Recombinant synthesis of mouse Zn 3 - ? and Zn 4 - ? metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Capdevila, Mercè; Cols, Neus; Romero‐Isart, Núria; Gonzàlez‐Duarte, Roser; Atrian, Sı́lvia; González-Duarte, Pilar

doi:10.1007/s000180050088

Cited by 84 publications

(82 citation statements)

References 28 publications

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“…First, the in vivo synthesized M-MT4, M-␣MT4, and M-␤MT4 (where M represents zinc, cadmium, and copper) aggregates and the recombinant Cd-MT1 and Cd-␣MT1 species, not previously studied, were characterized. Second, the reaction pathways of the in vitro zinc/cadmium and zinc/copper replacement processes undergone by the three Zn-MT4 peptides at pH 7 were determined using the rationale previously applied for MT1 (21)(22)(23)(24). Detailed analysis of the spectroscopic (CD and UV-visible electronic absorption) sets of data recorded during these titrations provided information on the number of metal-MT species generated, their stoichiometry, and their degree of folding.…”

Section: Resultsmentioning

confidence: 99%

“…According to the composition of the recombinant samples reported here, only the CD spectra of the Cd 4 -␣MT4, Cd 7 -MT1, and Cd 4 -␣MT1 solutions can be considered representative of the CD fingerprints of these species (data included in Fig. 4), all showing the characteristic derivative-shaped band at ϳ250 nm of the Cd-MT aggregates (21,22). In contrast, the CD spectra of the recombinant Cd-MT4 and Cd-␤MT4 samples (data included in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The conditions for both PCRs were the same as for the entire MT4 cDNA. The construction of MT1 expression vectors has previously been described (21,22).…”

Section: Methodsmentioning

confidence: 99%

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Functional Differentiation in the Mammalian Metallothionein Gene Family

Tío

Villarreal

Atrian

et al. 2004

Journal of Biological Chemistry

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This paper reports on the characterization of the metal binding abilities of mammalian MT4 and their comparison with those of the well known MT1. Heterologous Escherichia coli expression in cultures supplemented with zinc, cadmium, or copper was achieved for MT4 and for its separate ␣MT4 and ␤MT4 domains as well as for MT1 and its ␣MT1 domain in cadmium-enriched medium. The in vivo conformed metal complexes and the in vitro substituted zinc/cadmium and zinc/copper MT4 aggregates were characterized. Biosynthesis of MT4 and ␤MT4 in Cd(II)-supplemented medium revealed that these peptides failed to form the same homometallic species as MT1, thus appearing less effective for cadmium coordination. Conversely, the entire MT4 and both of its domains showed better Cu(I) binding properties than MT1, affording Cu 10 -MT4, Cu 5 -␣MT4 and Cu 7 -␤MT4, stoichiometries that make the domain dependence toward Cu(I) clear. Overall results allow consideration of MT4 as a novel copper-thionein, made up of two copper-thionein domains, the first of this class reported in mammals, and by extension in vertebrates. Furthermore, the in silico protein sequence analyses corroborated the copper-thionein nature of the MT4 peptides. As a consequence, there is the suggestion of a possible physiological role played by MT4 related with copper requirements in epithelial differentiating tissues, where MT4 is expressed.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Functional Differentiation in the Mammalian Metallothionein Gene Family

Tío

Villarreal

Atrian

et al. 2004

Journal of Biological Chemistry

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“…1-ml fractions were collected and analyzed for protein content by their absorbance at 254 nm. Validation of these recombinant synthesis procedures has been fully confirmed for mammalian MT (32). Aliquots of the protein-containing fast performance liquid chromatography fractions were analyzed by 15% SDS-PAGE and stained by Coomassie Blue.…”

Section: Mt Expression and Analysis Of The Recombinant Zinc And Coppementioning

confidence: 98%

A New Insight into Metallothionein (MT) Classification and Evolution

Valls¹,

Bofill²,

Gonzàlez‐Duarte³

et al. 2001

Journal of Biological Chemistry

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We report the synthesis and characterization of a Homarus americanus MT-cDNA (MTH) through retrotranscription of MTH-mRNA from metal-injected lobsters. Heterologous Escherichia coli expression in zinc-and copper-supplemented medium was achieved for MTH, the two domains ␤␤MTH and ␤␣MTH and three site-directed mutants, ␤␤C9H, ␤␣C37H, and ␤␣E31C/T34C. The in vivo conformed metal complexes and the in vitro substituted cadmium aggregates were characterized. Major stoichiometries of M The metallothionein (MT) 1 superfamily comprises a wide range of low molecular weight polypeptides, sharing the ability to chelate heavy metal atoms (1). MT are present in all animal phyla and in most fungi and plants and have been related to metal homeostasis and detoxification, although their definite physiological function is still a matter of debate (2-5). To classify these extremely heterogeneous amino acid sequences, three different classes were proposed: Class I, comprising all the MT with clear sequence similarity to horse liver MT 1; Class II, including all the sequences with no clear similarity to horse liver MT 1; and Class III for the phytochelatins and vegetal ␥-glutamyl peptides of enzymatic origin (1). More recently, a new classification consisting of a singular set for each individual eukaryotic taxon has been proposed (www.unizh.ch/ ϳmtpage/classif.html). Both systematizations are based on primary structure data but do not include any functional or evolutionary information. Conventionally, terminology and standards corresponding to the canonical mammalian MT molecule have been extended to describe amino acid sequence and metal cluster features of newly described MT forms. Mammalian MT folds into a twodomain structure when coordinating divalent metal ions. The N-terminal segment (␤ domain), with 9 cysteines in Cys-XaaCys arrays, binds three M(II) ions, and the C-terminal segment (␣ domain), with 11 cysteines, some of which in Cys-Cys tandems binds four M(II) ions (6, 7). A higher binding affinity for monovalent (copper) than for divalent (zinc and cadmium) ions was described for the mammalian ␤ domain, with an opposite trend for the ␣ domain (8). Contemporarily, Saccharomyces cerevisiae MT CUP1 was characterized. The synthesis of this protein was induced by copper, and the MT recovered contained copper ions exclusively (9). Because the CUP1 cysteine distribution aligned well to the ␤ domain of mammalian MT, it was assumed that proteins with a primary structure similar to the mammalian ␤ domain would exhibit copper binding rather than zinc binding preferences. On the basis of their amino acid alignments, further application of the ␤/␣ domain terminology led to the consideration of crustacean peptides as ␤-␤ MT (10) and thus putative copper thioneins. This was corroborated by the three divalent ions contained in each domain of the crustacean Cd-MT structures solved by NMR (11, 12) and thus comparable with the Cd 3 -␤MT fragment in mammalian MT.A survey of the literature on naturally occurring and metalinduced crustacean MT ...

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“…It was found that MT levels in mammalian tissues under physiological conditions could be rather high to harbour important implications for MTs operation in Zn and redox metabolism (Capdevila et al, 1997;Capasso et al, 2005;Kelly et al, 2006). Different substances besides metals, such as fungicides fenhexamid, mancozeb, and also hydrogen peroxide, induce the elevation of MT content in fish (Viarengo et al, 1999;Cavaletto et al, 2002;Paris-Palacios et al, 2003;Mosleh et al, 2005;Kang, 2006).…”

Section: Non-enzymatic Antioxidants: Glutathione and Other Scavengersmentioning

confidence: 99%