Cloning and sequencing of cDNAs encoding for a novel copper-specific metallothionein and two cadmium-inducible metallothioneins from the blue crab Callinectes sapidus

Syring, Rachel; Brouwer, Thea Hoexum; Brouwer, Marius

doi:10.1016/s0742-8413(99)00114-0

Cited by 34 publications

(32 citation statements)

References 22 publications

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“…The strict requirement of stabilizing zinc ions in copper clusters is consistent with our observations that the copper content of copper,zinc-␤␤MTH is higher when zinc, as well as copper, is present in the culture medium, and also with the reported extreme susceptibility to oxidation of the copper-treated C. maenas MT (16). The poorer response to copper-rich physiological environments of MTH if compared with mammalian MT would be overcome by the presence of another copper-specific form, as has been found in C.sapidus (21). The zinc thionein would then be released from the selective pressure of efficient copper binding.…”

Section: Recombinant Synthesis and Characterization Of Zn-␤␤mth And Zsupporting

confidence: 92%

“…Furthermore, the number of MT genes varies in the analyzed species: Cancer pagurus (13,14), Carcinus maenas (15,16), Homarus americanus (17,18), and Callinectes sapidus (19,20), ranging from one to three MT encoding polypeptides, with substantially different metal affinities. This has recently been substantiated by the identification in C. sapidus of a copper-specific MT, in addition to two cadmiuminducible MT forms (21), according to a previous report by Dallinger et al (22). Second, characterization of the proteins induced in Crustacea by high copper and cadmium concentrations has yielded a wide range of metal stoichiometries.…”

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confidence: 64%

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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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Section: Recombinant Synthesis and Characterization Of Zn-␤␤mth And Zsupporting

confidence: 92%

mentioning

confidence: 64%

A New Insight into Metallothionein (MT) Classification and Evolution

Valls¹,

Bofill²,

Gonzàlez‐Duarte³

et al. 2001

Journal of Biological Chemistry

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“…Ten genes (Table 1: genes 1 to 10), including heat shock protein 70 (Hsp70), mitochondrial and cytosolic manganese superoxide dismutase (mit-MnSOD and cyt-MnSOD) (Brouwer et al 2003), hemocyanin 1 (Brouwer et al 2002), Cu and Cd metallothionein (CuMT3 and CdMT1) (Syring et al 2000), beta-actin, and ribosomal proteins S15, L23 and S20, were cloned and sequenced from hepatopancreas tissue of blue crab. The blue crab genes were PCRamplified and then robotically spotted in duplicate onto neutral nylon membranes together with various controls, including exogenous Arabidopsis 'spiking' genes, as described elsewhere (Larkin et al 2003).…”

Section: Methodsmentioning

confidence: 99%

Molecular indicators of hypoxia in the blue crab Callinectes sapidus

Brown‐Peterson¹,

Larkin²,

Denslow³

et al. 2005

Mar. Ecol. Prog. Ser.

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Occurrence of hypoxia in estuarine waters is increasing, and recovery of impacted estuaries is slow. Detection of early effects of hypoxia is needed for timely remedial action to be taken. Here we examine the use of hypoxia-responsive gene and protein expression profiles in the blue crab Callinectes sapidus as early indicators of impacts of hypoxia. We cloned 23 potential hypoxiaresponsive genes, which were used to construct gene macroarrays. Crabs exposed to chronic hypoxia (2.5 ppm dissolved oxygen [DO]) for 15 d showed significant (p < 0.05) changes in gene expression of heat shock protein 70 (Hsp70), copper metallothionein (CuMt3), cytosolic MnSOD (cyt-MnSOD) and ribosomal proteins S15 and L23. In all cases except for CuMt3, gene expression decreased after 5 d exposure to hypoxia. Expression of Hsp70, CuMt3 and cyt-MnSOD also increased (p < 0.05) in normoxic crabs held for 15 d, suggesting confounding effects from confinement stress. Hemocyanin protein concentrations changed significantly (p = 0.005) across the 15 d chronic hypoxia exposure. Hemocyanin in crabs exposed to 10 d intermittent hypoxia (2.5 to 8 ppm DO over a 24 h cycle) did not change, but cyt-MnSOD gene expression increased significantly (p = 0.037), whereas cytochrome c oxidase subunit 1 (ccox1) showed a 2.2-fold downregulation. Blue crabs collected from Pensacola Bay, Florida, showed significant (p < 0.006) downregulation of ccox1 and cyt-MnSOD gene expression as well as hemocyanin protein levels at a diurnally hypoxic marsh site. Several hypoxiaresponsive genes (elongation factor 2, cryptocyanin, hemocyanin) were also significantly elevated (p < 0.006) in intermolt versus premolt normoxic crabs. The identification of hypoxia-responsive genes and proteins in the blue crab is a promising first step towards the development of sensitive molecular tools for the detection of sublethal effects of hypoxia in estuarine-resident species.KEY WORDS: Dissolved oxygen · Hypoxia · Crustacea · Molecular biomarker · Gene arrays Resale or republication not permitted without written consent of the publisher

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“…Thus, on the basis of structure/function relationships, we identified MT1 and ␣MT1 as Zn-thioneins (21-23) and the independent ␤MT1 as a Cu-thionein (24,25). Currently known Cu-thioneins belong to arthropoda (Drosophila (26,27) and crustacea (28)) and unicellular eukaryota (yeasts (29) and ciliate protozoa (30)). …”

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confidence: 99%

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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Cloning and sequencing of cDNAs encoding for a novel copper-specific metallothionein and two cadmium-inducible metallothioneins from the blue crab Callinectes sapidus

Cited by 34 publications

References 22 publications

A New Insight into Metallothionein (MT) Classification and Evolution

A New Insight into Metallothionein (MT) Classification and Evolution

Molecular indicators of hypoxia in the blue crab Callinectes sapidus

Functional Differentiation in the Mammalian Metallothionein Gene Family

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