The effect of the EAAEAE insert on the property of human metallothionein-3

Qi Zheng1, Wan-Ming Yang1,2, Wen-Hao Yu1, Bin Cai1, Xin-Chen Teng1, Yi Xie3, Hong-Zhe Sun4, Ming-Jie Zhang5 and Zhong-Xian Huang1,6

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MT3 shows apparently different properties and function from MT1 even though they have 70% sequence homology. Possibly the two inserts, Thr5 and a negatively charged hexapeptide at position-55 in MT3, play important roles. A series of MT3 variants around the EAAEAE hexapeptide have been prepared by site-directed mutagenesis and their properties and reactivity towards pH, EDTA and DTNB have been studied. Our detailed studies revealed that the EAAEAE insert is essential to the property of MT3. It is the hexapeptide insert, to some extent, making the MT3 {alpha}-domain looser and lower stability of the metal–thiolate cluster, which could be accessed more easily.

Keywords: EAAEAE insert/human metallothionein-3/neuron growth inhibitory factor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Metallothionein-3 (MT3) is a brain-specific isoform of MTs. Besides being expressed restrictedly in the central nervous system (CNS), MT3 is functionally unique—it inhibits the elevated neurotrophic activity of Alzheimer’s disease (AD) brain extracts (Uchida et al., 1991Go). Although the exact molecular mechanism has not been well understood, MT3 seems to play an important role in the process of neuronal regeneration and degeneration. It has been reported that the MT3 level decreases in astrocytes in lesioned areas of degenerative diseases such as AD, Parkinson’s disease, amyothrophic lateral sclerosis and progressive supranuclear palsy (Uchida, 1994Go). MT3-deficient mice exhibit more susceptibility to seizures induced by kainic acid with greater neuron injury in the CA3 field of hippocampus and an enhanced increase in glia fiber acid protein (GFAP) expression associated with aging (Erickson et al., 1997Go). Exogenous MT3 prevents the initial formation and extension of neurite of cortical neurons in the early period of differentiation (Chung et al., 2002Go; Uchida et al., 2002Go) and the death of neurons caused by high oxygen exposure (Uchida et al., 2002Go).

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., 1991Go; Tsuji et al., 1992Go). The peptide loop (amino acids 52–63) of MT3 reveals ~85% {alpha}-helical structure according to the secondary structure prediction program SSCP (Eisenhaber et al., 1996Go). 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., 1995Go; Hasler et al., 2000Go). Some studies reported that the clusters in MT3 were quite flexible (Faller and Vasak, 1997Go; Hasler et al., 1998Go; Faller et al, 1999Go). 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., 2001Go). Surprisingly, it was reported that the inhibitory activity of MT3 arises from the N-terminal ß-domain (Sewell et al., 1995Go). 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., 2002Go). 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, {Delta}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)–{alpha}(MT1). After characterization by ESI–MS, 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents

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, glutathione–Sepharose 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. (1988Go). 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 52–63 reveals ~85% {alpha}-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 {alpha}-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)–{alpha}(MT1), has also been prepared here.

Expression and purification

The expression and purification procedures for the wild-type hMT3 and its mutants, {Delta}E(55)–E(60), E55/58/60Q, ß(MT3)–{alpha}(MT1) and E55D/A56G/A57G/E58D/A59G/E60D/A61G/E62D, were carried out as described in the instructions for glutathione–Sepharose 4B (Amersham Pharmacia Biotech) with some modifications (Yu et al., 2002Go). 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 ({phi} 1.6x55 cm). The main eluted peak was concentrated, desalted and lyophilized, and the proteins were stored at –20°C.

ESI–MS 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, 3000–10 000.

pH titration

Spectrophotometic pH titration was carried out according to the literature (Winge and Miklossy, 1982Go). 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 ~200–400 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 Tris–HCl, pH 7.5, 100 mM KCl buffer, as previously described (Li et al., 1980Go). The reaction of the proteins with DTNB was performed according to the method of Shaw et al. (Shaw et al., 1991Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein characterization

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 (~6–14 mg of protein per liter of culture). Since these proteins were cleaved from GST–MT3 fusion protein by thrombin, they had an additional Gly–Ser dipeptide in the N-terminus and their molecular weights were confirmed by ESI–MS. The molecular weights of apo-MT measured are listed as below: MT3, 7070.69 Da; {Delta}E(55)–E(60), 6469.94 Da; E55/58/60Q, 7067.57 Da; ß(MT3)–{alpha}(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 ESI–MS 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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Fig. 1. ESI–MS of wild-type MT3. Protein was dissolved in 3 mM NH4HCO3 (pH 7.4); the molecular weights of apo-MT3 and Cd-MT3 under these conditions were 7063.96 and 7843.79 Da, respectively.

 
pH titration

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, 1984Go; Cismowski and Huang, 1991Go; Chang et al., 1998Go). 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)–{alpha}(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 {alpha}-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. (1994Go). The results are listed in Table I.



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Fig. 2. UV absorption at 250 nm of the pH titration of the wild-type MT3 (squares), {Delta}E(55)–E(60) (--), ß(MT3)–{alpha}(MT1) (triangles), mkMT1(····), E55/58/60Q (diamonds) and D55G56G57D58G59D60G61D62 (—). The protein samples were 6 µM in a solution of 5 mM Na2HPO4 and 100 mM KCl and acidified stepwise with concentrated HCl solution.

 

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Table I. The association constants determined by pH titration and observed rate constants of reaction of MTs with EDTA and DTNB
 
Compared with MT1, MT3 shows a major stability difference in its Cd4S11 cluster, whose association constant is calculated to be 8.4x1019 (versus 5.6x1020 in MT1). When the acidic hexapeptide was deleted, the midpoint value of the Cd4S11 cluster was moved to pH 3.2 and the association constant was raised to 2.6x1020. This result undoubtedly confirms that the EAAEAE insert made the Cd4S11 cluster looser. It is interesting to note that the replacement of acidic residue into amides did not lead to the change of stability of the Cd4S11 cluster significantly (Figure 2). This result implies that the negative charges of the hexapeptide insert did not exert too much influence on the cluster. It is probable that the QAAQAQ hexapeptide still kept its original conformation. The pH titration plot of the {alpha}-helix-broken mutant, E55D/A56G/A57G/E58D/A59G/E60D/A61G/E62D, was also interesting. Its ß-domain was identical to that of wild-type MT3, as predicted, but its {alpha}-domain was more stable than that of the wild-type hMT3. The association constant was 2.6x1020, equal to that of {Delta}E(55)–E(60). The reason for this phenomenon is discussed in more detail below.

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., 1995Go), this reaction could be divided into two phases: the fast phase and the slow phase. By plotting ln(At A{infty}) 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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Fig. 3. Logarithmic plots of the reaction rates of EDTA with wild-type MT3 (--), ß(MT3)–{alpha}(MT1) (– – –), {Delta}E(55)–E(60) (····), mkMT1(– – –), E55/58/60Q (–··–) and D55G56G57D58G59D60G61D62 (—). Concentration changes of product were measured through absorbance at 265 nm; 8 µM protein reacted with 1 mM EDTA in 10 mM Tris–HCl, 100 mM KCl buffer (pH 7.5) at 25°C.

 
The reaction of MT with DTNB was usually used to describe the accessibility of DTNB to sulfhydryl groups. Under pseudo-first-order conditions, it is also a biphasic process. Figure 4 displays the ln(A{infty}At) versus time plots and the observed rate constants are also listed in Table I. The results show that the rates of the fast phase of all variants do not change significantly; however, the rates of the slow phase of MT1, the ß(MT3)–{alpha}(MT1) and the {Delta}E(55)–E(60) mutants have similar values, which are almost the half of the other three mutants.



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Fig. 4. Logarithmic plots of the reaction rates of DTNB with wild-type MT3 (), ß(MT3)–{alpha}(MT1) (– –), {Delta}E(55)–E(60) (····), mkMT1 (—), E55/58/60Q (–··–) and D55G56G57D58G59D60G61D62 (– – –). Concentration changes of products were measured through absorbance at 412 nm; 3 µM protein reacted with 1 mM DTNB in 10 mM Tris–HCl, 100 mM KCl buffer (pH 7.5) at 25°C.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The expression of MT3 has been reported in several ways (Sewell et al., 1995Go; Wang et al., 1997Go; Wu et al., 1998Go). In general, protein yields were not so high. Besides, it could not be used for some systems when unstable proteins such as the sole ß-domain is needed (Sewell et al., 1995Go). Here we choose a GST-fusion protein system to express MT3. All proteins, including the sole {alpha}-domain and the sole ß-domain (data not shown), could be obtained with high yields. Moreover, the purification of various mutants was successfully completed in the same protocol. The only deficiency was that the two additional amino acid residues, Glu and Ser, were added at the N-terminus of the protein owing to the cleavage site of thrombin. However, this addition does not affect the structure and property of MT significantly. It was reported that the recombinant mouse MT1 expressed in the same way shows similar structural characteristics and properties to native rabbit MT1 (Xiong and Ru, 1997Go). Recently, we have obtained hMT3’s solution structure by NMR, and the result corresponds well with the published data for mouse MT3 (Q.Zheng et al., to be submitted).

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., 2000Go), 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. (1980Go) except that the reactions were monitored at 265 nm because EDTA absorbs at 254 nm (Gan et al., 1995Go). 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 {alpha}-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., 1994Go). 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., 2002Go).

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., 1981Go; Bernhard et al., 1986; Savas et al., 1991Go; Zhu et al., 1995Go; Munoz et al., 1999Go). Shaw and coworkers (Savas et al., 1991Go; Zhu et al., 1995Go) 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 {alpha}-domain and ß-domain, respectively. However, in the case of MT3, our separate study on the individual {alpha}- or ß-domains reacted with DTNB proved that these reactions were a single-phase process; the slow phase was closely related to the {alpha}-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)–{alpha}(MT1) and the {Delta}E(55)–E(60) mutants implies that these variants have the same {alpha}-domain structure, showing identical reaction activity with DTNB. Whereas for the rest of the variants, which maintained the similar structure of the {alpha}-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 inserts—conserved 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 {alpha}-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 {alpha}-domain was raised markedly. The variants at this site exhibit different behaviors towards pH, EDTA and DTNB.


    Acknowledgement
 
This project was supported by the National Science Foundation of China.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received March 7, 2003; revised October 10, 2003; accepted October 21, 2003