Báo cáo khoa học: Comparative metal binding and genomic analysis of the avian (chicken) and mammalian metallothionein potx

Comparative metal binding and genomic analysis of the avian (chicken) and mammalian metallothionein Laura Villarreal 1 , Laura Tı ´o 2 , Merce ` Capdevila 1 and Sı ´lvia Atrian 2 1 Departament de Quı ´ mica, Facultat de Cie ` ncies, Universitat Auto ` noma de Barcelona, Bellaterra, Spain 2 Departament de Gene ` tica, Facultat de Biologia, Universitat de Barcelona, Spain Metallothioneins (MTs), the ubiquitous metal-binding proteins first described by Vallee in 1957 [1] constitute a large superfamily of small, cysteine-rich peptides present in some prokaryotes and in all eukaryotes (protista and fungi, plants and animals) examined so far (http://www.expasy.org/cgi-bin/lists? ⁄ metallo.txt). The current limited knowledge of their origin and differentiation patterns can mainly be attributed to the lack of detailed, comparative studies involving MTs other than mammalian isoforms. Besides, any homol- ogy-driven structural, biochemical or functional infer- ence using mammalian data makes little sense when considering MTs other than those belonging to the unquestionable family of homology of the vertebrate forms. Precisely, invertebrate, fungal and plant MTs exhibit a high sequence heterogeneity, both among Keywords dimerization; Gallus gallus; metal binding; metallothionein; molecular evolution Correspondence S. Atrian, Department of Genetics, Faculty of Biology, University of Barcelona, Avenue Diagonal 645, 08028-Barcelona, Spain Fax: +34 934034420 Tel: +34 934021501 E-mail: satrian@ub.edu Note L. Villarreal and L. Tı ´ o made equal contribu- tions to this work. (Received 20 October 2005, revised 1 December 2005, accepted 2 December 2005) doi:10.1111/j.1742-4658.2005.05086.x Chicken metallothionein (ckMT) is the paradigm for the study of metallo- thioneins (MTs) in the Aves class of vertebrates. Available literature data depict ckMT as a one-copy gene, encoding an MT protein highly similar to mammalian MT1. In contrast, the MT system in mammals consists of a four-member family exhibiting functional differentiation. This scenario prompted us to analyse the apparently distinct evolutionary patterns fol- lowed by MTs in birds and mammals, at both the functional and structural levels. Thus, in this work, the ckMT metal binding abilities towards Zn(II), Cd(II) and Cu(I) have been thoroughly revisited and then compared with those of the mammalian MT1 and MT4 isoforms, identified as zinc- and copper-thioneins, respectively. Interestingly, a new mechanism of MT dime- rization is reported, on the basis of the coordinating capacity of the ckMT C-terminal histidine. Furthermore, an evolutionary study has been per- formed by means of in silico analyses of avian MT genes and proteins. The joint consideration of the functional and genomic data obtained questions the two features until now defining the avian MT system. Overall, in vivo and in vitro metal-binding results reveal that the Zn(II), Cd(II) and Cu(I) binding abilities of ckMT lay between those of mammalian MT1 and MT4, being closer to those of MT1 for the divalent metal ions but more similar to those of MT4 for Cu(I). This is consistent with a strong func- tional constraint operating on low-copy number genes that must cope with differentiating functional limitation. Finally, a second MT gene has been identified in silico in the chicken genome, ckMT2, exhibiting all the features to be considered an active coding region. The results presented here allow a new insight into the metal binding abilities of warm blooded vertebrate MTs and their evolutionary relationships. Abbreviations FPD, flame photometric detector; ICP-AES, inductively coupled plasma-atomic emission spectroscopy; MRE, metal-response-element; MT, metallothionein. FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS 523 them and in relation to the vertebrate peptides. Since no definite physiological roles have been assigned to these proteins, functional constraints or adaptative trends modulating their evolutionary history are also difficult to envisage. Some attempts to characterize the evolution of vertebrate MTs have been carried out through the analysis of the maximum parsimony trees construc- ted with protein and cDNA sequences (http:// www.biochem.unizh.ch/mtpage/poster/posterevol.html). These studies not only locate fish, amphibian and avian MTs in increasing proximity to the mammalian MT radiation, but also propose that all vertebrate MTs originated from a single ancestor. Unfortunately, the amount of experimental information on nonmam- malian MT genes and proteins is still too scarce to support any hypothesis. In this scenario, avian MTs gather patent interest, as birds represent a class of warm-blooded vertebrates that evolved in parallel to mammals for nearly 310 million years, after the late Palaeozoic divergence of the respective lineages [2]. Chicken (Gallus gallus) is the model organism for avian molecular biology, and ckMT has also been the paradigm for the study of avian MTs (reviewed in [3]). CkMT was isolated and first characterized in the early 1970s as not significantly different from the mammalian (mouse) MT1-MT2 forms [4,5] and was further identified as a 63-residue long polypeptide, with 68% sequence similarity and two amino acid insertions in relation to mouse MT1 [6]. CkMT cDNA [7] and gene [8] features again suggested great structural and functional resemblance to the mamma- lian MT system, since the ckMT gene showed the same exon ⁄ intron distribution and was apparently regulated by the same cis elements, responding to the same stimuli: metal overdose, oxidative stress, gluco- corticoids and lipopolysaccharides [9,10]. Only two differences were mentioned, the ontogenic expression pattern of liver ckMT, acutely increasing after hatch- ing [11], and the apparently solid evidence that MT was a one-copy gene in birds [6,7]. Studies on other genera (Meleagris gallopavo (turkey), Phasianus colchi- cus (pheasant), Colinus virginianus (new world quail) [11]; Cairina moschata and Anas platyrhyncos (ducks) [12]; and Coturnix coturnix (quail) [13]) showed an exceptional conservation rate for the unique MT form isolated in each of them: no amino acid substitutions and 97% identity at cDNA level, features which were readily justified by the functional constraint imposed on a single copy gene. Description in Columba livia (pigeon) of two MT isoforms, neither of them coinci- dent with the previously reported MT sequence [14], has been the unique evidence of MT multiplicity in an avian genome, and also of sequence diversity among avian MTs. This apparent simplicity of the MT system in birds contrasts with its complexity in mammals, where duplication events originated a four- member cluster (MT1 to MT4), with a further 13-fold amplification of MT1 in humans. Physiological differ- entiation has been shown for the four isoproteins: the MT1-MT2 ubiquitous, metal-induced forms have been related to homeostasis, transport and detoxification of metal ions; MT3, only synthesized in neural tissues, has been related to neuronal growth; and a role for MT4 in the differentiation of stratified squamous epi- thelia, the only tissue in which it is expressed, has been suggested. We have proposed a further differen- tiation between MTs, which has ended in the classifi- cation of mammalian MT1 as optimum for divalent metal coordination (zinc-thionein behaviour [15]), and of MT4 as prone for copper-binding, or with a copper-thionein character [16]. In view of the distinct evolutionary patterns followed by MTs in birds and mammals, we decided to focus our interest on the determination of the metal coordination features of ckMT, using the same meth- odological approach previously applied for mammalian MTs. By this study, we aimed to answer two main questions. First, to determine the metal binding beha- viour, preferences and peculiarities of the single avian MT form, since absence of duplication may have prevented metal binding specialization. and second, to elucidate if the avian MT appeared functionally closer to MT1, as previously reported in the literature, or to MT4, its closest neighbour according to phylogenetic and protein distance trees mentioned above. CkMT characterization was accomplished by determination of the spectroscopic and spectrometric features of the Zn-, Cd- and Cu- complexes rendered by the recom- binant full-length protein and its separate b and a domains, as well as of the metal species obtained by Zn ⁄ Cd or Zn ⁄ Cu in vitro replacement. In the course of this research, the annotation of the complete chicken genome was released [2], allow- ing us an exhaustive in silico search for MT-like sequences as well as the determination of synteny relationships between the MT gene containing regions in the human, rat and mouse genomes. Thus, evalua- tion of the avian versus mammalian MT functional differentiation trends could be completed by compar- ative genomics analyses. Remarkably, the joint con- sideration of all the data here reported basically reformulates the two main features defining until now avian MTs: the full functional equivalence with mam- malian MT1 and the singularity of MT genes in avian genomes. Chicken metallothionein L. Villarreal et al. 524 FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS Results and discussion Metal binding analysis rationale The metal binding abilities of ckMT were analysed fol- lowing a two-step strategy. First, the in vivo synthes- ized M-ckMT, M-ackMT and M-bckMT complexes (M ¼ Zn II ,Cd II or Cu I ) were characterized. Second, the in vitro Zn ⁄ Cd and Zn ⁄ Cu replacement processes of the three Zn-ckMT peptides, at pH 7, were analysed as previously described for MT1 and MT4 [16–20]. Spectroscopic data provided information on the num- ber of in vivo and in vitro metal–MT generated species, their stoichiometry, and their degree of folding. Addi- tionally, the spectrometric measurements revealed the composition of the recombinant samples and the molecular distribution [21,22] of the various complexes present at each point of the titrations. Although it was possible to determine the Zn:Cd:MT ratio in the heter- ometallic Zn,Cd–ckMT species, the proximity between the atomic weights of zinc and copper and the ESI-MS experimental error range prevented determination of the Zn:Cu ratio in the heterometallic Zn,Cu–ckMT species. Zn(II) and Cd(II) binding abilities of ckMT and its separate domains Synthesis of ckMT, ackMT and bckMT both in Zn- and in Cd- supplemented media yields as major species the canonical complexes expected for a vertebrate MT, i.e. M 7 –ckMT, M 4 –ackMT and M 3 –bckMT, where M ¼ Zn or M ¼ Cd (analytical results included in Table 1 and Table S1). This behaviour coincides with that reported for mammalian MT1 and differs from that regarding MT4. The additional presence of minor sulphide-containing species in all preparations except in Zn 4 –ackMT is in accordance with a recent study reporting that most recombinant MT samples include these acid-labile ligands [23]. Quantification by GC- FPD confirms the presence of sulphide in all the prep- arations, even in Zn 4 –ackMT (Table 1), and suggests that they may harbour a more significant role in the Cd- than in the Zn- complexes. The CD spectra of the M–ckMT and M–ackMT preparations (M ¼ Zn, Fig. 1A; M ¼ Cd, Fig. 1B) clo- sely resemble those of the corresponding MT1 com- plexes [17,18] and provide evidence that the degree of folding of ckMT and ackMT upon Zn(II) and Cd(II) Table 1. Molecular masses and metal (Zn, Cd or Cu) to protein ratios found for the in vivo synthesized ckMT, ackMT and bckMT metal aggregates. A comprehensive table including the theoretical m calculated from the metal-MT composition, and the metal : MT molar ratios measured from conventional and acid ICP-AES is available as Supplementary Table S1. Metal supplemented in culture media Protein m exp a Da M ⁄ MT b S 2– ⁄ MT c M ¼ Zn ckMT 7050.9 ± 0.8 Zn 7 –ckMT (S) 2.5 7083.5 ± 1.4 Zn 7 S 1 –ckMT (s) ackMT 3744.5 ± 0.7 Zn 4 –ackMT 1.1 bckMT 3597.7 ± 0.8 Zn 3 –bckMT (S) 3.1 3692.6 ± 1.7 Zn 3 S 3 –bckMT (s) M ¼ Cd ckMT 7378.9 ± 2.4 Cd 7 –ckMT (S) 4.7 7332.4 ± 2.5 Cd 6 S 2 –ckMT 7284.4 ± 5.5 Cd 5 S 4 –ckMT (s) ackMT 3932.5 ± 0.9 Cd 4 –ackMT (S) 2.9 3884.7 ± 0.0 Cd 3 S 2 –ackMT (s) bckMT 3738.6 ± 0.3 Cd 3 –bckMT (S) 5.6 3692.7 ± 0.6 Cd 2 S 2 –bckMT (s) M ¼ Cu ckMT 7231.7 ± 2.1 M 10 –ckMT (S) N ⁄ D d 7292.0 ± 3.5 M 12 –ckMT 7358.3 ± 1.7 M 11 –ckMT ackMT 3862.7 ± 2.6 M 6 –ackMT (S) N ⁄ D 3925.2 ± 0.0 M 7 –ackMT 3807.4 ± 0.0 M 5 –ackMT (s) bckMT 3783.6 ± 1.0 Cu 6 –bckMT (S) N ⁄ D 3845.3 ± 0.9 Cu 7 –bckMT 3719.9 ± 1.0 Cu 5 –bckMT a Experimental molecular masses for the Zn–, Cd– and Cu–MT complexes. b Metal per MT molar ratio calculated from the mass difference between holo- and apo-protein. (S) denotes a major species; (s) denotes a minor species. c S 2– to MT ratio measured by GC-FPD. d N ⁄ D, Non- detectable. L. Villarreal et al. Chicken metallothionein FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS 525 coordination is closer to that of MT1 than to that of MT4 [16]. In these samples, the absorptions of the metal-sulphide chromophores are of such a low inten- sity that they do not significantly contribute to the final CD spectra (Type A according to the classifica- tion proposed in [23]). Conversely, the CD spectra of the in vivo M–bckMT samples (M ¼ Zn, Fig. 1C; M ¼ Cd, Fig. 1D) differ significantly from those obtained for the corresponding mammalian bMT1 and bMT4 complexes. To analyse the chromophores that may contribute to these new CD fingerprints, recom- binant Zn–bckMT was further purified either in Tris ⁄ chloride or Tris ⁄ perchlorate buffer [24], these two preparations were, respectively, titrated with Cd(II) chloride or Cd(II) perchlorate, and all the metal–bckMT species formed were characterized. The comparison of the CD fingerprints of the Zn– and Cd– bckMT species formed in the presence or absence of chloride ions (data not shown) revealed that, at the assayed concentrations, these anions have no spec- troscopically detectable contribution either to the metal-cluster structure or to its chirality. Then, the presence of S 2– ligands in the in vivo Zn–bckMT and Cd–bckMT samples was considered another plausible explanation for their uncommon CD fingerprints. To test this, both preparations were acidified to pH 1.5 and reneutralized to pH 7.5. As there was no signifi- cant difference between the initial and the final CD spectra (shown in Fig. 1C for Zn–bckMT) we conclu- ded that this was not the case. Consequently, the char- acteristic CD fingerprint of Zn– and Cd–bckMT should only be attributed to the peculiarities of their M(SCys) 4 chromophores, with perhaps some contribu- tion of protein conformation at the lowest wavelengths [25]. Full comparison of the spectroscopic Cd–bMT1, Cd–bMT4 and Cd–bckMT features is provided in Fig. 1D and in note 1 of the supplementary material. Finally, it is worth noting that in spite of the unusual Zn– and Cd–bckMT CD fingerprints, summation of the CD spectra of in vivo M–bckMT and M–ackMT affords spectra that closely resemble those of the full length M–ckMT (M ¼ Zn, Cd) (Fig. 1A and 1B), sug- gesting that both fragments behave independently when binding Zn(II) or Cd(II), as was the case for MT1 [18]. This independent behaviour can be extended to the binding capacity of each domain not only for the major (Zn 3 –orCd 3 –bckMT + Zn 4 –orCd 4 – ackMT ¼ Zn 7 –orCd 7 –ckMT) but also for the minor species (i.e. Cd 2 S 2 –bckMT+Cd 3 S 2 –ackMT ¼ Cd 5 S 4 – ckMT) (Table 1). Fig. 1. Comparison of the CD spectra of the biosynthesized (A) Zn–ckMT (solid grey line), Zn 4 –ackMT (dotted line) and Zn–bckMT (dashed line); (B) Cd–ckMT (solid grey line), Cd–ackMT (dotted line) and Cd–bckMT (dashed line). The spectra depicted in a solid black line in (A) and (B) represent the sum of the CD spectra of M–ackMT and M–bckMT; (C) Zn–bckMT (solid black line), Zn–bMT4 (solid grey line), Zn 3 –bMT1 (dashed line) and Zn–bckMT reneutralized (dotted line); (D) Cd–bckMT (solid black line), Cd–bMT4 (solid grey line) and Cd 3 –bMT1 (dashed line). Chicken metallothionein L. Villarreal et al. 526 FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS Two main results concerning the in vitro binding abilities of the ckMT peptides deserve explicit com- ment. First, analysis of the Zn ⁄ Cd replacement in bckMT (Fig. 2A and 2B, Table S2) further corrobor- ates the peculiar CD fingerprint of the in vivo Cd– bckMT sample, since the addition of 3 Cd(II) to Zn– bckMT renders a mixture of the same composition (Table S2B), and accordingly equivalent CD spectra (Fig. 2C) to that of the biosynthesized Cd–bckMT. Addition of further Cd(II) causes a red shift and a decrease in the intensity of the main CD signals (Fig. 2B), giving rise to a spectrum that could be considered characteristic of the Cd 3 –bckMT species, with contribution of the Cd 3 S 2 –bckMT complex (Table S2B), and that is clearly similar to that of Cd 3 – bMT1 and Cd 3 –bMT4 (Fig. 2D). Second, titration of Zn–ckMT and Zn 4 –ackMT with Cd(II) renders some unexpected results suggesting a new MT dimerization process. During these titrations, the CD spectra of the samples evolve similarly to those observed for MT1 [17,18] and MT4 [16] in analogous reactions until 7 and 4 Cd(II) respectively added to Zn–ckMT and Zn 4 –ackMT (Fig. 3A). But after these steps, the addi- tion of further Cd(II) leads to a marked development of positive shoulders at  250 nm in both sets of CD spectra. Since previous studies of Cys-to-His site- directed MT1 mutants [26] related this absorption to Cd(II)–NHis coordination, it was reasonable to hypothesize a possible Cd(II) binding role of the ckMT C-terminal histidine, obviously also present in its a fragment. To investigate this, the final solutions of the Cd(II) titrations of Zn 4 –ackMT and Zn 7 –ckMT were acidified from pH 7 to pH 4 (Fig. 3B), which caused the disappearance of the 250 nm absorptions and the preservation of all the other CD features in both cases. This result is consistent with Cd–His coordination being responsible for the 250-nm shoulder, since this amino acid protonates at a 4–5 pH range, while the lower pK a of the cysteinic thiolates still allows for maintenance of the Cd–SCys bonds at this pH. Inter- estingly, the appearance of these 250-nm absorptions is accompanied by a gradual decrease in the intensity of the CD envelopes. This variation has been related to MT dimerization events [27], and coincidently the ESI- MS results of our samples (Table S2A; Fig. 3C) reveal the presence of dimeric Cd 8 –(ackMT) 2 species whose abundance increase with the addition of further Cd(II) equivalents to Zn 4 –ackMT. Unfortunately, dimers of the full-length ckMT could not be detected (Table S2C). Furthermore, acidification of the sample corresponding to the final step of the Cd(II) titration of Zn 4 –ackMT give rise to ESI-MS data that show the disappearance of the Cd 8 –(ackMT) 2 species, thus con- firming that the dimeric forms are responsible for the 250 nm CD absorption. Two types of dimerization processes have been reported in MT literature: oxida- tive dimerization upon disulfide bridge formation [28] and metal-mediated dimer formation [29]. Our results are consistent with a third mechanism of MT dimeriza- tion, mediated by the C-terminal histidine. As once the dimerization process starts being spectroscopically detectable the formation of new chromophores stops, as shown in the CD and UV–vis difference spectra (Fig. 3A), we may assume a dimerization model (Scheme 1) involving a simultaneous intermolecular formation (N a1 –Cd a2 and N a2 –Cd a1 ) and intramolecu- lar loss (N a1 –Cd a1 and N a2 –Cd a2 ) of two Cd–NHis bonds, via the second nitrogen donor atom of two His residues. Therefore, in the dimeric species, two histi- dines would bridge two a fragments, in a similar way Fig. 2. (A) and (B) CD spectra corresponding to the titration of Zn–bckMT with Cd(II) at pH 7. The arrows show the evolution of the spectra when the indicated number of Cd(II) equivalents were added. (C) Superimposi- tion of the spectra recorded after 3 Cd(II) added to Zn–bckMT (solid grey line) and the spectra of the biosynthesized Cd– bckMT (solid black line). (D) Comparison of the in vitro constituted Cd 3 –bMT complexes of bckMT (solid black line), bMT4 (dashed line [16]), and bMT1 (dotted line [18] and note 2 in supplementary material). L. Villarreal et al. Chicken metallothionein FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS 527 Fig. 3. (A) CD and UV–vis difference spectra corresponding to the titrations of Zn–ckMT and Zn 4 –ackMT with Cd(II) at pH 7. Arrows show the evolution of the spectra when the indicated number of Cd(II) was added. (B) CD spectrum of the acidification of the final solution of the Cd(II) titration of Zn–ckMT, the arrow shows the evolution from pH 7 to 4. (C) ESI-MS spectra of an aliquot of the solution obtained after adding 7 Cd(II) to Zn 4 –ackMT. N N S S S Cd HOOC N N S S S Cd α 2 α 2 COOH N N S S S Cd α 1 α 1 HOOC N N S S S Cd COOH (1) (2) Scheme 1. Possible mechanism of dimerization. The Cd-NHis dashed bonds in (1) are broken when those of (2) are formed. Chicken metallothionein L. Villarreal et al. 528 FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS to that described for the active centre of superoxide dismutase [30]. Cu(I) binding abilities of ckMT and its separate domains The analytical characterization of the ckMT species biosynthesized in Cu-supplemented media is shown in Tables 1 and S1, none of the Cu–ckMT preparations showing evidence of sulphide ligands in their com- plexes. bckMT renders a mixture of homometallic Cu-species analogous to that obtained for bMT1 [20,31] and bMT4 [16]. However, Cu 6 –bckMT is the major species, contrasting with the Cu 7 –bMT major stoichiometry reported for bMT1 and bMT4. This is consistent with the comparable but nonidentical CD spectra of these three samples (Fig. 4C). bckMT shows absorptions in the 330–400 nm range, which suggests that it would offer the particular Cu(I) binding site previously reported for bMT4 [16] and absent in bMT1. In contrast to the b domain, the recombinant syntheses of ckMT and ackMT in Cu-supplemented media yield mixtures of heterometallic Zn,Cu–ckMT species. This behaviour is similar to that of MT1 [20] and differs from that of the MT4 counterparts [16]. The entire ckMT yields a major M 10 –ckMT species (M ¼ Zn and ⁄ or Cu) with a Zn 3 Cu 7 –ckMT stoichio- metry (Table 1), as also reported for MT1 [20] and Type 2 MT4 [16]. Coincidently, the three Cu–MT com- plexes afford comparable CD spectra (Fig. 4A). The ackMT peptide renders M 6 –ackMT as the most abun- dant species, contrasting with the biosynthesized major M 5 –aMT1 complex [20]. On the basis of the plasma- atomic emission spectroscopy (ICP-AES) data (0.5 Zn and 5.7 Cu for ackMT versus 0.5 Zn and 4.5 Cu for aMT1), the difference in the M ⁄ aMT stoichiometry could be explained by the presence of one additional Cu(I) ion in ackMT. Comparison of CD spectra (Fig. 4B) reveals that in the presence of Cu(I), ackMT folds more similarly to aMT4 than to aMT1, in spite of the hetero versus homometallic nature of both com- plexes. In spite of the structural dissimilarities between Zn– bckMT, Zn–bMT1 and Zn–bMT4 (Fig. 1C) and as otherwise expected from their similar in vivo Cu(I) binding behaviour, all three peptides give rise to anal- ogous Zn ⁄ Cu replacement reactions, with only a slight difference in the number of Cu(I) required to achieve some particular titration stages (the full set of spectro- scopic data and the spectrometric data recorded at some titration stages are given in Fig. S1 and Table S3, respectively). Particularly, it is worth noting that Cu 7 –bckMT is formed after the addition of only 5 Cu(I) to Zn–bckMT, while species of the same Cu(I):MT ratio and close peptidic fold are obtained only after the addition of 6 or 7 Cu(I) to Zn 3 –bMT4 [16] and Zn 3 –bMT1 [20], respectively (Fig. S1B). This suggests a Cu(I) in vitro binding ability higher for bckMT than for bMT1 or bMT4. The Zn ⁄ Cu replace- ment reaction on Zn–ackMT shares more similarities with that on Zn–aMT1 than with that on Zn–aMT4, although parallel evolutions until 5 Cu(I) are observed in the three cases. After that, for 6 Cu(I) added, the chirality of the aMT4 sample suffers a change that ackMT and aMT1 also endeavour but after the addi- tion of 7 or 8 Cu(I), respectively. These results allow consideration of a Cu(I) binding ability for ackMT intermediate between those of aMT1 and aMT4. Finally, the titrations of the full-length Zn–ckMT, Zn– MT1 and Zn–MT4 also evolve similarly until 7 Cu(I), a step that leads in all cases to the formation of Zn 3 Cu 7 –MT complexes, which are also equivalent to the corresponding in vivo-conformed Zn 3 Cu 7 –MT spe- cies (Fig. 4A). The differences observed at this stage should be attributed to the dissimilarity of the starting species CD spectra, and in the case of MT4, to the minor contribution at  350(+) nm assigned to the binding of Cu(I) to the bMT4 domain [16]. This par- ticular MT4 absorption, which intensifies with the for- mation of Cu 10 –MT4, is never observed during the Fig. 4. CD spectra of the recombinant (A) Zn 3 Cu 7 –ckMT (black solid line), Zn 3 Cu 7 –MT4 (dashed line) and Zn 3 Cu 7 –MT1 (dotted line); (B) Cu–ackMT (black solid line), Cu–aMT4 (dashed line) and Cu–aMT1 (dotted line); and (C) Cu–bckMT (black solid line), Cu–bMT4 (dashed line) and Cu–bMT1 (dotted line). L. Villarreal et al. Chicken metallothionein FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS 529 Cu(I) titration of Zn–MT1, but develops after the addition of 12 Cu(I) to Zn–ckMT, thus revealing that ckMT can also provide this special coordination envi- ronment when large amounts of Cu(I) are present. Regarding domain dependency ⁄ independency, it was observed that the separate a and b domains do not interact with Cu(I) in the same way as they do when linked together in the full-length ckMT, therefore exhibiting a dependent Cu-binding behaviour. This observation becomes obvious from the results of the biosyntheses in Cu-rich media, as M 10 –ckMT com- plexes are obtained instead of the theoretical M 12 – ckMT that would result from the addition of the major M 6 –ackMT and Cu 6 –bckMT species. Thus, the ckMT separate domains are characterized by a higher in vivo Cu(I) binding capacity than when constituting the entire polypeptide, as was also the case for MT1 [20] and MT4 [16]. Chicken genome search, comparative genomics and protein sequence analysis In order to investigate the syntenic relationships between the chromosomal regions containing the MT gene ⁄ s in birds and mammals, the recently annotated chicken genome (v.29.1) was searched using as a query the ckMT cDNA sequence cloned and heterol- ogously expressed in this work [7]. Surprisingly, homology is detected in two unlinked genomic loca- tions (Fig. 5). One of them (chromosome 11, contig 100.32) contains the expected ckMT gene. Although the clones of the Data Bank are discontinuous, gene misidentification was ruled out because the DNA sequence of exon 3 exactly corresponds to that in the ckMT cDNA, and besides, the size of the contiguous nonsequenced segments is sufficient to contain the remaining gene regions, in view of their small size [3]. The other MT-like sequence includes a complete MT ORF, which we call ckMT2, and whose translation fully matches the Columba livia form 1 MT. Conse- quently, the original ckMT will hereafter be called ckMT1. Analysis of the chicken, mouse and human ge- nomes clearly identifies synteny between a 50-kb ge- nomic region comprising ckMT1 and the mammalian MT clusters (111 kb in mouse and 200 kb in humans), bordered in the three cases by the Bbs2 and Nup93 genes, that we adopted as flanking markers (Fig. 5). Therefore, the initial proposal of a unique MT gene in chicken instead of a MT gene cluster, remains valid only if considering its mammalian syntenic region, but not for the whole genome. The following pieces of evidence highly favour the consid- eration of ckMT2 as a functional gene: the sequence identity shared by the putative ckMT2 and Columba MT1 proteins [14], the integrity of the ckMT2 gene structural elements, the scarcity of retrogenes in the chicken genome [2], the existence of a corresponding chicken EST in the databases, and the identification in the 5¢-ckMT2 region of canonic cis regulatory sequences, including those of three metal response ele- ments (MREs) at positions )269, )130 and )96. A low and ⁄ or time- and tissue-restricted ckMT2 expres- sion pattern would plausibly be the reason why its Fig. 5. Structure and synteny of the chicken, mouse and human genomic regions containing the MT genes. The chromosome number and bands enclosing the MT cluster are indicated for the mammalian genomes. For the chicken genome, the name of the corresponding contig is shown, as well as of the known chromosome. Two genes external to the MT cluster have been used as flanking synteny markers. Chicken metallothionein L. Villarreal et al. 530 FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS cDNA has remained undetected and thus the corres- ponding gene gone unnoticed. In fact, the expression levels of the ortologous Columba MT1 are reported to be fivefold lower than those of the main avian MT forms [14]. Finally, the features of the three currently known avian MT protein sequences (Fig. 6A) were com- pared to those of mammalian MT1 and MT4 and to representatives of lower vertebrate MTs. From the protein distance relationships (Fig. 6B), it is reason- able to consider Columba MT2 (avMTb in Fig. 6A) as a slight variant of the predominant avMTa avian MT form. If so, an early duplication in the avian lineage would have yielded one MT ( ckMT1) clo- ser to the amphibian and mammalian MT1–MT2 system and syntenic to the latter, and a second MT ( ckMT2) more similar to the hypothetically pri- meval mammalian MT4, and chromosomically non- linked to the previous one. What can no longer be assumed as a general rule is the alleged single-copy composition for the avian MT system, although, and in reflection of the general avian versus mammalian genome features, it is clear that the MT family has not expanded in birds as much as in mammals [2]. Conclusions The main goal of this work was the characterization of the ckMT metal binding abilities, as a model for avian MTs, mainly in comparison with the mammalian MT1 and MT4 forms. In mammals, the paradigmatic MT1 has been identified as a Zn-thionein and MT4 as a Cu-thionein, through the analysis of their in vivo and in vitro metal binding preferences and in silico consid- eration of their protein sequences [15]. The main factors for this classification are the capacity of Cu-thioneins versus the incapacity of Zn-thioneins to render homometallic copper complexes when heterolo- gously synthesized in Cu-supplemented media, as well as the clustering with known Cu-thioneins (as yeast Cup1 protein) in protein distance trees. Here we show that ckMT1 exhibits a Zn-thionein behaviour accord- ing to these criteria. Nevertheless, the Zn(II) and Cd(II) binding abilities of ckMT1 are intermediate between those of MT1 and MT4, but closer to MT1 than to MT4 (cf. stoichiometry, folding and domain dependency results). The Cu(I) binding studies of ckMT1 reveal also an intermediate character, but in this case closer to MT4. A B Fig. 6. (A) ClustalW alignment of the known avian MT sequences, and the MT1 (P02802) and MT4 (P47945) mammalian forms. The shaded boxes indicate the cysteine resi- dues. The avMTa sequence corresponds to the MT of Gallus gallus 1 (P68497), Cairina moschata (P68495), Meleagris gallopavo (P68498), Anas platyrhynchos (P68494) and Coturnix japonica (Q5G1K5). The avMTb is the Columba livia 2 isoform (P15787). The avMTc sequence is found in the Columba livia 1 (P15786) and Gallus gallus 2 isoforms. For avMTb and avMTc only the residues dif- fering from avMTa are shown. (B) Protein distance tree constructed with the amino acid sequences of the avian MT isoforms, the mammalian MT1, MT2 (P02798) and MT4 isoforms, the fish MTs of genera Ruti- lus (P80593), Danio (P52722) and Barbatula (P25128), and the amphibian Ambystoma MT (O42152). The bootstrap values of the branching points are indicated. The sequence accession numbers in the Uni- ProtKB ⁄ Swiss-Prot databank are indicated in parentheses. L. Villarreal et al. Chicken metallothionein FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS 531 ! Cu(I) MT1 ckMT1 MT4 Zn(II); Cd(II)  In conclusion, although a Zn-thionein, ckMT1 is a better copper-coordinating protein than MT1, which would allow a compensation for the absence of a specific Cu-thionein in chicken. The classification of ckMT1 as a Zn-thionein is in full concordance with the location of its sequence in the protein distance trees, closer to the syntenic mammalian MT1 form. Surprisingly, in silico analyses identify a second MT gene in chicken, unlinked with the known one, and with all the features to be considered as a functional coding region. Investigation on this gene ⁄ protein should be performed to fully understand the MT system in birds. Finally, a new dimerization mechan- ism in MTs, related to Cd(II) coordination by the C-terminal histidine of ckMT1, has been proposed. This is noteworthy because a histidine is not only the most common C-terminal residue among avian MTs, but also in significant invertebrate representatives, such as Caenorhabditis elegans MT, the characterization of which is currently being performed. Experimental procedures Bacterial strains and plasmids The Escherichia coli strains DH5a [32] and JM105 [33] were used for DNA manipulation and sequencing, and BL21 was used [34] for recombinant protein synthesis. pGEX-4T- 1 (GE-Amersham Biosciences, Europe GmbH, Barcelona, Spain) was used as expression vector. Recombinant E. coli strains were grown at 37 °C in Luria–Bertani media with ampicillin at a final concentration of 100 lgÆmL )1 . Protein synthesis was induced by adding 10 mm isopropyl-1-thio- b-d-galactopyranoside (final concentration) to the cultures. ZnCl 2 , CdCl 2 or Cu 2 SO 4 were added to final concentrations of 300 lm, 300 lm and 500 lm, respectively. Cloning of the chicken MT cDNA and its separate a and b domains for recombinant expression The ckMT coding sequence, kindly provided by Dr G.K. Andrews of the University of Kansas Medical Center [7] as a pSP6 clone, was amplified by PCR using the fol- lowing oligonucleotides: upstream primer, ckMT-BamHI (5¢-GCC GGATCCATGGACCCTCAGGA-3¢) and down- stream primer, ckMT-SalI(5¢-GCGCGC GTCGACTCAG TGGCAGCA-3¢). Through this reaction, a BamHI restric- tion site (underlined) was introduced before the ATG initi- ation codon and a SalI site (underlined) immediately after the stop codon. A 35-cycle PCR profile of 30 s at 94 °C (denaturing), 30 s at 60 °C (annealing) and 30 s at 72 °C (extension) was carried out in a total volume of 100 lL, comprising 2 lLof25mm dNTP mixture, 2 lLof20lm primer solution, 1 U of DeepVent DNA polymerase (New England Biolabs, Hitchin, England) and 100 ng of the tem- plate DNA. The cDNAs encoding the separate ckMT domains were obtained by mutagenic PCR on the initial clone. To amplify the ckMT a fragment, encompassing from the 32nd MT residue (Lys) to the C terminus, a PCR reaction was performed with the ackMT-BamHI primer (5¢- CGCGGATCCATGAAGAGCTGCTGCTC-3¢, upstream) and the ckMT-SalI primer (downstream). The ckMT b fragment extends from the ATG initiation codon to the 31st residue (Arg). The primers used for its PCR amplifica- tion were: ckMT-BamHI (upstream) and bckMT-SalI (5¢-CCGCGCGTCGACCTAGCGGCAGCTCCCGGCAGC GG-3¢, downstream). Both PCR reactions were performed using the same conditions than for the entire ckMT cDNA. In all cases, the PCR products were isolated from 2% ag- arose gels with the Gel Band Purification kit (GE-Amer- sham Biosciences), digested with BamHI and SalI (Takara Shuzo Co. Ltd, Kyoto, Japan) and subsequently ligated into the same sites of pGEX-4T-1. All the DNA constructs were confirmed by automatic DNA sequencing (ABI 370, Perkin Elmer, Wellesley, MA, USA), using the Dye Termi- nator Cycle Kit (GE-Amersham Biosciences), to rule out the presence of PCR induced substitutions. Synthesis and purification of the ckMT, ackMT and bckMT metal complexes Recombinant bacteria were grown both in small-scale cul- tures (1.5 L in Erlenmeyer flasks) and large-scale cultures (at least 10 L in a Microferm Fermentor (New Bruns- wich) coupled to a Westfalia CSA-1-06-475 centrifuge and controlled by a TVE-OP 76 ⁄ 0 programmer (Braun Biotech). In both cases, the transformed E. coli cells were grown as described [16], supplementing the medium either with ZnCl 2 , CdCl 2 or CuSO 4 as explained above. Purifi- cation of all the metal–MT complexes was performed as described for mouse MT1 [17,18], starting from the recovered GST–MT fusions. At the end, the MT-contain- ing fractions eluted from a FPLC Superdex TM 75 column (GE-Amersham Biosciences) in 50 mm Tris ⁄ HCl buffer pH 7.0, were analysed by SDS ⁄ PAGE (15% acrylamide). Samples were pooled, aliquoted and kept at )80 °C under argon until required. Analysis of the ckMT, ackMT and bckMT metal content Inductively coupled ICP-AES was used to determine the amount of protein present in the different preparations Chicken metallothionein L. Villarreal et al. 532 FEBS Journal 273 (2006) 523–535 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... 309–313 Wei D & Andrews GK (1988) Molecular cloning of chicken metallothionein Deduction of the complete amino acid sequence and analysis of expression using cloned cDNA Nucleic Acids Res 16, 537–553 Fernando LP & Andrews GK (1989) Cloning and expression of an avian metallothionein- encoding gene Gene 81, 177–183 Fernando LP, Wei D & Andrews GK (1989) Structure and expression of chicken metallothionein. .. BC, Fernando LP, Huet-Hudson YM, Dey SK & Andrews GK (1997) Activation of the chicken metallothionein promoter by metals and oxidative stress in cultured cells and transgenic mice Comp Biochem Physiol 116, 75–86 Shartzer KL, Kage K, Sobieski RJ & Andrews GK (1993) Evolution of avian metallothionein: DNA sequence analyses of the turkey metallothionein gene and metallothionein cDNAs from pheasant and quail... 7.5 The molecular mass of the apo-forms was determined as for the Cu-containing species, except that the carrier was a 1 : 1 mixture of acetonitrile and trifluoroacetic acid, pH 1.5 Masses for the holo-species were calculated as described in [35] Chicken metallothionein GC determination of the sulphide content in the ckMT, ackMT and bckMT metal complexes The sulphide presence in the ckMT, ackMT and. .. performed under argon, and all the titrations were carried out at least twice The pH for all experiments remained constant throughout without the addition of buffers, and the temperature was kept at 25 °C by means of a Peltier PTC-351S apparatus (TE Technology Inc., Traverse City, MI, USA) MS characterization of the ckMT, ackMT and bckMT metal complexes The molecular mass of the Zn-, Cd-, and Cu-MT species... Evolutionary Genetics Chicken metallothionein Analysis and Sequence Alignment Brief Bioinformatics 5, 150–163 Supplementary material The following supplementary material is available online: Note S1 Comparison of the CD fingerprints of the three in vivo Cd–bMT samples: Cd–bckMT, Cd3– bMT1 and Cd3–bMT4 Note S2 Assignment of the CD spectrum of the Cd3– bMT1 species Table S1 Molecular masses and metal (Zn, Cd or Cu)... or Cu) to protein ratios found for the in vivo synthesized ckMT, ackMT and bckMT metal aggregates Table S2 Distribution of the metal aggregates present in solution, according to ESI-MS data, during the titration of Zn4–ackMT (A), Zn3–bckMT (B) and Zn7–ckMT (C) with CdCl2 at pH 7 as a function of the number of Cd(II) equivalents added Table S3 Distribution of the metal aggregates present in solution,... (1997) Binding of excess Cadmium (II) to Cd7 -metallothionein from recombinant mouse Zn7- metallothionein 1 UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis J Inorg Biochem 68, 157–166 18 Capdevila M, Cols N, Romero-Isart N, Gonzalez-Duarte R, Atrian S & Gonzalez-Duarte P (1997) Recombinant synthesis of mouse Zn3-b and Zn4-a metallothionein. .. according to ESI-MS data, during the titration of Zn4–ackMT (A), Zn3–bckMT (B) and Zn7–ckMT (C) with [Cu(MeCN)4]ClO4 at pH 7 as a function of the number of Cu(I) equivalents added Fig S1 CD spectra corresponding to the titrations of (A) Zn4–ackMT (B) Zn3–bckMT, and (C) Zn7–ckMT with Cu(I) at pH 7 The arrows show the evolution of the spectra when the indicated number of Cu(I) equivalents were added This... copper metallothionein studies? The binding features of Ag (I) to mammalian metallothionein 1 J Biol Inorg Chem 8, 831–842 23 Capdevila M, Pagani A, Domenech J, Tio L, Villarreal L & Atrian S (2005) Zn- and Cd -Metallothionein recombinant species from the most diverse phyla may contain sulfide ligands Angew Chem Int Ed 44, 4618–4622 24 Villarreal L, Tio L, Atrian S & Capdevila M (2005) Influence of chloride... ckMT, ackMT and bckMT metal complexes was quantified by heavy acidification of the MT preparation, followed by GC and detection of the volatile H2S generated through a flame photometric detector-GC coupled system (FPD-GC) Analysis conditions are detailed in [23] In silico analysis of the chicken genome and avian MT protein sequences The last annotated chicken Genome version (29.1e, the Wellcome Trust Sanger . Comparative metal binding and genomic analysis of the avian (chicken) and mammalian metallothionein Laura Villarreal 1 , Laura Tı ´o 2 , Merce ` Capdevila 1 and Sı ´lvia Atrian 2 1. evolution of the spectra when the indicated number of Cd(II) was added. (B) CD spectrum of the acidification of the final solution of the Cd(II) titration of Zn–ckMT, the arrow shows the evolution. the num- ber of in vivo and in vitro metal MT generated species, their stoichiometry, and their degree of folding. Addi- tionally, the spectrometric measurements revealed the composition of the