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

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],

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.

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

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

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

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

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,

. 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
˚

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-

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

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-

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
*
**
*
*
**
**
**
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

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

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,

-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,

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

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

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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.
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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