1Chemical Biology Laboratory, Department of Chemistry and 3Institute of Genetics, Life School, Fudan University, Shanghai 200433, 2Department of Chemistry, Kunming University of Science and Technology, Kunming, 4Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong and 5Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, PR China
6 To whom correspondence should be addressed. e-mail: zxhuang{at}fudan.edu.cn
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Abstract |
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Keywords: EAAEAE insert/human metallothionein-3/neuron growth inhibitory factor
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Introduction |
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Like other members of the MT family, MT3 contains 20 cysteine residues at conserved positions. In addition, it conserves most lysine residues in other mammalian MTs. However, there are two inserts in the MT3 sequence which show a prominent difference from MT1/2: a single Thr in the N-terminal region and an acidic hexapeptide in the C-terminal region (Uchida et al., 1991; Tsuji et al., 1992
). The peptide loop (amino acids 5263) of MT3 reveals
85%
-helical structure according to the secondary structure prediction program SSCP (Eisenhaber et al., 1996
). Additionally, MT3 sequences contain the conserved C(6)-P-C-P(9) motif, which has proved to be essential for the inhibitory activity (Sewell et al., 1995
; Hasler et al., 2000
). Some studies reported that the clusters in MT3 were quite flexible (Faller and Vasak, 1997
; Hasler et al., 1998
; Faller et al, 1999
). Very recently, the spatial structure of the C-terminal region in mouse MT3 has been established by an NMR technique. Noteworthy, it revealed a tertiary fold very similar to MT1/2, except for a loop that accommodates the acidic hexapeptide insert (Oz et al., 2001
). Surprisingly, it was reported that the inhibitory activity of MT3 arises from the N-terminal ß-domain (Sewell et al., 1995
). The functional difference between MT3 and MT1/2 implies the uniqueness of MT3 in structure and property. It was reported that MT3 shows higher metal-binding capacity than MT1/2 in the gas phase (Palumaa et al., 2002
). However, so far, the property of MT3 has not been well studied.
In this communication, we focus on the EAAEAE insert of MT3 and a series of mutants at this site have been generated. These mutants include the EAAEAE-deleted mutant, E(55)E(60), acidic residues replaced mutants E55/58/60Q, helix-broken mutant E55D/A56G/A57G/E58D/A59G/E60D/A61G/E62D, and the domain-replaced mutant ß(MT3)
(MT1). After characterization by ESIMS, the properties of MT3 and its mutants have been investigated by pH titration and reactions with EDTA and DTNB. As a comparison, the properties of monkey MT1 (mkMT1) have been studied under the same conditions.
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Materials and methods |
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Human MT3 (hMT3) cDNA was prepared from cells by reverse transcription followed by polymerase chain reaction (PCR). It was then cloned into vector Bluescript KS(M13-) as a BamHI/EcoRI fragment. Fusion expression vector pGEX-4T-2, of Escherichia coli strain BL21, glutathioneSepharose 4B, Superdex 75 and Sephadex G-25 were the products of Pharmacia Biotech. The restriction enzymes, T4 DNA ligase and DNA polymerase were purchased from New England Biolabs. The gel extraction kit was purchased from Qiagen. Isopropyl ß-D-thiogalactoside, Pfu DNA polymerase, Triton X-100 and cell culture reagents were purchased from Sangon (Shanghai, China). The mkMT1 was expressed and purified in the same way and stored in the chemical biology laboratory of the Chemistry Department of Fudan University. Thrombin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were the products of Sigma. The other reagents were of analytical grade.
Cloning strategy
Site-directed mutagenesis was performed with the overlap extension PCR according to Higucchi et al. (1988). To study the role of the hexapeptide insert, we have prepared a deleted mutant, in which the acidic hexapeptide E(55)-A-A-E-A-E(60) has been deleted from the wild-type protein. Since the hexapeptide insert includes continuous glutamate residues, more attention should be paid to the negative charge. The negative charges have been eliminated in the E55/58/60Q mutant to examine the effect on properties of MT3. As we have mentioned above that the fragment 5263 reveals
85%
-helical structure, we have changed all Glu to Asp and Ala to Gly in preparing the E55D/A56G/A57G/E58D/A59G/E60D/A61G/E62D mutant. In this mutant the ability of helical formation of the segment has been weakened because both Asp and Gly are the
-helix breaker. In this situation we have maintained the characteristic groups and charges of each amino acid residue. Since MT3 differs from MT1 in both domains, to clarify the effect of the EAAEAE insert on the inter-domain interaction, a domain replaced mutant, ß(MT3)
(MT1), has also been prepared here.
Expression and purification
The expression and purification procedures for the wild-type hMT3 and its mutants, E(55)E(60), E55/58/60Q, ß(MT3)
(MT1) and E55D/A56G/A57G/E58D/A59G/E60D/A61G/E62D, were carried out as described in the instructions for glutathioneSepharose 4B (Amersham Pharmacia Biotech) with some modifications (Yu et al., 2002
). After digestion by thrombin, the elution containing thrombin and recombinant hMT3 or its mutants was concentrated and applied to FPLC (AKÄT Purifier100; Pharmacia Biotech) using a Superdex 75 column (
1.6x55 cm). The main eluted peak was concentrated, desalted and lyophilized, and the proteins were stored at 20°C.
ESIMS mass spectroscopy
Molecular weight was measured on a Bruker Esquire 3000 Electrospray Mass Spectrometer (Bruker Daltonicsk, Germany). Desalted proteins were dissolved in 0.1% formic acid (v/v). The measuring conditions were: HV, 4 kV; dry gas, 5 l/min; nebulizer gas, 15 p.s.i.; infusion flow rate, 3 µl/min; m/z, 300010 000.
pH titration
Spectrophotometic pH titration was carried out according to the literature (Winge and Miklossy, 1982). In brief, the samples were dissolved in 5 mM phosphate buffer, pH 8.5, containing 100 mM KCl, then the HCl solution was added stepwise. The ultraviolet (UV) absorption of the protein was measured in the region of
200400 nm on an HP8453 spectrophotometer. The protein concentration was
6 µM.
Reaction with EDTA and DTNB
To characterize the metal-binding ability of MT3, its mutants and mkMT1, the reaction of 8 µM protein with 1 mM EDTA was carried out in a 10 mM TrisHCl, pH 7.5, 100 mM KCl buffer, as previously described (Li et al., 1980). The reaction of the proteins with DTNB was performed according to the method of Shaw et al. (Shaw et al., 1991
).
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Results |
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Plasmid M13mp18 containing the gene of hMT3 or its mutants was sequenced. The results confirmed the correct sequences of hMT3 and its variants. After expression and purification, we obtained quite high yields of MT3 and the mutants (614 mg of protein per liter of culture). Since these proteins were cleaved from GSTMT3 fusion protein by thrombin, they had an additional GlySer dipeptide in the N-terminus and their molecular weights were confirmed by ESIMS. The molecular weights of apo-MT measured are listed as below: MT3, 7070.69 Da;
E(55)E(60), 6469.94 Da; E55/58/60Q, 7067.57 Da; ß(MT3)
(MT1), 6333.89 Da; E55D/A56G/A57G/E58D/A59G/E60D/A61G/E62D, 6956.14 Da. These results agree quite well with the calculated values. We have also measured the ESIMS spectra of metalloforms. Under pH 7.4, the molecular weight of apo-MT3 is 7063.96 Da and the molecular weight of Cd-MT3 is 7843.79 Da (Figure 1). Therefore, the Cd content is (7843.79 7063.96)/112 = 7.0.
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The pH titration results are shown in Figure 2. In the case of mkMT1, there is a clear indication of two independent titration stages resulting from the release of the bound cadmium ions, which presumably is caused by the different stability of the two clusters (Nielson and Winge, 1984; Cismowski and Huang, 1991
; Chang et al., 1998
). It is of note that one could hardly divide one stage from another in the pH titration plot of hMT3, this being different from the case found in MT1/2. On the other hand, in the case of the ß(MT3)
(MT1) mutant, the plot could be obviously divided into two parts: above pH 3.5, it duplicated MT3 accurately; below that pH, it imitated the
-domain of mkMT1 identically. This result proved the assumption that the two stages in the pH titration curve originated from the different stability of the two clusters. The stage around pH 3.95 reflects the character of the Cd3S9 cluster, and the stage around pH 3.0 reflects the character of the Cd4S11 cluster. The association constant KCd could be estimated from the midpoint value according to the method of Wang et al. (1994
). The results are listed in Table I.
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Reactions with EDTA and DTNB
The reaction of MT with EDTA reflects the stability of the metal-thiolate cluster. The kinetics of these reactions were studied under pseudo-first-order conditions (Figure 3). As described (Gan et al., 1995), this reaction could be divided into two phases: the fast phase and the slow phase. By plotting ln(At A
) versus time, the observed rate constants were obtained and are listed in Table I. As shown, the kf values of MT3 reacted with EDTA are three times faster than MT1, while the ks values are similar. These results are consistent with the pH titration results.
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Discussion |
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Although with 70% amino acid sequence identical to that of human MT1/2, hMT3 decreases the survival of rat neonatal cortical neurons in vitro, a property not shared by hMT1/2. A reasonable deduction should be bound up with the unique structure and/or property of MT3. In fact, we found that the Stokes radius of MT3 was larger than that of MT1, which means that the structure of MT3 was looser. This was confirmed by the pH titration and by its reactions with EDTA and DTNB. In all these studies, it was shown the clusters in MT3 collapsed easier. It is interesting to note that in MT3 the dissociation of the Cd4S11 cluster could not be separated from that of the Cd3S9 cluster clearly (Figure 2). This result is consistent with the literature (Hasler et al., 2000), implying that the replacement process of protons for metal ions of MT3 due to the change of cluster stability could not be described as a simple two-domain process, as we observed in the case of MT1/2. Here, we call special attention to the relationship between the stability of the Cd4S11 cluster and the hexapeptide insert in the C-terminal. When this motif was deleted or demolished, the association constant of the Cd4S11 cluster elevated markedly. This indicates that the existence of the acidic insert exerts some restriction on the metal-thiolate cluster, leading to a decrease of its stability. Thus, the hexapeptide insert could be an important structural factor for the difference between MT3 and MT1. On the other hand, the mutation from glutamate to glutamine did not affect the stability of the cluster significantly. This result implies that the negative charge in this fragment does not play an important structural role.
The reaction of metallothionein with EDTA reflects the competition between the sulfhydryl group and the exogenous ligands for the binding of metal ions. The conditions we used here were similar to those of Li et al. (1980) except that the reactions were monitored at 265 nm because EDTA absorbs at 254 nm (Gan et al., 1995
). This reaction is a typical biphasic process. The exact mechanism is unclear yet. According to our results, the three times higher rate constants of kf for all MT3 variants indicate that it is nothing to do with the
-domain, more likely it is related to the existence of Thr5 in the ß-domain. The study on the mutation of Thr5 does support this conclusion (Q.Zheng et al., to be submitted). Our previous study of metal transfer between Cd5Zn2-MT and apo-carbonic anhydrase also verifies this result (Huang et al., 1994
). Very recently, it has been proved by 113Cd-NMR that the TCPCP motif could lead to a structure destabilization in MT (Romero-Isart et al., 2002
).
Different from EDTA, DTNB could react with the nucleophilic sulfhydryl groups in MTs. This reaction is related closely to the solvent accessibility of the clusters. The reaction of MT with DTNB was also studied by several authors (Li et al., 1981; Bernhard et al., 1986; Savas et al., 1991
; Zhu et al., 1995
; Munoz et al., 1999
). Shaw and coworkers (Savas et al., 1991
; Zhu et al., 1995
) indicated that the biphasic reaction arose from independent reaction of each cluster with DTNB; in the case of MT2, the faster and the slower phase correspond to the reactions of DTNB with the
-domain and ß-domain, respectively. However, in the case of MT3, our separate study on the individual
- or ß-domains reacted with DTNB proved that these reactions were a single-phase process; the slow phase was closely related to the
-domain, and the faster phase to the ß-domain (data not shown). This conclusion is consistent with the results shown above because for the slow phase the similar ks values of the wild-type MT1 with the ß(MT3)
(MT1) and the
E(55)E(60) mutants implies that these variants have the same
-domain structure, showing identical reaction activity with DTNB. Whereas for the rest of the variants, which maintained the similar structure of the
-domain of MT3, the ks values were doubled. Thus, the rates in the slower phase reflected, at least partly, the solvent accessibility of the Cd4S11 cluster. For the faster phase, the kf values of these variants are similar because of their identical ß-domain structures. The character of the ß-domain could be attributed to the existence of the continual proline residues in the C(6)-P-C-P(9) motif, which distorted the peptide chain, leading to the Cd3S9 cluster being more exposed and, therefore, being more easily attacked by DTNB.
In summary, even though MT3 shows 70% sequence homology with MT1, these two members of the metallothionein family present apparent differences in property and function. It is reasonable to consider whether the difference is related to the remarkable two insertsconserved threonine at position 5 and a negatively charged hexapeptide at position 55 in MT3. Our detailed studies here obviously revealed that the EAAEAE insert is essential to the property of MT3. It is the hexapeptide insert, to some extent, that makes the MT3 -domain looser with lower stability of the metal-thiolate cluster, resulting in a cluster that can be accessed more easily. When this insert was deleted, the stability of both the Cd4S11 cluster and the
-domain was raised markedly. The variants at this site exhibit different behaviors towards pH, EDTA and DTNB.
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Acknowledgement |
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Received March 7, 2003; revised October 10, 2003; accepted October 21, 2003