neutral pH for Escherichia coli cells
grown in Zn(II)-supplemented medium (Fig. 1).
To allow quantification of metal affinities and their
comparison, it was very important to obtain clearly
defined homo-metallic species, and the data compiled
for Zn
6
-CeMT-2, Cd
6
-CeMT-2 and Zn
7
-CeMT-1 in
Fig. 1 and Table 1 show that this was achieved by
expression in the presence of the desired metal ion.
However, the CeMT-1 form isolated from Cd(II)-sup-
plemented cultures was Cd
6
Zn-CeMT-1 (Fig. S2), and
incorporation of seven cadmium ions was only possible
by reconstitution of metal-free CeMT-1 with rigorous
exclusion of Zn(II), using an established protocol [33].
Although the Cd
7
-CeMT-1 species was the major form
in this preparation, we observed a loss of definition in
metal binding stoichiometry despite extensive gel filtra-
tion and washing, as Cd

8
-CeMT-1 and Cd
9
-CeMT-1,
as well as a very small amount of Cd
6
Zn
1
-CeMT-1 me-
talloforms, were present as minor species (Fig. 1B).
The contribution of these over-metallated species
is also apparent in the stoichiometry determined by
ICP-OES given the larger than expected stoichiometry
for cadmium-bound CeMT-1.
The overall in vitro affinities of CeMT-1 and CeMT-2
towards Zn(II) and Cd(II) were determined by comp-
etition experiments using the metal chelator 5F-BAPTA
[34] and
19
F-NMR spectroscopy under conditions that
allow direct comparison with literature values. The
stability constants obtained for the homo-metallic zinc
and cadmium complexes of CeMT-1 and CeMT-2 are
given in Table 2, and represent means over all six
(CeMT-2) or seven (CeMT-1) binding sites. As
expected for predominantly thiol coordination, the sta-
bility constant for cadmium binding in CeMT-2 was
significantly larger than that for zinc binding, and was
close to the value for human MT-2 measured under
similar conditions [34]. Remarkably, this was not the

case for CeMT-1. Although both isoforms displayed
identical affinities for Zn(II), cadmium binding in the
CeMT-1CeMT-2
Relative Intensity
8000 8400 8800 9200
[–Met]
[–Met]
[–Met]
[–Met]
[–Met]
C
D
6600 7000 7400
7800
0
50
100
50
100
A
B
[–Met]
[+Met]
Cd
6
Cd
6
Zn
6
Zn

7
Cd
6
Zn
Cd
8
Cd
7
Cd
9
[–Met]
Mass (Da)
Fig. 1. Deconvoluted ESI mass spectra of
the various metalloforms of Caenorhabd-
itis elegans MTs. Holo zinc (A) and cadmium
(B) species of CeMT-1; zinc (C) and cad-
mium (D) species of CeMT-2 obtained at
neutral pH (10 m
M ammonium acetate, 10%
methanol). Samples (A), (C) and (D) result
from expression in the presence of the
respective metal ion; sample (B) was
obtained by expression in presence of Zn(II)
and reconstitution of the apoprotein with
Cd(II). )Met and +Met annotations refer to
the absence or presence of the N-terminal
methionine residue for each species. The
peaks in (A), (C) and (D) to the right of the
main peaks correspond to Na
+

adducts.
Table 1. Metal to protein stoichiometries for CeMT-1 and CeMT-2
metalloforms determined by mass spectrometry and elemental
analysis. Theoretical and observed mass are given for the major
species in each mass spectrum. )MET, without Met; +Met, includ-
ing Met.
Metalloform
Mass spectrometry
Stoichiometry
(ICP-OES)
Theoretical
mass (Da)
Observed
mass (Da) Zn Cd
CeMT-1
Zn
7
8402.7 ()Met) 8401.9 ± 0.7 6.6 ± 0.7 8.8 ± 0.8
Cd
7
8731.9 ()Met) 8731.5 ± 0.5
CeMT-2
Zn
6
6843.1 ()Met) 6843.3 ± 0.7 5.6 ± 0.6 6.0 ± 0.6
Cd
6
7256.5 (+Met) 7257.0 ± 0.6
S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between metals
FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2533

Cd
7
-CeMT-1 complex was dramatically weaker than
that in Cd
6
-CeMT-2 and other MTs (Table 2), even
when the effect of the over-metallated species is taken
into account. To date, the overall stability of cadmium
binding to CeMT-1 is the lowest value reported for
any MT. However, it is important to note that, despite
this, the overall affinity of CeMT-1 towards Cd(II) is
still an order of magnitude larger than that for Zn(II),
so it would be inappropriate to claim that CeMT-1 is
a Zn(II)-specific MT, although one particular site does
indeed appear to have an absolute preference for
Zn(II).
In vivo metal speciation in wild-type C. elegans:
excess zinc and cadmium are handled differently
To investigate the native organism-wide responses to
cadmium and zinc exposure, the wild-type (N2) C. ele-
gans strain was grown on supplemented media, and
the collective ligand environment of intracellular cad-
mium and zinc was analysed by X-ray absorption
near-edge structure (XANES) and extended X-ray
absorption fine structure (EXAFS) spectroscopy.
Because the low-energy photoelectrons have a long
mean free path, XANES spectroscopy is strongly
affected by multiple scattering, which means that it is
very sensitive to differences in geometry as well as
coordination number and oxidation state. Although

this complexity complicates the analysis of XANES
data, it is valuable as a ‘fingerprint’ technique, com-
paring unknowns with model compound spectra.
Indeed, XANES and EXAFS spectroscopy have previ-
ously been used successfully on rat liver samples to
distinguish different binding modes in Cd–S clusters
and metallothionein [35]. The cadmium XANES spec-
tra (Fig. 2A) show that the edge shape and position
are distinct from Cd–O-bonded complexes, and display
features that are more similar to S-coordinated cad-
mium models (Cd–S and rat Cd
7
-MT). The EXAFS
results and associated Fourier transforms, together
with the best possible fits, are shown in Fig. S3, and
indicate a single major transform peak at
R+D = 2.5 A
˚
(other fits gave higher residuals, data
not shown). When modelled, the cadmium EXAFS
data produce a best fit with one shell of four sulfur
scatterers at 2.49 A
˚
(Table 3). However, due to the
small size of the nematodes, even 300 000–500 000 syn-
chronized nematodes generated only a dilute sample
that, although sufficient for analysis, produced a short
data range and had a poor signal-to-noise ratio, thus
precluding fitting of further shells of scatterers.
Although the EXAFS data were admittedly noisy, they

Table 2. Zinc and cadmium binding affinities for MTs in Caenor-
habditis elegans and other species. Log K binding constants for
zinc and cadmium metalloforms of C. elegans MTs were deter-
mined and compared to those of other MTs. The log K for
cadmium binding was determined by competition between protons
and metal ions for complexed thiolate ligands [33]. In all cases, the
log K of zinc binding was measured by competition for metal ions
between the MTs and 5F-BAPTA (ionic strength 4 m
M and pH 8.1)
[34,48,53].
MT isoform log K
Zn
log K
Cd
CeMT-1 (C. elegans) 12.0 ± 0.1
a
13.1 ± 0.3
CeMT-2 (C. elegans) 12.0 ± 0.1 15.0 ± 0.1
MT-2 (human) [34] 11.5 14.8
MT-3 (human) [34] 10.8 14.3
SmtA (bacterial) [53] 13.0 –
E
C
(plant) [53] 10.6 –
a
Recalculating this value to account for over-metallation yields a
log K value of 13.4.
Wildtype
Cd-S
Cd-MT

Cd(OH)
2
CdSO
4
26 680 26 84026 80026 76026 720
Energy (eV)
Normalised signal
Wildtype
Zn-S
Zn foil
ZnSO
4
. H
2
O
Zn
3
(PO
4
)
2
Normalised signal
9600 9700 9800 9900 10 000
Ener
gy
(eV)
A
B
Fig. 2. XANES profiles in wild-type nematodes and standards. Cd
XANES spectra (A) and Zn XANES spectra (B). For cadmium,

a minor monochromator drift during data collection made it
necessary to correct the edge position using reference spectra,
therefore the error in the absolute position of the edge was
marginally larger than the station benchmark.
C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al.
2534 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS
are of sufficient quality to justify the conclusion that
sulfur coordination gives the best single shell fit, which
is also consistent with XANES results.
Previously recorded Zn(SO
4
)ÆxH
2
O, ZnS and
Zn
3
(PO
4
)
2
spectra were used to model the zinc
XANES spectra of the wild-type (N2) nematode. The
spectra show that, of all reference compounds, the
wild-type spectrum displayed features most similar to
those of the zinc phosphate standard (Fig. 2B). This
was corroborated by EXAFS spectra (Table 3 and
Fig. S4), which indicated the best fit to be four oxygen
atoms surrounding zinc in the first coordination shell,
with a mean Zn–O distance of 1.97 ± 0.03 A
˚

. This is
consistent with the tetrahedral coordination of zinc
phosphate [36].
Although, it may not be technically possible to distin-
guish between N ⁄ O ⁄ F or between P ⁄ S ⁄ Cl as a scatterer,
the difference between O and S is substantial. Therefore,
these data suggest that the mechanisms to deal with zinc
and cadmium employed by C. elegans are separate and
distinct, as accumulated cadmium is predominantly
S-bound and zinc is predominantly O-bound.
C. elegans metallothioneins are not the only
players in metal detoxification and homeostasis
The effects on the ligand environments of cadmium
and zinc upon deletion of metallothioneins were inves-
tigated by comparative analysis of XANES spectra
(Fig. 3) and EXAFS data (Table 3, Figs S3 and S4).
Cadmium XANES spectra (Fig. 3A) for the MT
knockout strains do not show features significantly dif-
ferent from those observed for the wild-type (N2),
which suggests that the cadmium ions are still predom-
inantly coordinated by sulfur atoms. However,
a broader edge and lower starting energy (1.5–2 eV)
were observed in spectra of the CeMT-1 KO and the
double knockout, both were observed in spectra of the
CeMT-1 knockout mtl-1 (tm1770) and the CeMT-1
Table 3. Cd ⁄ Zn EXAFS parameters. Best fit of the Cd ⁄ Zn K-edge data for Caenorhabditis elegans wild-type and metallothionein knockout
strains, where r is the absorber–scatterer distance in A
˚
(± 0.02 A
˚

, inner shell; ± 0.05 A
˚
, outer shell), N is the number of scatterers around
the central atom, 2d
2
is the the Debye–Waller factor in A
˚
2
, ± 25%, and the R factor is the least-squares residual, which indicates goodness
of fit.
Strain
Cadmium Zinc
Scatterer Nr(A
˚
)2d
2
(A
˚
2
) R factor Scatterer Nr(A
˚
)2d
2
(A
˚
2
) R factor
Wild-type S 4 2.49 0.026 74.3 O 4 2.00 0.010 39.1
CeMT-1 KO S 4 2.53 0.017 48.6 O 4 2.00 0.015 30.9
O 1 1.98 0.015 40.5

S 3.3 2.52 0.013
37.6
CeMT-2 KO S 4 2.48 0.028 78.8 O 4 1.97 0.014
P 2 3.27 0.008
S 4 2.51 0.020 67.6 O 4 1.98 0.012 26.8
Double KO O 1 1.98 0.016 61.6
S 3.3 2.49 0.015
Energy (eV)
Normalised signal
26 700
26 780
26 820
26 860
26 740
26 700 26 710 26720
Wildtype
CeMT-1 KO
CeMT-2 KO
Double KO
Wildtype
CeMT-1 KO
CeMT-2 KO
Double KO
9630 9670 9710 9750 9790
Energy (eV)
9657 9658 9659 9660
Normalised signal
A
B
Fig. 3. Metal speciation in Caenorhabditis elegans strains. Compari-

son of Cd XANES spectra (A) and Zn XANES spectra (B) obtained for
C. elegans wild-type and metallothionein knockouts. The inserts
show the cadmium and zinc energy shifts between samples.
S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between metals
FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2535
and CeMT-2 double knockout mtl-1;mtl-2 (zs1). Such
features are characteristic of a Cd–O phase. This
observation was supported by EXAFS analysis, for
which data fitting was improved by addition of a shell
of oxygen scatterers and refining the distances, the De-
bye–Waller factors, and the ratio of S-bound to O-
bound cadmium (Table 3). The refined Cd–O distance
of 1.98 A
˚
is arguably very short compared to crystallo-
graphic values for Cd–O in phosphates, carbonates,
etc. However, this may be due to the Cd being four-
coordinate rather than six-coordinate, or may reflect a
larger than normal error in the EXAFS distance due
to weaker scattering of the oxygen than of the sulfur,
making the Cd–O contribution to the total EXAFS
spectra much smaller than the Cd–S contribution.
Nevertheless, this confirms that the cadmium coordina-
tion environments of the CeMT-1 KO and double
KO differ from those of the wild-type (N2) and the
CeMT-2 KO, although it should be emphasized that
the majority of the cadmium remained bound to sulfur
in all strains (including the metallothionein deletion
stains) (Table 3).
The absence of either or both MT(s) had no observa-

ble effect on zinc speciation. All XANES spectra
(Fig. 3B) were similar, and EXAFS data analysis
(Table 3 and Fig. S4) identified a common first shell
scatterer peak at 1.97 A
˚
, characteristic of O-coordina-
tion. Adding a second shell of phosphorus scatterers
improved the fit for all four spectra, but this shell was
statistically significant only in the case of mtl-2 (gk125).
Although superbly fitted Zn and Cd XANES and
EXAFS data have previously illustrated that isolated
mammalian metallothioneins bind metals [37,38], the
data presented here reveal that the MT status of the
nematode does not significantly alter the overall speci-
ation of zinc and cadmium in cells, as the principal
ligand environment for both metals is similar to that
of the wild-type (N2) strain. Nevertheless, the data
provide insights about the ultimate fate of each
metal ion. As Cd–S bonds were maintained in the
double knockout strain, it is clear that the Cd–S spe-
cies observed do not correspond to metallothionein-
bound Cd. Instead, it is likely that phytochelatins
dominate Cd speciation. Excess zinc in C. elegans is
clearly not MT- or phytochelatin-bound, but may be
sequestered through other means such as deposition in
phosphate-rich granules [39], possibly synonymous to
those found in earthworms [40,41].
However, these facts do not preclude a role for MTs
in metal handling, as binding of zinc and cadmium by
MTs may be transient, particularly as MTs are capable

of releasing metal ions relatively rapidly [42–44], possi-
bly to molecules downstream in the detoxification
pathway. We therefore next address the question of
whether MTs in C. elegans influence overall zinc and
cadmium levels at all.
Metal levels in metal-exposed worms: CeMT-2 is
important with regard to cadmium accumulation
In the wild-type (N2) strain, low levels of cadmium
accumulation were observed when nematodes were
grown (from L1 larval to pre-adult stage L4) on
Cd-supplemented medium (Fig. 4A and Table S1). An
equivalent cadmium body burden was also observed in
the CeMT-1 knockout. This suggests that, upon cad-
mium exposure, the CeMT-1 KO strain responds ‘as
wild-type’, and the mechanism of this response is not
hindered by lack of CeMT-1 in the cytosol. In con-
trast, the CeMT-2 KO strain shows an approximately
twofold increase in cadmium levels compared to the
wild-type (N2) strain, indicating that one of the mech-
anisms by which C. elegans normally responds to cad-
mium exposure has been disrupted. This is exacerbated
in the double knockout strain, in which the cadmium
burden is significantly increased. These data suggest
that (a) if CeMT-2 is expressed, then CeMT-1 does
not play a significant role in the cadmium response,
(b) if CeMT-2 is absent, then CeMT-1 can fulfil the
role carried out by CeMT-2, but not as effectively, and
(c) if both metallothioneins are absent, the ‘normal’
and ‘back-up’ MT-mediated pathways of dealing with
cadmium exposure are impaired, leading to hyperaccu-

mulation of cadmium compared to the wild-type
strain.
Both CeMT-1 and CeMT-2 are important in
maintaining physiological zinc levels
Under control (non-metal-supplemented) conditions in
the wild-type (N2), zinc was maintained at basal
physiological levels (Fig. 4B and Table S1). For the
CeMT-2 and double mutant strains, no significant dif-
ference from wild-type (N2) was observed; however,
the CeMT-1 mutant accumulated slightly more Zn(II).
Under Zn-supplemented conditions, all three knockout
strains accumulated significantly more zinc compared
to the wild-type (N2) strain. Of the single knockout
strains, deletion of CeMT-1 resulted in accumulation
of the highest zinc concentration; however, deletion of
CeMT-2 also led to a moderate increase in zinc levels.
The double knockout did not differ significantly from
the CeMT-1 knockout. This indicates that (a) CeMT-1
has a more significant role than CeMT-2 in the regula-
tion of zinc levels, (b) both CeMT-1 and CeMT-2 are
required to maintain physiological zinc levels, as lack
C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al.
2536 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS
of CeMT-2 also disrupts the mechanism that prevents
zinc accumulation, and (c) CeMT-1 and CeMT-2 oper-
ate in a synergistic manner in zinc trafficking.
Figure 4B also includes data for Zn(II) levels after
Cd exposure, and these data offer further interesting
insights. In the CeMT-1 knockout, which showed only
basal Cd levels, Zn levels were depressed, but were ele-

vated under Zn exposure. This observation can only
be rationalized if we consider that the two isoforms
are regulated differently, and that Cd(II) strongly
induces CeMT-2. Hence, in the CeMT-1 knockout,
induction of CeMT-2 may have led to enhanced excre-
tion (or reduced uptake) of not only Cd(II), but also
some of the basal Zn(II), possibly mediated by the
same CeMT-2-dependent pathway. No difference in
Zn levels was observed for the CeMT-2 knockout
mutant, indicating that zinc homeostasis functioned
normally even in the presence of Cd(II). Finally, in the
double knockout, a significant increase in Zn(II) levels
was observed, indicating significant disruption of
Zn(II) homeostasis.
CeMT-1 and CeMT-2 provide a system for
discrimination between essential Zn(II) and toxic
Cd(II)
The question of how cells select the correct metal ions
is of current interest [45]. One emerging concept holds
that it is not the absolute but the relative affinity of
various metal-trafficking proteins towards various
metal ions in a common cytosol that governs metal ion
selection and distribution. The in vitro and in vivo data
presented here are consistent with this concept, and
allow development of a framework that helps to
understand the discrimination between Zn(II) and
Cd(II) by the two metallothioneins in C. elegans,as
well as at a more general level.
To illustrate this idea, we have used the in vitro
(Table 2) and in vivo (Fig. 4) data to approximate

the proportion of metal ions bound to CeMT-1 and
CeMT-2 if presented with Zn : Cd ratios as encountered
by C. elegans. Using a Cd : Zn ratio of 33 : 1 [21 nm
Cd(II) and 0.7 lm Zn(II)] and 0.1 lm of CeMT-1 and
CeMT-2 each, and the stability constants given in
Table 2, it can be calculated that 98.6% of Cd(II) is
bound to CeMT-2, and only 1.4% to CeMT-1. Zn(II) is
more evenly distributed (45 : 55%) between CeMT-1
and CeMT-2. When equimolar amounts of Zn(II) and
Cd(II) are used (0.65 lm each), 93% of Zn(II) is bound
to CeMT-1, and 85% of Cd(II) is bound to CeMT-2.
With a 10-fold excess of MTs and the same metal con-
centrations, 98.4% of Cd are bound to CeMT-2, and the
Zn(II) distribution is 57 : 43% for CeMT-1 : CeMT-2.
These numbers have been calculated based on two
relatively crude simplifications: first that all binding sites
in CeMT-1 and CeMT-2 are equivalent, and second that
no other competing ligands are present. It is conceivable
that the overall reduction in Cd(II) affinity is to a con-
siderable extent, but not exclusively, due to weaker
binding to the histidine-rich site. It is therefore likely
that the difference in affinities for binding to the
Cadmium
Concentration (ng·L
–1
)Concentration (ng·L
–1
)
Zn-exposed
a

a
b
c
ND ND ND ND ND ND ND ND
WT CeMT-1
KO
CeMT-2
KO
Double
KO
0
500
1500
1000
2000
2500
3000
c
b
a
a
a
b
c
b
a
3500
Zinc
a
a

a
*
**
*
*
**
**
**
Control
0
Cd-exposed
20
40
60
80
100
Double
KO
CeMT-2
KO
CeMT-1
KO
WT
A
B
Fig. 4. Metal accumulation in nematodes. Levels of cadmium (A)
and zinc (B) were quantified by ICP-OES in Caenorhabditis elegans
wild-type and metallothionein deletion strains cultured in the pres-
ence or absence of cadmium (25 l
M) or zinc (340 lM). Values are

the means ± SEM of five replicates. Different letters above bars
indicate statistical significance compared with each other.
*P < 0.05;**P < 0.01. ND, not detectable (below detection limits).
S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between metals
FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2537
all-thiolate sites in the two proteins is < 1.9 orders of
magnitude. However, even a difference of only 0.3 log
units would achieve a 66 : 34% distribution of Cd(II) in
CeMT-1 and CeMT-2, and, given the presence of fur-
ther mechanisms, this sorting level may be sufficient to
ensure tolerable management of both Zn(II) and Cd(II)
in C. elegans. Our simplistic model demonstrates that,
even though both MTs show an overall preference for
Cd(II) over Zn(II), as expected for predominantly thio-
late coordination, the decrease in affinity of Cd for
CeMT-1 may allow segregation of Cd(II) into predo-
minantly CeMT-2 in a common cytosol.
Discussion
Like other soil-dwelling organisms, C. elegans nema-
todes are constantly exposed to and ingest varying lev-
els of essential and toxic metal ions present in the
surrounding medium. Consequently, such organisms
require mechanisms that capture and redistribute the
correct amounts of biologically essential metal ions
whilst preventing the accumulation of harmful levels of
toxic metal ions. Within this framework, mechanisms
must exist that allow the cell to distinguish between
closely similar essential and toxic metal ions, such as
Zn(II) and Cd(II), respectively. The predominating
Cd–S and Zn–O forms observed by X-ray absorption

analysis suggest that separate pathways exist for traf-
ficking of these two metal ions. These pathways do not
appear to be MT-mediated, and the negligible effect on
in vivo speciation for either Cd(II) or Zn(II) in knockout
mutants has excluded the possibility that MTs function
as metal storage proteins in C. elegans. In contrast, the
reduced accumulation, or excretion, of cadmium and
zinc is MT-mediated, as there was a large effect on the
levels of accumulated zinc and cadmium when CeMT-1
and CeMT-2 were deleted. We interpret this observation
as an indication that some processes, possibly excretion
of excess zinc and cadmium, do not function normally
in the double knockout strain. Furthermore, and most
importantly, the extents to which these MT-
mediated processes are disrupted are isoform- and
metal-ion specific. We have shown that CeMT-2 plays a
more significant role in preventing hyperaccumulation
of cadmium. Conversely, both CeMT-1 and CeMT-2
are important in maintaining physiologically acceptable
zinc levels, and the lack of CeMT-1 had a more deleteri-
ous effect. These metal-specific preferences at the cellu-
lar level are mirrored in the relative affinities of the
individual CeMT-1 and CeMT-2 proteins towards
Zn(II) and Cd(II). The thermodynamic data suggest
that, when presented with both MT isoforms, cadmium
ions preferentially bind to CeMT-2, thus leaving CeMT-
1 to deal with zinc. The origin of this differential affinity
is most likely rooted in the structure of the two isoforms.
It is conceivable that the differences in specificity are, at
least to a considerable extent, associated with the four

additional metal ligands in CeMT-1, particularly the his-
tidine residues (see Fig. S1A). Previous studies on both
zinc fingers [46] and metallothioneins [47–50] have dem-
onstrated that an increasing number of histidine residues
in a metal binding site shifts the preference towards
Zn(II). Further studies, including determination of 3D
structures for CeMT-1 and CeMT-2, are required to
determine the precise cause of the observed metal speci-
ficities.
In conclusion, the nematode C. elegans exhibits both
MT-mediated and non-MT-mediated pathways to deal
with cadmium and zinc. We have shown for the first
time that the responses to cadmium and zinc ions at
the cellular level are isoform-specific, and that this
specificity is reflected at the protein level.
Experimental procedures
Cloning of MT constructs
Total RNA was isolated from nematodes using TRI
reagent (Sigma, St Louis, MO, USA) and reverse tran-
scribed into cDNA from 1 lg RNA using oligo(dT) primers
and MMLV reverse transcriptase (Stratagene, La Jolla,
CA, USA), all according to the supplier’s protocols.
C. elegans metallothioneins were amplified by PCR from
cDNA using isoform-specific primers containing SalI and
NdeI restriction site extensions (mtl-1_fwd: 5¢-TATACAT
ATGGCTTGCAAGTGTGACTGC-3¢; mtl-1_rev: 5¢-AGC
TTGTCGACGTTAATGAGCCGCAGCAGTTCCC-3¢;
mtl-2_fwd: 5¢-TATACATATGGTCTGCAAGTGTGACT
GC-3¢ and mtl-2_rev: 5¢-AGCTTGTCGACGTTAATGA
GCAGCCTGAGCACAT-3¢), generating DNA fragments

of 247 and 211 bp for isoform 1 and isoform 2, respec-
tively. The purified PCR products, as well as the plasmid
pET29a, were digested using SalI and NdeI (Promega,
Madison, WI, USA) at 37 °C for 3 h, and ligated overnight
at 4 °C using T4 ligase (Promega). The ligations were trans-
formed into DH5a-competent cells (Invitrogen, Carlsbad,
CA, USA) and positive clones were identified by PCR
screening. The identity of the insert was confirmed by
sequencing both strands of the cloned inserts.
In vitro protein expression and purification
Plasmids containing the respective metallothionein isoform
were transformed into E. coli Rosetta TM2 (DE3)pLysS
(Merck, Nottingham, UK) using standard molecular clon-
ing techniques. Expression cultures (1 L) selective for kana-
mycin and chloramphenicol (50 and 34 lgÆmL
)1
,
C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al.
2538 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS
respectively) were induced using isopropyl b-d-1-thiogalac-
topyranoside (500 lm final concentration). Following
induction, ZnSO
4
or CdCl
2
(both Sigma) were added to a
final concentration of 500 lm. Protein expression was per-
formed for up to 6 h at 30 °C before harvesting the cells by
centrifugation at 5000 g. Cell pellets were resuspended in
ice-cold sonication buffer (50 mm Tris ⁄ Cl, 0.1 m KCl,

3mm dithiothreitol, 1 mm ZnSO
4
, pH 8.5), and ruptured
by sonication. This was followed by centrifugation at
45 000 g for 45 min to remove cell debris. The resulting
lysate was subjected to a chemical fractionation similar
to that described by You et al. [32]. Briefly, strepto-
mycin (10% w ⁄ v solution) was added to the lysate at
0.375 mLÆg
)1
wet cell weight. To this mixture, one volume
of chilled ethanol ⁄ chloroform (100 : 8) solution was added
dropwise with continuous stirring. The mixture was centri-
fuged for 5 min at 5000 g. A further three volumes of the
ethanol ⁄ chloroform solution was added dropwise to the
resulting supernatant, and this mixture was stored over-
night at )20 °C. The precipitate was collected by centrifu-
gation at 5000 g, resuspended in 20 mm ammonium
bicarbonate (pH 7.8), and purified by gel filtration (16 ⁄ 60
HiLoadÔ 75 SuperdexÔ prep grade, GE Healthcare, Little
Chalfont, UK). MT-containing fractions were pooled and
concentrated by ultrafiltration (Amicon Ultra; Millipore,
Billerica, MA, USA). The isolated proteins either retained
or did not retain the N-terminal methionine. The cleavage
efficiency of the E. coli Met aminopeptidase appeared to be
dependent on the metal ion supplied, such that MTs
expressed in the presence of Cd(II) mostly retained the initi-
ation methionine.
Preparation of Cd
7

-CeMT-1
Cd
7
-CeMT-1 was prepared using a modified procedure
based on the method reported by Vas
ˇ
ak [33]. Briefly, an ali-
quot of Zn
7
-CeMT-1 (50 mm Tris, 50 mm NaCl, pH 7.4)
was incubated at room temperature with dithiothreitol
(approximately 10 mm) for 1 h. This mixture was acidified
to a pH of approximately 1 using 2 m HCl, and applied to
a gel filtration column (Sephadex G25, PD10, Amersham
Biosciences). The demetallated protein was eluted under
nitrogen gas using 0.1 m HCl. CdCl
2
(7.5 molar equiva-
lents) was added to the eluate, and the pH was increased to
> 7.0 via addition of 2 m Tris base. Extensive washing by
ultrafiltration ensured removal of unbound metal ions.
Mass spectrometry
All isoforms (20 lm) were buffer exchanged into 10 mm
ammonium acetate (pH 7.2) by ultrafiltration. Prior to the
analysis, methanol was added to a final concentration of
10% v ⁄ v. Samples were infused directly via a syringe
pump operating at a rate of 250 lLÆh
)1
. Analyses were
performed using either ESI-TOF (MicrOTOF; Bruker,

Bremen, Germany) or ESI-ion trap (HCT-UltraTM Dis-
covery System; Bruker) mass spectrometers. Data were
acquired for 1.5 min in the positive mode over the m ⁄ z
range 500–3000 Th. Using data analysis software supplied
by Bruker Daltonics, smoothing and baseline subtraction
were applied to averaged data, which were subsequently
deconvoluted.
19
F-NMR spectroscopy
A sample of each C. elegans metalloform, approximately
480 lm with respect to metal ion concentration, was pre-
pared in 10 mm Tris ⁄ Cl (pH 8.1, 10% D
2
O). Accurate
determinations of metal ion content were performed using
ICP-OES (Optima 5300 DV ICP-OES; PerkinElmer,
Cambridge, UK). 5F-BAPTA [1,2-bis(2-amino-5-fluoro-
phenoxy)ethane-N,N,N¢,N¢-tetraacetic acid; 4 mm final con-
centration] was added to the sample, and incubated
overnight at room temperature. Direct observe 1D
19
F-NMR spectroscopy was performed using a DRX400
spectrometer (Bruker) fitted with a quadruple nuclei probe
(QNP) probe head operating at 375.91 MHz for
19
F nuclei.
Chemical shifts are reported with respect to the signal
for CCl
3
F [51]. Spectra were acquired at 298 K with a

spectral width of 50 p.p.m., an acquisition time of 3.48 s
and a relaxation delay of 1.0 s, with 12 288 scans. Fre-
quency Induction Decay (FID)s were apodized using
squared-sine bell functions, Fourier-transformed using
65 536 complex data points, and baseline-corrected. Spectra
were processed using topspin version 2.1 software (Bruker
Biospin). The value for K
Cd(BAPTA)
(at 30 °C and
I = 138 mm) was corrected for temperature (25 °C) and
ionic strength (4 mm) as described by Hasler et al. [34] to
give a log K value of 11.75. Calculations of apparent stabil-
ity constants for metal–MT complexes were performed
using a published procedure [34].
Sample preparation for in vivo studies
Wild-type (N2) and the CeMT-2 knockout strain mtl-2
(gk125) were obtained from the Caenorhabditis Genetics
Center (CGC) at the University of Minnesota, Minneapolis,
MN, USA, and the CeMT-1 knockout strain mtl-1
(tm1770) was obtained from the Mitani Laboratory at the
Tokyo Women’s Medical University School of Medicine,
Japan. The metallothionein double knockout mtl-1;mtl-2
(zs1) was generated previously [25]. Each strain was syn-
chronized (bleach prepped), and 300 000–500 000 L1 nema-
todes were cultured on nematode growth medium
containing sub-lethal concentrations of either CdCl
2
(25 lm) or ZnCl
2
(340 lm). A maximum of 2000 nematodes

were cultured per plate (90 mm diameter), and grown at
20 °C to pre-adult stage (L4), then harvested and quench-
frozen by immersion in liquid nitrogen.
S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between metals
FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2539
Metal quantification
Nematodes were digested in 1 n concentrated nitric acid,
and metal concentrations were quantified by inductively
coupled plasma optical emission spectrometry (ICP-OES)
using standard methods [52].
X-ray absorption spectra collection and analysis
The samples were ground to a fine powder under liquid
nitrogen, and stored as fully hydrated deep-frozen samples
at )80 °C. The X-ray absorption spectra at the cadmium
K-edge (approximately 26 710 eV) and the zinc K-edge
(approximately 9660 eV) were collected on station 16.5 of
the Synchrotron Radiation Source (now closed) at the
Science and Technology Facilities Council Daresbury Labo-
ratory, Warrington, UK. The ring operated at 2 GeV with
a mean current of 140 mA: the station was equipped with a
vertically focusing mirror and a flat Si (220) double crystal
monochromator detuned to 70% transmission to minimize
harmonic contamination. The monochromator was cali-
brated at each energy value using a 15 lm cadmium foil or
a10lm zinc foil. Data were collected with the station
operating in fluorescence mode using an Ortec 30 element
solid-state Ge detector. The samples were mounted onto
aluminium sample holders, and X-ray absorption spectros-
copy measurements were performed at cryogenic tempera-
ture (approximately 20 K) using an Oxford Instruments

helium closed-cycle cryostat. The standard samples were
prepared by grinding in an agate pestle and mortar, diluted
with boron nitride to give an edge step of approximately 1,
and mounted in 1 mm thick aluminium sample holders with
Sellotape windows. Single scans were collected for the
model compounds in the transmission mode, and 16–23
scans were collected and summed for each experimental
sample. Background subtraction and analysis of EXAFS
spectra were performed as described previously [36].
Acknowledgements
This work was supported by the Biotechnology and
Biological Sciences Research Council (BBSRC grant
BB ⁄ E025099), the Science and Technology Facilities
Council (STFC grant BB ⁄ E05099), an Altajir Trust
PhD studentship (to S.Z G.), and the Royal Society
(Olga Kennard Fellowship to C.A.B.). The X-ray
absorption spectroscopy was performed at the Dares-
bury Synchrotron Radiation Source (station 16.5),
managed and kindly assisted by Mr Bob Bilsborrow.
We wish to acknowledge Dr Suresh Swain (King’s
College London) and Dr Samantha Hughes (King’s
College London, now at Oxford University) for valu-
able advice and resources provided throughout the
project, and finally the Caenorhabditis Genetics Centre
(CGC), which is funded by the National Institutes of
Health National Centre for Research Resources, for
the supply of Caenorhabditis elegans wild-type (N2)
and mtl-2 ( gk125) and Escherichia coli OP50, and the
Mitani Laboratory at the Tokyo Women’s Medical
University School of Medicine, Japan, for the supply

of mtl-1 (tm1770).
References
1 Van Straalen NM & Roelofs D (2008) Genomics
technology for assessing soil pollution. J Biol 7, 19.
2 Timmermans MJTN, de Boer ME, Nota B, de Boer
TE, Marie
¨
n J, Klein-Lankhorst RM, Van Straalen NM
& Roelofs D (2007) Collembase: a repository for
springtail genomics and soil quality assessment. BMC
Genomics 8, 341.
3 Bundy JG, Sidhu JK, Rana F, Spurgeon DJ, Svendsen
C, Wren JF, Stu
¨
rzenbaum SR, Morgan AJ & Kille P
(2008) Systems toxicology approach identifies coordi-
nated metabolomic responses to copper in a terrestrial
non-model invertebrate, the earthworm Lumbricus
rubellus. BMC Biol 6, 25.
4 Owen J, Hedley A, Svendsen C, Wren JF, Jonker M,
Hankard KP, Lister L, Stu
¨
rzenbaum SR, Morgan AJ,
Spurgeon DJ et al. (2008) Transcriptome profiling of
developmental and xenobiotic responses in a keystone
soil animal, the oligochaete annelid Lumbricus rubellus.
BMC Genomics 9, 226.
5 Menzel R, Swain SC, Hoess S, Claus E, Menzel S,
Steinberg CE, Reifferscheid G & Stu
¨

rzenbaum SR (2009)
Gene expression profiling to characterize sediment
toxicity – a pilot study using Caenorhabditis elegans
whole genome microarrays. BMC Genomics 10, 160.
6 Galay-Burgos M, Spurgeon DJ, Weeks JM,
Stu
¨
rzenbaum SR, Morgan AJ & Kille P (2003)
Developing a new method for soil pollution
monitoring using molecular genetic biomarkers.
Biomarkers 8, 229–239.
7 Spurgeon DJ, Stu
¨
rzenbaum SR, Svendsen C, Hankard
PK, Morgan AJ, Weeks JM & Kille P (2004) Toxico-
logical, cellular and gene expression responses in earth-
worms exposed to copper and cadmium. Comp Biochem
Physiol C Toxicol Pharmacol 138, 11–21.
8 Newman MC & McIntosh AW (1991) Metal Ecotoxi-
cology: Concepts and Applications. Lewis Publishers,
Chelsea, MI.
9 Rainbow PS & Dallinger R (1986) Strategies of metal
detoxification in terrestrial invertebrates. In Ecotoxicolo-
gy of Metals in Invertebrates (Dallinger R & Rainbow
PS eds), pp. 245–290. Lewis Publishers, Boca Raton,
FL.
10 Hamer DH (1986) Metallothionein. Annu Rev Biochem
55, 913–951.
C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al.
2540 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS

11 Nordberg M & Nordberg GF (2009) Metallothioneins
and related chelators. In Metal Ions in Life Sciences
(Sigel A, Sigel H & Sigel RKO eds), pp. 1–29. The
Royal Society of Chemistry, Cambridge, UK.
12 Margoshes M & Vallee BC (1957) A cadmium protein
from equine kidney cortex. J Am Chem Soc 79,
4813–4814.
13 Coyle P, Philcox JC, Carey LC & Rofe AM (2002)
Metallothionein: the multipurpose protein. Cell Mol
Life Sci, 59, 627–647.
14 Klaassen CD, Liu J & Diwan BA (2009) Metallothion-
ein protection of cadmium toxicity. Toxicol Appl
Pharmacol 238, 215–220.
15 Maret W (2009) Molecular aspects of human cellular
zinc homeostasis: redox control of zinc potentials and
zinc signals. Biometals 22, 149–157.
16 Hidalgo J, Aschner M, Zatta P & Vasak M (2001)
Roles of the metallothionein family of proteins in
the central nervous system. Brain Res Bull 55, 133–145.
17 Chabicovsky M, Niederstatter H, Thaler R, Hodl E,
Parson W, Rossmanith W & Dallinger R (2003)
Localisation and quantification of Cd- and Cu-specific
metallothionein isoform mRNA in cells and organs of
the terrestrial gastropod Helix pomatia. Toxicol Appl
Pharmacol 190, 25–36.
18 Stu
¨
rzenbaum SR, Georgiev O, Morgan AJ & Kille P
(2004) Cadmium detoxification in earthworms: from
genes to cells. Environ Sci Technol 38, 6283–6289.

19 Guo W, Meetam M & Goldsbrough PB (2008)
Examining the specific contributions of individual
Arabidopsis metallothioneins to copper distribution and
metal tolerance. Plant Physiol 146, 1697–1706.
20 Bofill R, Capdevila M & Atrian S (2009) Independent
metal-binding features of recombinant metallothioneins
convergently draw a step gradation between Zn- and
Cu-thioneins. Metallomics 1, 229–234.
21 Moilanen LH, Fukushige T & Freedman JH (1999)
Regulation of metallothionein gene transcription.
Identification of upstream regulatory elements and
transcription factors responsible for cell-specific expres-
sion of the metallothionein genes from Caenorhabditis
elegans. J Biol Chem 42, 29655–29665.
22 Williams PL, Ma H, Glenn TC, Jagoe CH & Jones KL
(2009) A transgenic strain of the nematode
Caenorhabditis elegans as a biomonitor for heavy metal
contamination. Environ Toxicol Chem 10, 496.
23 Stu
¨
rzenbaum SR (2009) Earthworm and nematode
metallothioneins. In Metal Ions in Life Sciences (Sigel
A, Sigel H & Sigel RKO eds), pp. 183–197. The Royal
Society of Chemistry, Cambridge, UK.
24 Freedman JH, Slice LW, Dixon D, Fire A & Rubin CS
(1993) The novel metallothionein genes of
Caenorhabditis elegans. Structural organization and
inducible, cell-specific expression. J Biol Chem 268,
2554–2564.
25 Hughes S & Stu

¨
rzenbaum SR (2007) Single and double
metallothionein knockout in the nematode C. elegans
reveals cadmium dependent and independent toxic
effects on life history traits. Environ Pollut 145, 395–
400.
26 Vatamaniuk OK, Bucher EA, Ward JT & Rea PA
(2001) A new pathway for heavy metal detoxification in
animals: phytochelatin synthase is required for cadmium
tolerance in Caenorhabditis elegans.
J Biol Chem 276,
20817–20820.
27 Clemens S, Schroeder JI & Degenkolb T (2001)
Caenorhabditis elegans express a functional phytoche-
latin synthase. Eur J Biochem 268, 3640–3643.
28 Hughes SL, Bundy JG, Want EJ, Kille P &
Stu
¨
rzenbaum SR (2009) The metabolomic responses of
Caenorhabditis elegans to cadmium are largely
independent of metallothionein status, but dominated
by changes in cystathionine and phytochelatins. J Prot
Res 8, 3512–3519.
29 Swain SC, Keusekotten K, Baumeister R &
Stu
¨
rzenbaum SR (2004) C. elegans metallothioneins:
new insights into the phenotypic effects of cadmium
toxicosis. J Mol Biol 341, 951–959.
30 Bofill R, Orihuela R, Romagosa M, Dome

`
nech J,
Atrian S & Capdevila M (2009) Caenorhabditis elegans
metallothionein isoform specificity – metal binding
abilities and the role of histidine in CeMT1 and
CeMT2. FEBS J 276, 7040–7056.
31 Blindauer CA, Harrison MD, Robinson AK,
Parkinson JA, Bowness PW, Sadler PJ & Robinson
NJ (2002) Multiple bacteria encode metallothioneins
and SmtA-like zinc fingers. Mol Microbiol 45, 1421–
1432.
32 You C, MacKay EA, Gehrig PM, Hunziker PE & Ka
¨
gi
JHR (1999) Purification and characterization of
recombinant Caenorhabditis elegans metallothionein.
Arch Biochem Biophys 375, 44–52.
33 Vasak M (1991) Metal removal and substitution in
vertebrate and invertebrate metallothioneins. Methods
Enzymol 205, 452–458.
34 Hasler DW, Jensen LT, Zerbe O, Winge DR & Vasak
M (2000) Effect of the two conserved prolines of human
growth inhibitory factor (MTIII) on its biological
activity and structure dynamics: comparison with a
mutant protein. Biochemistry 39, 14567–14575.
35 Chan J, Merrifield ME, Soldatov AV & Stillman MJ
(2005) XAFS spectral analysis of the cadmium
coordination geometry in cadmium thiolate clusters in
metallothionein. Inorg Chem 44, 4923–4933.
36 Fomina M, Charnock JM, Hillier S, Alexander IJ &

Gadd GM (2006) Zinc phosphate transformations by
the Paxillus involutus ⁄ pine ectomycorrhizal association.
Microb Ecol 52, 322–333.
37 Jiang DT, Heald SJ, Sham TK & Stillman MJ (1994)
Structures of the cadmium, mercury, and zinc thiolate
S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between metals
FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2541
clusters in metallothionein: XAFS study of Zn7-MT,
Cd7-MT, Hg7-MT, and Hg18-MT formed from rabbit
liver metallothionein 2. J Am Chem Soc 116,
11004–11013.
38 Abrahams IL, Bremner I, Diakun GP, Garner CD,
Hasnain SS, Ross I & Vasak M (1986) Structural study
of the copper and zinc sites in metallothioneins by using
extended X-ray-absorption fine structure. Biochem J
236, 585–589.
39 Brown BE (1982) The form and function of
metal-containing granules in invertebrate tissues. Biol
Rev 57, 621–667.
40 Morgan AJ, Winters C & Stu
¨
rzenbaum SR (1999)
Electron microscopy: X-ray microanalysis techniques.
In Methods in Molecular Medicine Biology (Hajibagheri
N ed), pp. 245–276. Humana Press, Totowa, NJ.
41 Andre J, Charnock J, Stu
¨
rzenbaum SR, Kille P,
Morgan AJ & Hodson ME (2009) Metal speciation in
field populations of earthworms with multi-generational

exposure to metalliferous soils: cell fractionation and
high energy synchrotron analyses. Environ Sci Technol
43, 6822–6829.
42 Vallee BL (1995) The function of metallothionein.
Neurochem Int 27, 23–33.
43 Krezoski SK, Villalobos J, Shaw CF & Petering DH
(1988) Kinetic lability of zinc bound to metallothionein
in Ehrlich cells. Biochem J 255, 48–91.
44 Maret W & Li Y (2009) Coordination dynamics of zinc
in proteins. Chem Rev 109, 4682–4707.
45 Waldron KJ, Rutherford JC, Ford D & Robinson NJ
(2009) Metalloproteins and metal sensing. Nature 460,
823–830.
46 Krizek BA, Merkle DL & Berg JM (1993) Ligand vari-
ation and metal ion binding specificity in zinc finger
peptides. Inorg Chem 32, 937–940.
47 Blindauer CA (2008) Metallothioneins with unusual
residues: histidines as modulators of zinc affinity and
reactivity. J Inorg Biochem 102, 507–521.
48 Blindauer CA, Razi MT, Campopiano DJ & Sadler PJ
(2007) Histidine ligands in bacterial metallothionein
enhance cluster stability. J Biol Inorg Chem 12, 393–
405.
49 Zhou YJ, Zhang NJ, Li LY & Ru BG (2000) The
cysteine-to-histidine substitution in b domain of
metallothionein enhances its zinc binding ability.
Protein Pept Lett 7, 9–16.
50 Romero-Isart N, Cols N, Termansen MK, Gelpi JL,
Gonzalez-Duarte P, Atrian S & Capdevila M (1999)
Replacement of terminal cysteine with histidine in the

metallothionein a and b domains maintains its binding
capacity. Eur J Biochem 259, 519–527.
51 Harris RK, Becker ED, Cabral de Menezes SM,
Goodfellow R & Granger P (2001) NMR nomenclature.
Nuclear spin properties and conventions for chemical
shifts (IUPAC Recommendations 2001). Pure Appl
Chem 73, 1795–1818.
52 Langdon CJ, Hodson ME, Arnold RE & Black S
(2005) Survival, Pb-uptake and behaviour of three
species of earthworm in Pb treated soils determined
using an OECD-style toxicity test and a soil avoidance
test. Environ Pollut 138, 368–375.
53 Leszczyszyn OI, Schmid R & Blindauer CA (2007)
Toward a property ⁄ function relationship for
metallothioneins: histidine coordination and unusual
cluster composition in a zinc-metallothionein from
plants. Proteins 68, 922–935.
Supporting information
The following supplementary material is available:
Fig. S1. Amino acid sequence and expression ⁄
purification of recombinant Caenorhabditis elegans
MTs.
Fig. S2. Deconvoluted ESI mass spectra of Caenor-
habditis elegans Cd
6
Zn-CeMT-1 species (obtained
using the method described for Cd
7
-CeMT-2 in Experi-
mental procedures).

Fig. S3. Cd K-edge EXAFS spectra and Fourier
transforms of Caenorhabditis elegans strains.
Fig. S4. Zn K-edge EXAFS spectra and Fourier
transforms of Caenorhabditis elegans strains.
Table S1. Total Cd and Zn contents in acid-digested
nematodes.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al.
2542 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS