Báo cáo khóa học: Chemical foundation of the attenuation of methylmercury(II) cytotoxicity by metallothioneins pptx

Chemical foundation of the attenuation of methylmercury(II)
cytotoxicity by metallothioneins
A
`
ngels Leiva-Presa
1
, Merce
`
Capdevila
1
, Neus Cols
2
, Silvia Atrian
2
and Pilar Gonza
´
lez-Duarte
1
1
Departament de Quı
´
mica, Facultat de Cie
`
ncies, Universitat Auto
`
noma de Barcelona, Spain;
2
Departament de Gene
`
tica,
Facultat de Biologia, Universitat de Barcelona, Spain

To elucidate the chemical interactions underlying the role
of metallothioneins (MTs) in reducing the cytotoxicity
caused by MeHg(II), we monitored in parallel by electronic
absorption and CD spectroscopies the stepwise addition of
MeHgCl stock solution to mammalian Zn
7
-MT1 and the
isolated Zn
4
-aMT1 and Zn
3
-bMT1 fragments. The incor-
poration of MeHg
+
into Zn
7
-MT and Zn
3
-bMT entails
total displacement of Zn(II) and unfolding of the protein.
However, both features are only partial for Zn
4
-aMT. The
different behavior observed for this fragment, whether iso-
lated or constituting one of the two domains of Zn
7
-MT,
indicates interdomain interactions in the whole protein.
Overall, the binding properties of Zn
7

-MT, Zn
4
-aMT and
Zn
3
-bMT toward MeHg
+
are unprecedented. In addition,
the sequestration of MeHg
+
by Zn
7
-MT and the con-
comitant release of Zn(II) are probably two of the main
contributions in the detoxifying role of mammalian MT.
Keywords: methylmercury(II) binding; methylmercury(II)
toxicity; methylmercury(II)–metallothionein; a-metallothio-
nein; b-metallothionein.
Mercury is a widespread contaminant that enters the
environment from a variety of sources including industrial
processes and hazardous waste sites. The ability of aquatic
micro-organisms to convert metallic mercury into the
methylmercury(II) cation (MeHg
+
) is the key to its
accumulation in fish, which then become a common source
of exposure of humans to MeHg
+
[1,2]. Whereas the
damaging pathological and biochemical consequences of

MeHg
+
in humans have long been known, current studies
are focusing on the effects of MeHg
+
on the central nervous
system [3] and male fertility [4]. In both cases, a role for
metallothioneins (MTs) in attenuating the cytotoxicity
caused by MeHg
+
has been proposed [5–7]. A main feature
of MTs, a family of ubiquitous low molecular mass proteins,
is their extremely high content of cysteine residues. These
bind to metal centers enabling them to serve as a heavy-
metal-detoxification system [8]. Considering the abundance
of MTs in the central nervous system and the preference of
Hg(II) ions for soft sulfur ligands, the study of MeHg–MT
species from a chemical perspective is warranted.
Although the interaction of MTs with Hg(II) ions has
long been established [9], elucidation of the binding features
of Hg-MT species has been hampered by the inherent
difficulties of Hg(II) thiolate chemistry, which mainly arise
from the diverse coordination preferences of Hg(II) and the
various ligation modes of the thiolate ligands [10,11].
Nevertheless, the analysis of Hg(II) binding to MTs has
been intensively studied [9]. In contrast, the chemistry of
MeHg(II)–MT complexes has attracted much less attention.
Earlier studies found MT to have no significant role in the
detoxification of MeHg
+

[12]andtobeunabletobindto
MeHg
+
either in vivo or in vitro [13]. Subsequent attempts
to induce brain MT by exposure to MeHg
+
gave incon-
sistent results: MT concentrations remained unchanged
in rats [14,15], whereas MT and mRNA concentrations
increased in MeHg
+
-treated rat neonatal astrocyte cultures
[16]. However, there is mounting evidence that induction of
MTs in astrocytes attenuates and even reverses the
cytotoxicity caused by MeHg
+
[5,6], indicating binding of
MeHg
+
by an astrocyte-specific MT isoform, MT1 [17].
Existing data on Hg(II)–MT species cannot be extended
to MeHg–MT complexes mainly because of the different
coordination chemistry of Hg(II) and MeHg
+
towards
thiolate ligands and thus towards the cysteine residues
responsible for metal coordination in MTs. The coexistence
of digonal, trigonal-planar and tetrahedral coordination
geometries together with the presence of secondary mer-
cury–sulfur interactions are common features in the chem-

istry of Hg(II) thiolates [10,11]. In contrast, MeHg
+
shows
a clear preference to form essentially linear two-coordinate
Hg(II) complexes with thiolate ligands, even if, in some
cases, secondary interactions at the metal center are
observed [18,19]. As part of our development of the metal-
binding properties of MTs and with the aim of contri-
buting to the study of MeHg–MT species from a chemical
perspective, we investigated the behavior of MeHgCl
towards mammalian MT1 protein. We report the spectro-
scopic features of the species generated by replacing Zn(II)
with MeHg
+
in recombinant mouse Zn
7
-MT1, and in the
Correspondence to P. Gonza
´
lez-Duarte, Departament de Quı
´
mica,
Facultat de Cie
`
ncies, Universitat Auto
`
noma de Barcelona,
E-08193 Bellaterra, Barcelona, Spain.
Fax: + 34 935813 101, Tel.: + 34 935811 363,
E-mail:

Abbreviations: eq, equivalents; MT, metallothionein; ICP-AES,
inductively coupled plasma atomic emission spectrometry.
(Received 18 December 2003, revised 4 February 2004,
accepted 16 February 2004)
Eur. J. Biochem. 271, 1323–1328 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04039.x
corresponding Zn
4
-aMT1 and Zn
3
-bMT1 independent frag-
ments.Inaddition,thepossiblecorrelation between the results
described here and the protective role of MTs in MeHg-
induced cytotoxicity is discussed.
Materials and methods
Protein preparation and characterization
Fermentator-scale cultures, purification of the glutathione-
S-transferase–MT fusion proteins, and recovery and ana-
lysis of the recombinant mouse Zn
7
-MT1, Zn
4
-aMT1and
Zn
3
-bMT1 domains were performed and obtained in 50 m
M
Tris/HCl buffer (pH 7) as previously described [20–22]. The
molecular mass of the three Zn-proteins (Table 1) was
determined by electrospray ionization MS on a Fisons
Platform II Instrument (VG Biotech) calibrated using horse

heart myoglobin (0.1 mgÆmL
)1
). Assay conditions were:
source temperature, 120 °C; capillary counter electrode
voltage, 4500 V; lens counter electrode voltage, 1000 V;
cone potential, 35 V; m/z range, 1000–1800; scanning rate,
5 s per scan; interscan delay, 0.5 s. The running buffer was
an appropriate mixture of acetonitrile and 5 m
M
ammo-
nium/acetate ammonia, pH 7.5. The molecular mass of
the apo-forms was determined under the same conditions
except that the carrier was a 1 : 1 mixture of acetonitrile and
trifluoroacetic acid, pH 1.5. The total sulfur content of the
samples and their zinc content were also determined by
inductively coupled plasma atomic emission spectrometry
(ICP-AES) using a Thermo Jarrell Ash
1
Polyscan 61E
(Thermo Electron Corporation, Barcelona, Spain) at
182.0 nm (S) or 213.9 nm (Zn) without any previous
treatment of the samples [23]. Protein stock solution
concentrations were determined from measurement of
thiol groups over total sulfur using the reagent 5,5¢-dithio-
bis(nitrobenzoic acid) in 3
M
guanidine hydrochloride [24]
taking into account the details reported previously [20].
Very good agreement between total sulfur determination by
ICP–AES and SH content by Ellman’s method was

obtained. Protein solutions had a final concentration of
54.8 l
M
(MT), 127 l
M
(aMT fragment) and 302 l
M
(bMT
fragment). These were diluted to a final concentration of
 10 l
M
(MT) or 20 l
M
(a and b fragments) with Milli-Q-
purified and Ar-degassed water before being titrated with
MeHgCl solutions at 25 °C.
Metal solutions
CAUTION: Methylmercury compounds are extremely
toxic. All direct contact must be avoided by using suitable
protective measures such as wearing special gloves.
All solutions used in MeHg
+
binding were prepared with
Milli-Q-purified water and were either argon saturated or
vacuum degassed before use. Glassware was cleaned with
10% (v/v) nitric acid and repeatedly rinsed with ultrapure
water. A commercial MeHgCl standard solution of
1000 p.p.m. (pH 5–6) (Sigma-Aldrich) was used as titrating
agent.
Metal ion binding reactions

Metal-binding experiments were carried out by sequentially
adding molar-ratio aliquots of concentrated MeHgCl stock
solutions to single solutions of the Zn
7
-MT, Zn
4
-aMT and
Zn
3
-bMT proteins. Titrations were monitored in parallel by
optical CD and UV-vis spectroscopies, and, at each titration
point, the optical spectra were recorded every 10 min until
saturation of the spectral traces, before continuation with
the titration. Electronic absorption (UV) measurements
were performed on an HP-8452A diode array. A Jasco
spectropolarimeter (model J715) interfaced with a computer
was used for CD measurements. A Peltier PTC-351S
maintained the temperature at 25 °C. All spectra were
recorded with 1 cm capped quartz cuvettes, corrected for
the dilution effects, and processed using the program
GRAMS
32.
All manipulations involving the protein and metal ion
solutions were performed in Ar atmosphere, and titrations
were carried out at least in duplicate to ensure the
reproducibility of every single point.
Results and Discussion
The experimental results were obtained by monitoring by
CD and UV-vis spectroscopies the sequential addition
of MeHgCl stock solution to recombinant mammalian

Table 1. Amino-acid sequence of the three recombinant mouse MT1 peptides and molecular masses of the corresponding Zn and apo forms.
Experimental molecular masses were measured by electrospray ionization MS at pH 7.0 or 3.0, respectively. Calculated molecular masses for
neutral species with loss of two protons per zinc bound corresponded to the canonical Zn
7
-MT1, Zn
3
-bMT1 and Zn
4
-aMT1 aggregates [34]. The
recombinant proteins contained two extra N-terminal amino acids (N-GS) which have been shown not to interfere with the metal-binding features
of MT1 [20,21].
Molecular mass (Da)
Apo form Zn form
Expected Calculated Expected Calculated
Full-length MT1
GSMDPNCSCSTGGSCTCTSSC
ACKNCKCTSCKKSCCSCCPVGCSKCAQGCVCKGAADKCTCCA
6159.35 6162.13 6603.44 6605.72
bMT1 domain
GSMDPNCSCSTGGSCTCTSSCACKNCKCTSCK 3159.69 3158.58 3348.70 3348.70
aMT1 domain
GSKSCCSCCPVGCSKCAQGCVCKGAADKCTCCA 3296.82 3295.48 3550.80 3549.50
1324 A
`
. Leiva-Presa et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Zn
7
-MT. In addition, with the aim of facilitating knowledge
on the behavior of the whole protein, the MeHg
+

binding
abilities of the isolated Zn
4
-aMT and Zn
3
-bMT fragments
were also studied by analogous procedures. The two
spectroscopic techniques, CD and UV-vis, used in the study
of the metal-binding features of MT [8,9], have already been
used to analyse the binding features of the same Zn
7
-MT
protein in the presence of Cd(II) [20,21], Cu(I) [22,25], Ag(I)
[22,26] and Hg(II) [27]. These techniques provide informa-
tion on the stoichiometry and degree of folding of the
predominant metal–MT species present in solution at each
titration point as well as on the number of species formed
during the titration. In addition, similar CD features for
different metal–MT species indicate comparable 3D struc-
tures. However, the comparative analysis of the CD and
UV-vis spectra recorded during the titration of Zn
7
-MT
(Fig. 1), Zn
4
-aMT (Fig. 2) and Zn
3
-bMT (Fig. 3) with
MeHg
+

reveals that the behavior of these proteins in the
presence of MeHg
+
is unprecedented when compared with
previous findings with other metal centers [20–22,25–27],
including the Hg(II) ion [27].
CD spectra analysis
Consideration of the CD data recorded during the addition
of MeHg
+
to Zn
7
-MT (Fig. 1A,B), Zn
4
-aMT (Fig. 2A,B)
or Zn
3
-bMT (Fig. 3A,B) indicates that the Zn/MeHg
replacement in the three proteins essentially follows a
common pattern. The only exception is observed for the
Zn
4
-aMT fragment, which shows some differences from
the other two proteins in the last stages of the titration.
Overall, the addition of MeHg
+
equivalents (eq) to the
three proteins is accompanied by the gradual loss of the
characteristic CD fingerprint corresponding to zinc-loaded
mammalian MTs, which consists of an exciton coupling

with a crossover point at  240 nm [28]. Not only is the
decrease in intensity of this signal not concomitant with the
appearance of new bands, but the decrease continues to
the end of the titration, which is identified by the saturation
of the CD features. This occurs for the addition of 16
MeHg(II) eq to the aMT fragment, 14 to the bMT
fragment, and 22 to the whole MT. At this point, the shape
of the CD envelopes for the two latter proteins closely
resembles that of the corresponding apo-MT forms, which
have no 3D structure and thus show no CD features [28].
Accordingly, the absence of CD bands indicates that the
interaction of MeHg
+
has probably caused complete
unfolding of the whole protein (Fig. 1B) as well as of the b
fragment (Fig. 3B). However, this unfolding is only partial
for aMT, as shown by the maintenance of a low intensity
signal corresponding to Zn(SCys)
4
chromophores even
after the addition of 16 MeHg
+
eq to Zn
4
-aMT (Fig. 2B).
Overall, although the CD data show that the addition of
MeHg
+
entails complete loss of the Zn(II) ions initially
bound to Zn

7
-MT and Zn
3
-bMT, and only partial loss in
the case of Zn
4
-aMT, they do not provide direct evidence
of the incorporation of MeHg
+
into these proteins.
Moreover, the CD data indicate that the binding features
of the a domain are not coincident when it is part of the
whole protein or, alternatively, an isolated fragment. Thus,
Fig. 1. (A, B) Circular dichroism, (C) UV-vis absorption, and (D–F) difference absorption spectra recorded during the titration of a 9.993 l
M
Zn
7
-MT
solution with MeHgCl. The latter are obtained by subtracting the successive spectra of (C).
Ó FEBS 2004 Interaction of MeHg
+
with metallothionein (Eur. J. Biochem. 271) 1325
Fig. 2. (A, B) Circular dichroism, (C) UV-vis absorption, and (D–F) difference absorption spectra recorded during the titration of a 20.021 l
M
Zn
4
-aMT solution with MeHgCl. The latter are obtained by subtracting the successive spectra of (C).
Fig. 3. (A, B) Circular dichroism, (C) UV-vis absorption, and (D–F) difference absorption spectra recorded during the titration of a 20.230 l
M
Zn

3
-bMT solution with MeHgCl. The latter are obtained by subtracting the successive spectra of (C).
1326 A
`
. Leiva-Presa et al.(Eur. J. Biochem. 271) Ó FEBS 2004
the complete unfolding of the whole MT (Fig. 1A,B)
requires the loss of the 3D structure in both constitutive a
and b domains, but this does not occur for the isolated
aMT fragment. This different behavior is consistent with
the presence of interdomain interactions in the whole
protein.
UV-vis absorption spectra analysis
Evidence for the incorporation of MeHg
+
into Zn
7
-MT
(Fig. 1C), Zn
4
-aMT (Fig. 2C) or Zn
3
-bMT (Fig. 3C) is
provided by the UV-vis spectra. These show that the
addition of MeHg
+
to the protein-containing solutions is
accompanied by an increase in absorption covering the
wavelength range of the study, and thus by the formation
of new chromophores. However, more information about
this interaction can be obtained from the difference UV-vis

absorption spectra, which are obtained by subtracting
successive UV-vis absorption curves and thus provide
information on the chromophores appearing and/or disap-
pearing after each addition of MeHg
+
.Onthisbasis,the
evolution of Zn
7
-MT (Fig. 1D–F), Zn
4
-aMT (Fig. 2D–F)
and Zn
3
-bMT (Fig. 3D–F) in the presence of MeHg
+
follows a common pattern for the three proteins. Also, the
loss of absorption in the range 220–230 nm, as recorded
after addition of the first MeHg
+
eq to Zn
7
-MT (Fig. 1D),
Zn
4
-aMT (Fig. 2D) and Zn
3
-bMT (Fig. 3D), is indicative
of the loss of Zn(SCys)
4
chromophores and thus of the

removal of Zn(II) from the corresponding proteins.
Although these two features, the common evolution of the
three MTs and the loss of Zn(II) ions, are fully consistent
with those inferred from the CD data, evidence for the
binding of MeHg
+
to MT becomes apparent only through
the difference UV-vis absorption spectra.
Therefore, the binding of MeHg
+
to Zn
7
-MT, Zn
4
-aMT
and Zn
3
-bMT is evidenced by the difference UV-vis
absorption band centered at 250 nm together with a
shoulder at higher wavelengths, both features appearing
from the first stages of the titration. Remarkably, further
additions of MeHg
+
up to the end of the titration do not
give rise to new absorption bands. The maintenance of the
same contributions from the beginning to the end indicates
that only one main chromophore involving MeHg
+
is
formed during the three titrations. On the basis of the strong

preference of the MeHg
+
cation for digonal coordination
to thiolate ligands [29], it is reasonable to propose that this
linear geometry is prevalent in the (MeHg)
x
–MT species.
Linear coordination geometry would be compatible not
only for MT species with a molar MeHg
+
/SCys
–
ratio £ 1,
where the cysteine residues would behave as terminal
ligands, but also for those where this ratio is greater than 1,
as in this case the cysteine residues would behave as bridging
ligands. This behavior would be consistent with the striking
ability of thiolate sulfur to bridge two mercury atoms, as
found in thiolate complexes with R¢Hg
+
cations, R¢ ¼ Me
or Ph [19,30,31].
The above results on the binding of MeHg
+
to Zn
7
-MT,
Zn
4
-aMT and Zn

3
-bMT cannot be easily compared with
those obtained from the titration of the same proteins with
Hg(II), which is consistent with the different behavior of the
two cations toward thiolate ligands. Thus, displacement of
Zn(II) by the addition of HgX
2
(X ¼ Cl
–
,ClO
4
–
) entails
formation of a wide family of heterometallic Zn
x
Hg
y
–MT
and homometallic Hg
y
–MT aggregates, each enfolding
diverse coordination geometries, tetrahedral, trigonal-pla-
nar and digonal, about Hg(II) [10,11]. Moreover, the only
data in the literature on the spectroscopic fingerprints of the
species formed by the interaction of MeHg
+
with mam-
malian MT are difficult to compare because of the different
experimental conditions used [12]. The scarcity of data on
MeHg

+
–MT species is also noteworthy, which may be due
to the serious difficulties involved in the manipulation of
MeHg
+
compounds.
Overall, combination of CD and UV-vis data has allowed
us to establish that the MeHg
+
cation replaces Zn(II)
in recombinant mammalian Zn
7
-MT, Zn
4
-aMT and Zn
3
-
bMT with the concomitant unfolding of the MT proteins.
Earlier results indicating that the binding of MeHg
+
to MT
is either very weak [12] or even nonexistent in vivo and
in vitro [13] are not consistent with the data reported here.
Conversely, the interaction of MeHg
+
with zinc-loaded
mammalian MT species may account for the role of
metallothioneins in attenuating the cytotoxicity caused by
MeHg
+

. Thus, the Zn(II) ions released as a result of the
binding of MeHg
+
to Zn
7
-MT would enable them to
induce the synthesis of more protein, in agreement with the
function of Zn(II) as primary inductor of the synthesis of
MT [32,33]. High concentrations of MT should thus
contribute to the sequestration of MeHg
+
,preventingits
binding to membrane receptors and their subsequent
quenching.
Acknowledgements
Research reported from our laboratories was supported by grants from
the Spanish Ministerio de Ciencia y Tecnologı
´
a (BQU2001-1976 and
BIO2000-0910). We also acknowledge the Servei d’Ana
`
lisi Quı
´
mica,
Universitat Auto
`
noma de Barcelona (CD, UV-vis) and the Serveis
Cientı
´
fico-Te

`
cnics, Universitat de Barcelona (ICP-AES), for allocating
instrument time.
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