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Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.

ABSTRACT: A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

SUBMITTER: Morris CA

PROVIDER: S-EPMC1220085 | biostudies-other | 1999 Mar

REPOSITORIES: biostudies-other

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Project description:Marine coccolithophorid algae are thought to play a significant role in carbon cycling due to their ability to incorporate dissolved inorganic carbon (DIC) into both calcite and photosynthetic products. Among coccolithophorids, Emiliania huxleyi is the most prolific, forming massive blooms that affect the global environment. In addition to its ecological importance, the elaborate calcite structures (coccoliths) are being investigated for the design of potential materials for science and biotechnological devices. To date, most of the research focus in this organism has involved the partitioning of DIC between calcification and photosynthesis, primarily using measurements of an external versus internal carbonic anhydrase (CA) activity under defined conditions. The actual genes, proteins, and pathways employed in these processes have not been identified and characterized (see the work of Quinn et al. in this issue [P. Quinn, R. M. Bowers, X. Zhang, T. M. Wahlund, M. A. Fanelli, D. Olszova, and B. A. Read, Appl. Environ. Microbiol. 72:5512-5526, 2006]). In this study, the cloning and preliminary characterization of two genetically distinct carbonic anhydrase cDNAs are described. Phylogenetic analysis indicated that these two genes belonged to the gamma (gamma-EhCA2) and delta (delta-EhCA1) classes of carbonic anhydrases. The deduced amino acid sequence of delta-EhCA1 revealed that it encodes a protein of 702 amino acids (aa) (ca. 77.3 kDa), with a transmembrane N-terminal region of 373 aa and an in-frame C-terminal open reading frame of 329 aa that defines the CA region. The gamma-EhCA2 protein was 235 aa in length (ca. 24.9 kDa) and was successfully expressed in Escherichia coli BL21(DE3) and purified as an active recombinant CA. The expression levels of each transcript from quantitative reverse transcription-PCR experiments under bicarbonate limitation and over a 24-h time course suggest that these isozymes perform different functions in E. huxleyi.

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Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.

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