Although the interaction of MTs with HgII ions has long been established [9], elucidation of the binding features of Hg-MT species has been hampered by the inherent difficulties of HgII t
Trang 1Chemical foundation of the attenuation of methylmercury(II)
cytotoxicity by metallothioneins
A`ngels Leiva-Presa1, Merce` Capdevila1, Neus Cols2, Silvia Atrian2and Pilar Gonza´lez-Duarte1
1
Departament de Quı´mica, Facultat de Cie`ncies, Universitat Auto`noma de Barcelona, Spain;2Departament 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 CDspectroscopies the stepwise addition of
MeHgCl stock solution to mammalian Zn7-MT1 and the
isolated Zn4-aMT1 and Zn3-bMT1 fragments The
incor-poration of MeHg+into Zn7-MT and Zn3-bMT entails
total displacement of Zn(II) and unfolding of the protein
However, both features are only partial for Zn4-aMT The
different behavior observed for this fragment, whether
iso-lated or constituting one of the two domains of Zn7-MT,
indicates interdomain interactions in the whole protein Overall, the binding properties of Zn7-MT, Zn4-aMT and
Zn3-bMT toward MeHg+are unprecedented In addition, the sequestration of MeHg+ by Zn7-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] and to be unable to bind to 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-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: pilar.gonzalez.duarte@uab.es
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)
Trang 2corresponding Zn4-aMT1 and Zn3-bMT1 independent
frag-ments.Inaddition,thepossiblecorrelationbetweentheresults
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 Zn7-MT1, Zn4-aMT1 and
Zn3-bMT1 domains were performed and obtained in 50 mM
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, 120C; 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 mM
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
(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 3Mguanidine 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 lM(MT), 127 lM(aMT fragment) and 302 lM(bMT
fragment) These were diluted to a final concentration of
10 lM(MT) or 20 lM(a and b fragments) with
Milli-Q-purified and Ar-degassed water before being titrated with MeHgCl solutions at 25C
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 Zn7-MT, Zn4-aMT and
Zn3-bMT proteins Titrations were monitored in parallel by optical CDand 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 CDmeasurements A Peltier PTC-351S maintained the temperature at 25C All spectra were recorded with 1 cm capped quartz cuvettes, corrected for the dilution effects, and processed using the program GRAMS32
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 CDand 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)
Expected Calculated Expected Calculated Full-length MT1
GS MDPNCSCSTGGSCTCTSSC
ACKNCKCTSCKKSCCSCCPVGCSKCAQGCVCKGAADKCTCCA
6159.35 6162.13 6603.44 6605.72 bMT1 domain
GS MDPNCSCSTGGSCTCTSSCACKNCKCTSCK 3159.69 3158.58 3348.70 3348.70 aMT1 domain
GS KSCCSCCPVGCSKCAQGCVCKGAADKCTCCA 3296.82 3295.48 3550.80 3549.50
Trang 3Zn7-MT In addition, with the aim of facilitating knowledge
on the behavior of the whole protein, the MeHg+binding
abilities of the isolated Zn4-aMT and Zn3-bMT fragments
were also studied by analogous procedures The two
spectroscopic techniques, CDand 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 Zn7-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 CDfeatures for
different metal–MT species indicate comparable
3Dstruc-tures However, the comparative analysis of the CDand
UV-vis spectra recorded during the titration of Zn7-MT
(Fig 1), Zn4-aMT (Fig 2) and Zn3-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 CDdata recorded during the addition
of MeHg+to Zn7-MT (Fig 1A,B), Zn4-aMT (Fig 2A,B)
or Zn3-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-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 CDfingerprint 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 CDfeatures 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 CDenvelopes for the two latter proteins closely resembles that of the corresponding apo-MT forms, which have no 3Dstructure and thus show no CDfeatures [28] Accordingly, the absence of CDbands 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 Zn4-aMT (Fig 2B) Overall, although the CDdata show that the addition of MeHg+ entails complete loss of the Zn(II) ions initially bound to Zn7-MT and Zn3-bMT, and only partial loss in the case of Zn4-aMT, they do not provide direct evidence
of the incorporation of MeHg+ into these proteins Moreover, the CDdata 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).
Trang 4Fig 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 -bMT solution with MeHgCl The latter are obtained by subtracting the successive spectra of (C).
Trang 5the complete unfolding of the whole MT (Fig 1A,B)
requires the loss of the 3Dstructure 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 Zn7-MT
(Fig 1C), Zn4-aMT (Fig 2C) or Zn3-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+ On this basis, the
evolution of Zn7-MT (Fig 1D–F), Zn4-aMT (Fig 2D–F)
and Zn3-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 Zn7-MT (Fig 1D),
Zn4-aMT (Fig 2D) and Zn3-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 CDdata, evidence for the
binding of MeHg+to MT becomes apparent only through
the difference UV-vis absorption spectra
Therefore, the binding of MeHg+to Zn7-MT, Zn4-aMT
and Zn3-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 Zn7-MT,
Zn4-aMT and Zn3-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 (X¼ Cl–, ClO ) entails
formation of a wide family of heterometallic ZnxHgy–MT and homometallic Hgy–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 CDand UV-vis data has allowed
us to establish that the MeHg+ cation replaces Zn(II)
in recombinant mammalian Zn7-MT, Zn4-aMT and Zn3 -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 Zn7-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+, preventing its binding to membrane receptors and their subsequent quenching
Acknowledgements
Research reported from our laboratories was supported by grants from the Spanish Ministerio de Ciencia yTecnologı´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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