A New Pathway for Heavy Metal Detoxification in Animals

Abstract

Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms. Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, ␥-Glu-CysGly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins. Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH. Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that the ce-pcs-1 gene of the nematode worm Caenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism.Plants and some fungi post-translationally synthesize novel peptides termed phytochelatins (PCs) 1 when exposed to heavy metals. Fabricated from the ubiquitous thiol tripeptide GSH and related thiols in a novel transpeptidation reaction catalyzed by PC synthases (␥-glutamylcysteinyltransferase; EC 2.3.2.15), PCs have the general structure (␥-Glu-Cys) n -Xaa, contain 2-11 ␥-Glu-Cys repeats, chelate heavy metals at high affinity, and facilitate the vacuolar sequestration of heavy metals, most notably Cd 2ϩ (1-3). Although it is more than a decade since the first report of the partial purification of a heavy metal-, primarily Cd 2ϩ -activated PC synthase from plant extracts (1), it is only recently that the small family of genes encoding these enzymes has been identified in plants and the fission yeast Schizosaccharomyces pombe (4 -6). As exemplified by the clone from Arabidopsis thaliana (AtPCS1), these genes encode 45-55-kDa proteins that are sufficient for heavy metalactivated PC synthesis from GSH both in vivo and in vitro (6, 7).An unexpected outcome of the cloning of AtPCS1 and its equivalents from other plants and S. pombe was the identification of a single-copy gene homolog (accession number Z66513) in the nematode worm Caenorhabditis elegans (4 -6). Designated ce-pcs-1, this gene encodes a hypothetical 40.8-kDa protein (CePCS1) bearing 32% identity (45% similarity) to AtPCS1 in an overlap of 367 amino acid residues (6). Disclosure of a PCS1 homolog in the genome of C. elegans was surprising in that it raised for the first time the possibility that not only GSH and metallothioneins (8) but also PCs might participate in metal homeostasis in at least some animals.In the report that follows we demonstrate unequivocally that ce-pcs-1 encodes a bona fide PC synthase whose activity is necessary for the detoxification of heavy metals in the intact organism. Discovery of the PC synthase-dependent pathway in the model organism C. elegans establishes a firm basis for determining the ubiquity of this pathway in other animals and for elucidation of the identity and organization of the cellular machiner...

Identification of Mutations in Caenorhabditis elegans That Cause Resistance to High Levels of Dietary Zinc and Analysis Using a Genomewide Map of Single Nucleotide Polymorphisms Scored by Pyrosequencing

“…Phytochelatins are peptides that can bind metals and contribute to cadmium tolerance. C. elegans pcs-1 mutants (defective in phytochelatin synthesis) and hmt-1 mutants (defective in phytochelatin transport) display enhanced cadmium toxicity (Vatamaniuk et al 2001(Vatamaniuk et al , 2005. These results indicate that metallothioneins and phytochelatins are involved in cadmium detoxification.…”

CeHMT-1, a Putative Phytochelatin Transporter, Is Required for Cadmium Tolerance in Caenorhabditis elegans

“…Identification and Cloning of ce-hmt-1-The discovery of a functional PCS gene, ce-pcs-1 in C. elegans (11,12), and the results of earlier investigations suggesting that in S. pombe its homolog SpPCS acts upstream of SpHMT1 (15, 16) prompted a systematic search of worm sequence databases for HMT-1 homologs. Knowing that the C. elegans genome contains some 60 open reading frames for ABC proteins, of which 29 fall into the half-molecule transporter category (24), and that even ABC transporters with disparate capabilities can have a relatively high degree of sequence similarity (18), putative C. elegans HMT-1 homologs were identified according to two structural criteria in addition to overall sequence similarity.…”

“…It is now almost 15 years since the partial purification of the enzyme capable of catalyzing PC synthesis (5), yet it is only in the last several years that its molecular identity has been determined by the cloning and characterization of genes encoding PC synthases (PCSs). Originally isolated from Arabidopsis thaliana, S. pombe, and wheat (Triticum aestivum), and latterly from C. elegans, these genes (designated AtPCS1, SpPCS, TaPCS1 and ce-pcs-1, respectively) encode 40 -50% sequence-similar 41-55-kDa polypeptides that contribute to heavy metal detoxification by catalyzing the de novo synthesis of PCs (7)(8)(9)(10)(11)(12).…”

“…dsRNA Microinjection and Phenotypic Screens-The ce-hmt-1 RNAi experiments and phenotypic screens were performed as described for ce-pcs-1 (12). In all cases, the dsce-hmt-1 RNA-or control (injection buffer alone)-injected worms were first cultured for 12-18 h on NGM plates seeded with E. coli OP50 before transfer to plates supplemented with Cd 2ϩ .…”

“…RNA was transcribed from the T3 and T7 promoters of the construct using an in vitro RNA transcription kit (Stratagene). After DNase digestion of the template, the sense and antisense preparations were gel-purified, mixed in an equimolar ratio in 1 mM EDTA buffered to pH 7.5 with Tris-HCl, heated for 1 min in boiling water, and annealed at room temperature as described for ce-pcs-1 (12). To ensure that the ce-hmt-1 dsRNA preparations from the annealing step were devoid of residual single-stranded RNA, the mixtures were treated with RNase T1 (22).…”