Purification, characterization, and cDNA cloning of a novel metallothionein-like, cadmium-binding protein from Caenorhabditis elegans

Lee W. Slice, Jonathan H. Freedman, Charles S. Rubin

Research output: Contribution to journalArticle

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Abstract

Caenorhabditis elegans adapted for survival in high concentrations of Cd(II) express a heavy metal binding protein designated C. elegans metallothionein-like protein or MT-Ce. This protein was purified to homogeneity and characterized. MT-Ce binds 6 mol of Cd(II)/ mol protein. The sequence of 39 amino-terminal residues in MT-Ce was determined. A radiolabeled 41-mer oligonucleotide, designed from the partial MT-Ce sequence, was used in conjunction with sucrose gradient centrifugation to obtain size-fractionated poly(A+) RNA enriched in MT-Ce sequences. Subsequently, cloned cDNAs, corresponding to MT-Ce mRNA sequences, were isolated from a λZapII cDNA library prepared from the enriched template mRNA. cDNA and protein sequence analysis revealed that MT-Ce comprises 62 amino acid residues and has a predicted Mr of 6462. Seventeen of the 18 Cys residues in the nematode cadmium-binding protein are included in Cys-X-Cys and X-Cys-Cys-X motifs that are characteristic of mammalian metallothioneins (MTs). However, the resemblance of MT-Ce to mammalian MTs is superficial. The amino acid sequence of MT-Ce is unique, and neither its putative α and β domains nor its Cys residues can be readily aligned with the corresponding regions of other eukaryotic MTs. This suggests that MT-Ce is an example of convergent evolution. The MT-Ce mRNA level in nematodes that were selected and grown with Cd(II) concentrations that are lethal for wild-type worms, was 55-fold higher than the level of MT-Ce mRNA in wild-type C. elegans. Comparison of the sequences of MT-Ce cDNAs revealed the occurrence of two types of MT-Ce mRNA. Each contains an identical coding region, but the cDNAs diverge markedly in their 5′-untranslated regions. This suggests the possibilities of regulation by alternative splicing and/ or the presence of multiple MT-Ce genes encoding a single protein, but controlled by different regulatory elements.

Original languageEnglish (US)
Pages (from-to)256-263
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number1
StatePublished - Jan 5 1990

Fingerprint

Metallothionein
Cloning
Caenorhabditis elegans
Purification
Organism Cloning
Complementary DNA
Messenger RNA
Proteins
cysteinylcysteine
Amino Acids
Gene encoding
Centrifugation
5' Untranslated Regions
Protein Sequence Analysis
Alternative Splicing
Heavy Metals
Protein C
Gene Library
Oligonucleotides
Sucrose

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification, characterization, and cDNA cloning of a novel metallothionein-like, cadmium-binding protein from Caenorhabditis elegans. / Slice, Lee W.; Freedman, Jonathan H.; Rubin, Charles S.

In: Journal of Biological Chemistry, Vol. 265, No. 1, 05.01.1990, p. 256-263.

Research output: Contribution to journalArticle

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abstract = "Caenorhabditis elegans adapted for survival in high concentrations of Cd(II) express a heavy metal binding protein designated C. elegans metallothionein-like protein or MT-Ce. This protein was purified to homogeneity and characterized. MT-Ce binds 6 mol of Cd(II)/ mol protein. The sequence of 39 amino-terminal residues in MT-Ce was determined. A radiolabeled 41-mer oligonucleotide, designed from the partial MT-Ce sequence, was used in conjunction with sucrose gradient centrifugation to obtain size-fractionated poly(A+) RNA enriched in MT-Ce sequences. Subsequently, cloned cDNAs, corresponding to MT-Ce mRNA sequences, were isolated from a λZapII cDNA library prepared from the enriched template mRNA. cDNA and protein sequence analysis revealed that MT-Ce comprises 62 amino acid residues and has a predicted Mr of 6462. Seventeen of the 18 Cys residues in the nematode cadmium-binding protein are included in Cys-X-Cys and X-Cys-Cys-X motifs that are characteristic of mammalian metallothioneins (MTs). However, the resemblance of MT-Ce to mammalian MTs is superficial. The amino acid sequence of MT-Ce is unique, and neither its putative α and β domains nor its Cys residues can be readily aligned with the corresponding regions of other eukaryotic MTs. This suggests that MT-Ce is an example of convergent evolution. The MT-Ce mRNA level in nematodes that were selected and grown with Cd(II) concentrations that are lethal for wild-type worms, was 55-fold higher than the level of MT-Ce mRNA in wild-type C. elegans. Comparison of the sequences of MT-Ce cDNAs revealed the occurrence of two types of MT-Ce mRNA. Each contains an identical coding region, but the cDNAs diverge markedly in their 5′-untranslated regions. This suggests the possibilities of regulation by alternative splicing and/ or the presence of multiple MT-Ce genes encoding a single protein, but controlled by different regulatory elements.",
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