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: 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) 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). 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Chemical foundation of the attenuation of methylmercury(II) cytotoxicity by metallothioneins A ` ngels Leiva-Presa 1 ,. elucidate the chemical interactions underlying the role of metallothioneins (MTs) in reducing the cytotoxicity caused by MeHg(II), we monitored in parallel by