Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, 08071-Barcelona, Spain and 1 Departament de Química, Universitat Autònoma de Barcelona, 08193-Bellaterra, Barcelona, Spain
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Abstract |
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Keywords: ß domain/in vivo copper binding/in vivo cadmium binding/metallothionein/recombinant expression
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Introduction |
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Mammalian MT, the most evolved form, folds over metal atoms constituting two domains: ß, the N-terminal half, and , the C-terminal half. The ß domain includes nine Cys residues, mostly in an alternate pattern, -NCNCNor -NCNNCN-, and binds three divalent ions (Zn or Cd), giving rise to a M3(SCys)9 aggregate where each metal ion is tetrahedrally coordinated. The
domain comprises 11 Cys, in an -NCNCN- or -NCCNCC- array, and it is able to bind four divalent ions tetrahedrally, affording a M4(SCys)11 aggregate (Otvos and Armitage, 1980
; Furey et al., 1986
). These features are generally used to classify non-mammalian MT as
domain-like or ß domain-like peptides (Nemer et al., 1985
). According to this criterion, yeast and fungal MT, the most primitive eukaryotic forms, are constituted by single ß domains (Peterson et al., 1996
), crustacean MT has two (ßß) domains (Narula et al., 1995
), eqinodermal MT is made of two (
ß) domains (Wang et al., 1995
) and vertebrate MT shows two (ß
) domains. From these data, it seems plausible that duplication and divergence of an ancestral ß unit may have led to the present situation in crustacean, eqinodermal and vertebrates. If we consider their metal-chelating properties, fungal MT appears optimized for Cu-binding (Winge et al., 1985
), whereas divalent ions, especially cadmium, are preferentially bound by the mammalian forms. Not only has the high stability of the Cd4
MT mammalian cluster been assessed in vitro (Bernhard et al., 1986
; Capdevila et al., 1997
), but dramatic decreases in cadmium resistance in transformed cells has also been reported for mutants where the
domain is nonfunctional (Chernaik and Huang, 1991
). A housekeeping metal-regulatory role for MT in lower organisms is supported by the affinity of native ßMT forms for physiological metals (Cu, Zn). This function may have been extended to a detoxifying capacity, mainly acquired by the new
domain. Coordination studies have shown the differential chelating behaviour of both MT portions towards these metals in vitro (Nielson and Winge, 1984
; Nielson et al., 1985
; Okada et al., 1985
, 1986
; Li and Otvos, 1996
), but genetic engineering strategies allow characterization of the metal binding ability in vivo. We have previously shown that both mammalian recombinant domains and the entire MT are able to coordinate Zn, leading to Zn3ßMT, Zn4
MT and Zn7MT respectively (Capdevila et al., 1997
; Cols et al., 1997
), and time-coincident studies have also been reported for the same species (Xiong and Ru, 1997
). The corresponding MT aggregates were recovered from a GSTMT fusion construct rendered by a bacterial heterologous expression system, previously described for plant and cyanobacterial MT (Tommey et al., 1991
; Shi et al., 1992
). Although the recombinant Cd4
MT complex has been described, attempts to synthesize heterologous ßMT in Cd-supplemented media were reported unsuccessful (Sewell et al., 1995
; Kurasaki et al., 1996
; Kurasaki et al., 1997
). Furthermore, no data about in vivo mammalian
or ßMT copper binding have been reported up to now. Here we describe the synthesis of mouse ßMT in E.coli cells grown in the presence of Cu and Cd, and provide data on the corresponding metal clusters. The factors inhibiting expression when cadmium is present in the culture medium have also been addressed.
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Materials and methods |
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In order to express the ßMT domain as a Cu- and Cd-complex, E.coli strains JM105 (protease proficient) and BL21 (lon and ompT protease deficient) were transformed with the plasmid pGEX-4T-ßMT (Capdevila et al., 1997). To synthesize CußMT or CdßMT, E.coli cultures were supplemented with CuSO4 or CdCl2, to final concentrations of 300 and 500 µM Cu2+, or 100 and 300 µM Cd2+, respectively. To verify whether ßMT production was possible in non-metalsupplemented media, cultures without added metals were performed. As a control, the
MT domain was also assayed for expression in cadmium-supplemented media. Basic procedures for the expression and purification of the ßMTmetal complexes were as reported (Capdevila et al., 1997
). Aliquots of the protein-containing FPLC fractions were separated by 15% SDSPAGE, which were stained with Coomassie blue. Positive samples were pooled, and aliquots were stored at 70°C for further analysis.
Analysis of the binding properties of in vivo synthesized CußMT and CdßMT
Inductively coupled plasma-atomic emission spectrometry (ICP-AES), in a Thermo Jarrell Ash, Polyscan 61E (measuring S at 182.0 nm, Zn at 231.8 nm, Cd at 228.8 nm and Cu at 324.7 nm) was used to assess S, Zn, Cd and Cu contents, to calculate the amount of protein present in the preparation and the metal-to-protein ratios. Additionally, the reaction with DTNB [5,5'-dithiobis(nitrobenzoic acid)], as described elsewhere (Birchmeier and Christen, 1971), allowed us to determine the percentage of thiol groups over total sulfur in our samples except for those containing Cu, as it is widely accepted that the presence of this metal in MT prevents determination of SH content by Ellman's method (Winge, 1991
).
Electrospray ionization mass spectrometry (ESI-MS) was performed on a Platform II (Micromass) equipped with Max Lynx software, calibrated using horse heart myoglobin (0.1 mg/ml). Different sets of assay conditions were required depending on the sample. For apo-ßMT: source temperature, 60°C; capillary-counterelectrode voltage, 3.5 kV; lenscounterelectrode voltage, 0.5 kV; cone potential, 40 V; m/z range, 600 to 1700; scan rate, 4 s/scan; interscan delay, 0.2 s; and the carrier, a 1:1 mixture of acetonitrile and a 0.05% trifluoroacetic acid solution, at pH 2.5. For CußMT: source temperature, 120°C; capillary-counterelectrode voltage, 4.5 kV; lens-counterelectrode voltage, 1 kV; cone potential, 35 V; m/z range, 700 to 2000; scan rate, 4 s/scan; interscan delay, 0.2 s; and the carrier, a 5:95 solution of methanol and 3 mM ammonium formate/ammonia, at pH 7.0. To avoid the masking effect of Tris over CußMT, samples were dialyzed for 90 min against 2.5 mM TrisHCl, pH 7.0, using Pierce Slide-A-Lyzer membranes (cut-off: 2000) prior to injection. For CdßMT: source temperature, 120°C; capillary-counterelectrode voltage, 4.5 kV; lens-counterelectrode voltage, 1 kV; cone potential, 35 V; m/z range, 1000 to 1800; scan rate, 5 s/scan; interscan delay, 0.5 s; and the carrier, a 5:95 solution of methanol and 3 mM ammonium formate/ammonia at pH 7.5.
Electronic absorption measurements were performed on a HP-8452A Diode array UV-visible spectrophotometer. A Jasco Spectropolarimeter (J-715) interfaced to a computer (GRAMS/32 Software) was used for circular dichroism determinations.
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Results |
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We had previously shown that successful biosynthesis of the ßMT domain, as well as that of MT, was achieved in Zn-supplemented media using both JM105 and BL21 E.coli strains, and as expected the latter always rendered higher protein yield (Capdevila et al., 1997
). In the present study, however, markedly different results were obtained for the synthesis of ßMT in the presence of other metals. When the pGEX-4T-ßMT construct was expressed in E.coli JM105, the GSTßMT fusion protein was recovered at comparable yields in both non-metal and Zn2+-supplemented media (Figure 1
, lanes 2 and 3); showed significant levels of synthesis when the medium was Cu2+-supplemented (lane 5); and was almost negligible in the assays of Cd2+-supplemented media (lane 4). Unlike ZnMT and CdMT, the yield of Cu(I)MT synthesis is affected by the required reduction of the Cu(II) available to the E.coli cell prior to the formation of the corresponding clusters. To further test the behaviour of the recombinant ßMT in the presence of cadmium, the same construct was transformed and tested for expression in BL21 cells. Figure 2
shows the PAGE results for GSTßMT, and GST
MT as a control, for the two E.coli strains in Cd-supplemented media. It is well reported that pure, homogenous, metal-loaded MT migrates heterogeneously on SDSPAGE, often rendering diffuse bands, and shows an electrophoretic mobility much lower (apparent 14 kDa) than that corresponding to the molecular mass of the apo-form (3.5 kDa), due to the fact that the SDS sample and gel conditions are not able to cause full denaturation of the metalprotein clusters, and thus the protein migrates as a metal aggregate along the gel (McCormick et al., 1991). The behaviour of the
MT fusion protein was similar to that in Zn medium, with significant production from both strains (lanes 14), but higher amounts in BL21 (lanes 3 and 4), and a slight reduction at higher cadmium concentrations. However, while the ßMT fusion protein was again practically absent from the homogenates of JM105 cells grown in cadmium (lanes 5 and 6), amounts comparable to those obtained for GST
MT were recovered from the BL21 host (lanes 7 and 8). These results clearly indicate that recombinant synthesis of the ßMT domain in cadmium-supplemented media was essentially dependent upon the E.coli host strain.
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CußMT was obtained by thrombin digestion of the GSTßMT protein recovered from E.coli JM105 cells grown in the presence of 500 µM Cu2+. The metal content of the samples, determined by ICP-AES, was 6.3 to 6.8 Cu atoms per total sulfur content, which is consistent with the stoichiometry reported for the native mammalian ßMT (Nielson and Winge, 1985). Thus, our results showed that when the ßMT domain was recombinantly synthesized in the presence of copper, it was recovered as a fully loaded CußMT species. Comparison of the CD spectrum with those of the CußMT species generated in vitro by Zn/Cu replacement (Figure 3
) (Bofill et al., 1999
) showed that the sample recovered from biosynthesis of the ß domain in Cu-supplemented media contained Cu6ßMT and Cu7ßMT. Specifically, the CD spectral trace of the in vivo CußMT species precisely matched the computer-generated spectrum corresponding to 75% of Cu7ßMT plus 25% of Cu6ßMT. This explains the Cu/protein ratios of 6.3 and 6.8 found by ICP-AES. The molecular mass of the single peak detected by ESI-MS of the CußMT sample was 3599.27 (data not shown), which is consistent with the expected value of 3596.36 for the Cu7ßMT species (apoßMT + 7 Cu7H). The peak corresponding to Cu6ßMT probably remains undetectable due to the low contribution of this species (25%) and to the excessive background produced by the Tris buffer in the sample, which cannot be exhaustively dialyzed if integrity of the Cu(I) aggregates is to be preserved. Thus, our data pointed towards a major formation of a Cu7ßMT cluster inside the cell.
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In order to analyze the features of the in vivo-structured Cd-cluster, and compare it with the Cd3ßMT generated in vitro from Zn3ßMT by Zn/Cd replacement, fusion GSTßMT was purified from total protein extracts of BL21 cells grown in 300 µM cadmium medium, and used to recover the ßMT portion. Two types of culture were set up: small-scale 0.5 l cultures in Erlenmeyer flasks, grown in a New Brunswick Orbital Incubator, and large-scale 30 l cultures grown in a Biostat U (Braun Biotech) Fermentor. In both cases approximately 0.50.9 mg of the ß peptide were recovered per litre of culture after thrombin digestion and FPLC purification. ICP-AES measurements of the cadmium versus total sulfur content invariably yielded a ratio of 1.351.40, in contrast to the stoichiometric relationship of 3 reported for the Cd3ßMT species generated in vitro. Neither Zn nor Cu was detected in the sample. Cadmium content was consistent with a 6063% oxidation rate indicated by the DTNB reaction for samples purified from both types of culture. So far, the results point to the presence of undermetaled cadmium species, due to partial oxidation of the thiol groups. Surprisingly, further characterization revealed a different composition of the ßMT preparations obtained from the different culture conditions.
The CD spectrum of the CdßMT species obtained from small-scale cultures paralleled that of the Cd3ßMT species generated in vitro from Zn3ßMT (Figure 4), which indicated the presence of some well-structured Cd3ßMT clusters. The mass of these molecules was 3489.74 at pH 7.0, which coincides with the expected value of 3489.74 for Cd3ßMT (apo-ßMT + 3 Cd 6 H) (Figure 5A
). Acidification of this sample yielded an apo-form with a molecular mass of 3159.69, which agrees well with the calculated value of 3158.54 (data not shown).
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Discussion |
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In vivo, recombinant ßMT binds copper to form aggregates comparable with those obtained in vitro by Zn/Cu replacement from Zn3ßMT (Bofill,R., Capdevila,M., Gonzàlez-Duarte,P., Palacios,O., Cols,N., Gonzàlez-Duarte,R. and Atrian,S., manuscript submitted). The coexistence of Cu7ßMT (75%) and Cu6ßMT (25%) species in the samples recovered from the transformed E.coli cells could be due to several factors: (i) equilibrium between the two species inside the cell; (ii) a partial loss of the seventh copper ion during sample manipulation; or (iii) the restrictive source of Cu(I) ions. The formation of the Cu7ßMT cluster in vivo, as revealed by ESI-MS and CD spectroscopy, is consistent with previous in vitro copper-coordination data (Nielson and Winge, 1985), and reinforces the hypothesis of the primeval copper affinity of the ß domain.
In contrast, CdßMT aggregates were recovered only in special conditions: in a protease-deficient E.coli strain, BL21, and expressed as a fusion protein, GST-tailed. The almost complete absence of CdßMTGST fusion protein in JM105 cells (Figures 1 and 2) fully agrees with reported results (Sewell et al., 1995
; Kurasaki et al., 1996
, 1997
). In addition, the stability of the CdßMT aggregates was found to be dependent upon culture conditions. On the one hand, preparations obtained from small-scale cultures were a mixture of approximately one third Cd3ßMT, with identical structure to that generated in vitro through Zn/Cd replacement (Capdevila et al., 1997
), and two thirds of fully oxidized protein, devoid of Cd(II) ions. On the other, large-scale cultures did not produce the Cd3ßMT species, but probably less structured and partially oxidized CdßMT aggregates. Our data are also in accordance with several in vitro ßMT cadmium-binding studies, showing that the apo-ßMT peptide is not prone to form de novo stable Cd3 clusters (Nielson and Winge, 1985
; Kull et al., 1985; Stillman et al., 1987
).
It is tempting to speculate on the lack of ßMT recovery from cadmium cultures. The finding that a protease-deficient host could yield high amounts of the ß domain argues in favour of a post-translational proteolytic degradation of ßMT. Indeed, the evidence that ßMT mRNA is present in transformed E.coli JM105 cells, which never accumulates the recombinant peptide, was reported by Kurasaki et al. (1996). Furthermore, the low affinity of the ß peptide towards cadmium would impair correct folding or lead to conformational changes and so favour its vulnerability to proteolysis. Finally, it had been shown that hydrolysis of thioamide peptides by carboxypeptidase A increases 240970% in the cadmium-substituted enzyme (Bond et al., 1986). This is consistent with the hypothesis that cadmium, a thiophilic metal, increases the positive character of the carbonyl carbon and facilitates the nucleophilic attack of substrates by the proteolytic metalloenzymes.
From the present data, functional and evolutionary implications could also be drawn. If primeval ßMT forms were selected on the basis of their contribution to homeostasis of physiological metals, mainly Cu and Zn, this particular feature would have been retained by the present mammalian ß domain, although probably blurred by its interaction with the counterpart. This has previously been shown for Zn (Capdevila et al., 1997
), and is now confirmed for Cu. As a further evolutionary step, the duplication of the ßMT domain may have led to a derived
MT peptide, which conferred upon the organisms the capacity to respond to newly encountered toxic metals, such as cadmium. This would account for the poor in vivo reactivity of ßMT towards cadmium, as also described in this work, mainly if compared with its affinity for metals associated with life processes, such as Cu or Zn. Further in-depth investigation of the interaction mechanisms between different metal ions and the ßMT thiol groups should be now facilitated by the availability of functional recombinant ßMT peptide.
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Acknowledgments |
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Notes |
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* Made equal contributions to this study
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References |
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Received April 28, 1998; revised November 23, 1998; accepted December 11, 1998.