Worms take the ‘phyto’ out of ‘phytochelatins’

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26 Denyer, M.C. et al. (1998) Preliminary study on the suitability of a pharmacological bio-assay based on cardiac myocytes cultured over microfabricated microelectrode arrays. Med. Biol. Eng. Comput. 36, 638–644 27 Gourdie, R.G. et al. (1998) Endothelininduced conversion of embryonic heart muscle cells into impulse-conducting Purkinje fibers. Proc. Natl. Acad. Sci. U. S. A. 95, 6815–6818

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28 Wobus, A.M. and Hescheler, J. (1992) Development of an in vitro cardiomyocytes cell model for embryotoxicological and pharmacological studies. ALTEX 9, 29–42 29 Carrier, R.L. et al. (1999) Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterisation. Biotechnol. Bioeng. 64, 580–589 30 Akin, R.E. et al. (1997) Neonatal rat heart cells cultured in simulated microgravity. In Vitro Cell. Dev. Biol. Anim. 33, 337–343

Worms take the ‘phyto’ out of ‘phytochelatins’ Olena K. Vatamaniuk, Elizabeth A. Bucher, James T. Ward and Philip A. Rea Phytochelatin synthase is the enzyme responsible for the synthesis of heavy-metal-binding peptides (phytochelatins) from glutathione and related thiols. It has recently been determined that it is not only restricted to plants and some fungi, as was once thought, but also has an essential role in heavy-metal detoxification in the model nematode Caenorhabditis elegans. These findings and others that demonstrate phytochelatin synthase-coding sequences in the genomes of several other invertebrates, including pathogenic nematodes, schistosomes and roundworms, herald a new era in phytochelatin research, in which these novel post-translationally synthesized peptides will not only be investigated in the context of phytoremediation but also from a clinical parasitological standpoint.

Olena K. Vatamaniuk Philip A. Rea* Plant Science Institute, Dept of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. *e-mail: [email protected] Elizabeth A. Bucher James T. Ward Dept of Cell and Developmental Biology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Essential heavy metals, such as Cu2+ and Zn2+, are required as cofactors in redox reactions and ligand interactions, in addition to charge stabilization, charge shielding and water ionization during biocatalysis [1]. However, both essential and nonessential heavy metals can pose an acute problem for organisms. Supraoptimal concentrations of essential heavy metals and micromolar concentrations of nonessential heavy metals (e.g. arsenic, cadmium and mercury) are toxic: they displace endogenous metal cofactors, heavy or otherwise, from their cellular binding sites, undergo aberrant reactions with the thiol groups of proteins and coenzymes, and promote the formation of destructive active oxygen species [2]. The ‘haves’ and ‘have nots’

Although the inherent reactivity of thiol groups towards soft metals appears to be a common thread in biological heavy-metal detoxification, the notion has emerged (primarily by default) that there is a strict demarcation between organisms according to the types http://tibtech.trends.com

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31 Eschenhagen, T. et al. (1997) Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart model system. FASEB J. 11, 683–694 32 Mitrius, J.C. and Vogel, S.M. (1990) Doxorubicininduced automaticity in cultured chick heart cell aggregates. Cancer Res. 50, 4209–4215 33 Rothen-Rutishauser, B.M. et al. (1998) Different behaviour of the non-sarcomeric cytoskeleton in neonatal and adult rat cardiomyocytes. J. Mol. Cell Cardiol. 30, 19–31

of thiol compounds they deploy for this purpose. Considerations of heavy-metal detoxification in animals have focused almost exclusively on two main classes of thiol peptides: the ubiquitous tripeptide, glutathione (GSH, γ-Glu–Cys–Gly), and a family of small 5–6-kDa polypeptides containing multiple Cys–X–X–Cys motifs (where X denotes any amino acid), the metallothioneins (MTs) [3]. By contrast, considerations of plants and some fungi have not only been concerned with GSH and MTs but also another class of peptide, the phytochelatins (PCs) [4]. It is now evident from the results of recent investigations that such a ‘have’–‘have not’ division does not apply to PCs. Some animals contain the machinery for PC biosynthesis and in one animal model, the soil nematode Caenorhabditis elegans, this machinery is vital if exposure to certain heavy metals is not to be lethal. Heavy metal antidotes

PCs (poly-[γ-Glu–Cys]n–X polymers) were first discovered in the fission yeast Schizosaccharomyces pombe and termed cadystins [5]; since then, they have been found in all plant species investigated, a few species of fungi and some marine diatoms [6]. PCs act as high-affinity metal chelators and facilitate the vacuolar sequestration of heavy metals, most notably cadmium ions (Cd2+) (Fig. 1). PC-dependent vacuolar Cd2+ sequestration is best understood in S. pombe, in which the hmt1+ gene product, a PC-selective ATP-binding cassette (ABC) transporter, pumps Cd2+–PC complexes and apo-PCs from the cytosol into the vacuole at the expense of ATP [7,8]. PC synthase

PCs are synthesized post-translationally; PC synthases (γ-glutamylcysteine dipeptidyl transferases, EC 2.3.2.15) catalyze the net transfer of a γ-Glu–Cys unit from one GSH molecule to another [γ-Glu–Cys–Gly + γ-Glu–Cys–Gly → (γ-Glu–Cys)2–Gly + Gly], or to a previously synthesized PC molecule [(γ-Glu–Cys)n–Gly + γ-Glu–Cys–Gly → (γ-Glu–Cys)n+1– Gly + Gly] to generate polymers containing 2–11 γ-Glu–Cys repeats (Fig. 1). It is over a decade since pioneering investigations by Meinhardt Zenk, Erwin Grill and colleagues yielded partially purified preparations of an enzyme capable of mediating these reactions [9], but it was not until just over a year ago that its molecular identity was elucidated by the

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Fig. 1. PC synthase catalyses the synthesis of PCs, (γ-Glu–Cys)n–X polymers, by the net transfer of a γ-Glu–Cys unit from one thiol peptide (usually glutathione, GSH) to another, or to a previously synthesized PC molecule. The primary effect of heavy metals in activating this process is not to bind to and activate the enzyme directly, but to ensure that the thiol groups on one of the substrate molecules are blocked as a result of metal thiolate formation [14]. In media containing GSH and Cd2+, for example, PC2 is the product of a bisubstrate transpeptidation reaction in which both free GSH and bis(glutathionato)cadmium (Cd2+–GS2) participate. PCs thiol coordinate heavy metals and contribute to their detoxification. In plants and fungi, Cd2+–PCs are transported from the cytosol into the vacuole by ABC transporters (HMT1 in Schizosaccharomyces pombe) [7,8]. Red spheres denote the bulky anionic carboxylate groups that decorate the higher order Cd–PC complexes that accumulate.

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(γ-Glu_Cys)2_Gly (PC2)

γ-Glu_Cys_Gly (GSH) PC synthase

Cd2+ or

Activation γ-Glu_Cys_Gly (GSH)

Cd2+

Gly

S

S

S

Cd2+ S

S

Cd2+

S

S

S

(γ-Glu_Cys)n+1_Gly (PCn+1)

or

ATP

(γ-Glu_Cys)n_Gly (PCn)

ADP + Pi

HMT1 Vacuole

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independent cloning and characterization of genes encoding PC synthase for the first time by three groups [10–12]. PC synthase genes

Genes isolated from Arabidopsis thaliana, S. pombe and wheat (AtPCS1, SpPCS1 and TaPCS1, respectively) encode 50–55-kDa polypeptides bearing 40–50% sequence similarity [10–12]. According to genetic, molecular and biochemical criteria, these three genes encode the much-coveted enzyme PC synthase: Arabidopsis cad1 mutants (with a mutation in AtPCS1 [11]) are Cd2+-hypersensitive and PCdeficient [13]; SpPCS disruptants are hypersensitive to heavy metals and deficient in cellular PCs [10]; heterologous expression of AtPCS1 in Saccharomyces cerevisiae, an organism that lacks PCS homologs and does not otherwise synthesize PCs in appreciable amounts, confers increased heavy-metal tolerance concomitant with Cd2+-dependent intracellular PC accumulation [12]; cell-free extracts from AtPCS1- or SpPCS-transformed cells of Escherichia coli [11] and immunopurified epitope-tagged AtPCS1 catalyze the heavy-metal-activated synthesis of both short- and long-chain PCs from GSH in vitro [12,14]. In short, AtPCS1 and SpPCS, and by implication TaPCS1, are not only necessary but also sufficient for PC biosynthesis (i.e. they are PC synthases). A can of worms

Genetically, now that genes encoding PC synthases are readily available and it has been demonstrated that a single gene is sufficient to reconstitute core catalysis, investigations of strategies for enhancing the capacity of plants to tolerate and accumulate heavy metals will be expedited. Several laboratories in both the academic and commercial sectors are exploring this technology with a view to using plants for environmental clean-up (see below). Similarly, the ease with which recombinant AtPCS1 can now be purified to apparent homogeneity to yield PC synthase preparations with catalytic activities exceeding those http://tibtech.trends.com

of previous preparations from plant sources [9] by >1000-fold [12,14] has enabled detailed mechanistic analyses and exposed some of the basic deficiencies in our understanding of PC biosynthesis. Notable among these is the realization that heavy metals do not bind directly to the enzyme to activate PC biosynthesis but instead act as substrate ligands for a bisubstrate enzyme-substituted transpeptidation reaction in which free GSH and its corresponding heavy-metal thiolate are cosubstrates (Fig. 1) [14]. What this implies is that, contrary to the prevailing model for the self-termination of PC biosynthesis [15,16], arrest of the reactions catalyzed by PC synthases does not result from metal chelation by the products of the reaction, because GSH and PC complexes containing bound heavy metal are themselves active substrate species [14]. Instead, termination probably arises from diminution of the substrate-active thiolate pool, whether it be by the incorporation of heavy metals into higher order, substrate-inactive metal–PC complexes or by the removal of substrate-active metal–PC complexes from the PC synthase-accessible pool [14]. In short, the cytosolic concentration of free heavymetal ions need not increase even transitorily for net PC synthesis. The high values of the stability constants of heavy-metal–GSH complexes and the fact that the steady state concentration of GSH ( the most abundant intracellular thiol) is between 1 and 10 mM [17] mean that any soft metal that gains access to the cytosol will be rapidly converted to its corresponding thiolate. Because of the moderately high and constitutive expression of PCS genes, the GSH thiolates so formed will, in turn, be incorporated into derivatives, PCs, that also bind heavy metals but at higher affinity [14]. In other words, PC synthase is exquisitely tuned to deal not with free metals but with their dominant form in vivo, GSH and PC thiolates. These are important insights and emergent capabilities, but one of the most significant progressions from the cloning of plant and fungal PC synthase genes has been the discovery of a similar gene in an animal. Routine database searches disclosed a homologous

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Fig. 2. Defective PC biosynthesis results in impaired growth on Cd2+-containing media. (a) Arabidopsis (i) wild-type and (ii) cad1-3 mutant seedlings after germination and growth for 10 days on medium containing 50 µM CdCl2. (b) C. elegans (i) injection buffer-injected wild-type worms and (ii) doublestranded ce-pcs-1 (RNAi) mutants four days after hatching on growth medium containing 50 µM CdCl2. Scale bar = 0.1 mm. Abbreviations: PC, phytochelatin; RNAi, RNA interference technique.

single-copy gene in the genome of C. elegans. Tentatively named ce-pcs-1, this gene encodes a hypothetical 40.8-kDa polypeptide (CePCS1), with 30% identity (45% similarity) to AtPCS1 in an overlap of 367 amino acid residues [10–12]. This was completely unexpected because in over two decades of research into the biochemical basis of heavy-metal detoxification in animals, never before had the involvement of PCs been even cursorily mentioned or speculated. CePCS1 is another PC synthase

It was against this background that we sought to define the function of ce-pcs-1 [18]. The results were unequivocal: CePCS1 is a PC synthase. Heterologous expression of CePCS1 in yeast confers increased Cd2+tolerance concomitant with Cd2+-elicited intracellular PC accumulation. As is the case for AtPCS1 [14], the tolerance conferred by CePCS1 is not limited to Cd2+ but extends to other soft metals and metalloids, including mercury and arsenic. And, the in vivo deployment of CePCS1 for PC biosynthesis can be precisely replicated in vitro. Analyses of the capacity of cell-free yeast extracts expressing CePCS1 for the Cd2+-dependent incorporation of GSH into PCs demonstrate net synthesis of PC2 and PC3 at rates comparable to those measured for the corresponding fraction from AtPCS1-transformed cells [12,18]. Sick worms

Biochemical analyses based on the results of heterologous expression or in vitro measurements enable the feasibility of a process to be assessed but they do not tell us whether that process occurs in the intact organism or whether it is necessary for survival. Information of this type can only be acquired genetically. The finding that targeted suppression of ce-pcs-1 by the double-stranded RNA interference (RNAi) technique confers a cad1-like phenotype on C. elegans [18] was therefore crucial. PC-deficient Arabidopsis cad1 (AtPCS1) mutants exhibit a characteristic conditional phenotype: impaired growth and development, and extensive necrosis when exposed to Cd2+ concentrations tolerated by wild-type plants [13] (Fig. 2a). And, the same is true of C. elegans ce-pcs-1 RNAi mutants [18] (Fig. 2b). Wild-type worms and the progeny of worms injected with either injection buffer or double-stranded mock (GFP) RNA http://tibtech.trends.com

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are indistinguishable and tolerant of relatively high Cd2+ concentrations [18]. By contrast, the progeny of worms injected with double-stranded ce-pcs-1 RNA show severe growth retardation, developmental arrest, necrosis and sterility, and eventually die when exposed to Cd2+ concentrations tolerated by wild-type worms (Fig. 2b) [18]. CePCS1, like AtPCS1 or SpPCS1, is essential for heavy-metal detoxification in the intact organism. GSH and MTs [3] are clearly not the sole determinants of heavy-metal tolerance in C. elegans. As is often the case in exciting new research areas, a few months after the publication of our findings, another paper by Clemens et al. [19] independently demonstrated that ce-pcs-1 complements the Cd2+-hypersensitivity of SpPCS mutants and restores PC biosynthesis to near wild-type levels. Reverse osmosis

Because the experiments on C. elegans are the first to demonstrate PC synthase-mediated heavy-metal detoxification in an animal, it is inspiring for those of us who work on plants to consider that these studies owe their origins to fundamental research on plants. This is a reversal of the direction in which knowledge usually flows in the biological sciences. Many readers might also be intrigued to learn that although other gene products have been inferred to contribute to Cd2+ tolerance in C. elegans, CePCS1 is the first for which a firm biochemical basis has been established for the effects seen at the level of the whole organism. Elegant studies of C. elegans knockout mutants for a multidrug resistance-associated protein subclass ABC transporter [20] and for a mitogen-activated protein kinase kinase (MEK1) gene [21] have revealed an increase in Cd2+ sensitivity versus wild-type, but in neither case have the biochemical events underlying the mutant phenotypes been unraveled. Parasitic worms

Discovery of the PC-dependent metal detoxification pathway in C. elegans is of considerable strategic value. First, although the ubiquity of this pathway in animals needs to be determined (i.e. whether it is restricted to invertebrates or might extend to vertebrates), its existence in an organism of such genetic and molecular manipulability as C. elegans will greatly expedite investigations of the identity and organization of the cellular machinery that is probably involved in the eventual elimination, sequestration and/or metabolism of Cd2+–PCs and other PC thiolates in animals. For instance, it is still not known if animals like C. elegans sequester Cd2+–PCs in intracellular compartments, as is the case for plants and fungi (Fig. 1), or excrete them into the surrounding medium (which might explain why PCs have been overlooked in analyses of heavy-metal-binding peptides in animals). Second, the demonstration of strict orthology between CePCS1 and its homologs from plants and fungi increases our confidence that homologs from other sources will also prove to be PC synthases. With regard to an earlier

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Acknowledgements This work was supported by National Science Foundation Grant MCB0077838 awarded to P.A.R. and partially supported by National Institutes of Health Grant RO1-HL 59680-0 awarded to E.A.B. O.K.V. was sponsored by PlantGenix, Inc., Philadelphia, PA, USA.

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statement by Chris Cobbett [6] to the effect that although genes encoding PC synthase have been identified in some animals, their involvement in heavy-metal detoxification has yet to be demonstrated, we can now give a fairly definite answer: from what we have learned from C. elegans, participation of these genes in heavy-metal detoxification is highly likely. This is almost certainly the case for the PCR products from the aquatic midge Chironomus and a species of earthworm described by Cobbett [6]. In a broader context, it should be appreciated that C. elegans is only one of at least 100 000 nematode species on the planet, many of which are pathogens, and that PCS homologs are starting to appear in the EST databases of nematodes, and in those of their cousins, the schistosomes (trematodes) and roundworms (ascarids). The EST databases of two other nematodes – C. briggsae and Brugia malayi – contain partial PCS cDNAs, as do those of the schistosome Schistosoma mancons and the roundworm Parascaris univalens. C. briggsae is not a pathogen, but mosquito-borne B. malayi is responsible for deplorable diseases, such as elephantiasis and lymphatic filariasis, and species related to S. mancons (e.g. S. mansoni, S. haematobium and S. japonicum) and to P. univalens (e.g. P. equorum) are responsible for schistosomaniasis (blood flukes) and ascariasis (‘worms’). Thirteen million people worldwide are afflicted with elephantiasis and lymphatic filariasis, and it is estimated that an incredible 200 million people are infected with schistosomes, of which about one million die from the infestation each year. In the developing world, roundworms such as P. equorum cause massive livestock losses. An enzyme of wide toxicological significance

Several authors have discussed the potential value of PC synthase for phytoremediation (the use of native

References 1 Voet, D. and Voet, J.G. (1995) Biochemistry (2nd edn), Wiley, New York 2 Stadtman, E.R. (1990) Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic. Biol. Med. 9, 315–325 3 Klaassen, C.D. et al. (1999) Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu. Rev. Pharmacol. Toxicol. 39, 267–294 4 Grill, E. et al. (1985) Phytochelatins: the principal heavy-metal peptides of higher plants. Science 230, 674–676 5 Kondo, N. et al. (1985) Synthesis of metallothionein-like peptides Cadystin A and B occurring in a fission yeast, and their isomers. Agric. Biol. Chem. 49, 71–83 6 Cobbett, C.S. (1999) A family of phytochelatin synthase genes from plant, fungal and animal species. Trends Plant Sci. 9, 335–337 7 Ortiz, D.F. et al. (1992) Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter. EMBO J. 10, 3491–3499 8 Ortiz, D.F. et al. (1995) Transport of metal-binding peptides by HMT1, a fission yeast ABC-type vacuolar membrane protein. J. Biol. Chem. 270, 4721–4728 http://tibtech.trends.com

plant species or genetically engineered plants to remediate [remove and/or neutralize] xenobiotics, particularly heavy metals, from soils [22]). Indeed, although the isolation of other genes, the development of better soil amendments and improvements in the disposal of heavy metal-laden plants will also be required before phytoremediation is commonplace, it is likely that AtPCS1 or its equivalents from other plant species will be at least one ingredient in the cocktail of biological reagents that will be brought to bear on the development of this technology. AtPCS1 has three crucial properties for this purpose: (1) it is a single polypeptide coded by a single gene; (2) it feeds directly into the intracellular landfill capabilities of plant cells because it catalyzes the synthesis of heavy-metal-binding polymers destined for vacuolar sequestration; and (3) it confers resistance to arsenic, mercury and cadmium, the first-, thirdand fourth-ranked heavy-metal contaminants on the ASTDR/EPA priority list for US Superfund sites, respectively. By the same token, the discovery of functional PCS genes in organisms other than plants and fungi might prove to be of comparable importance in the years to come. On the one hand, its characterization in C. elegans, together with the prominence of heavy metals as environmental toxins in many disease states including human cancers, might mean that PC synthase-dependent metal detoxification in animals will assume wide environmental toxicological significance. On the other hand, the likely operation of equivalent metal detoxification pathways in pathogenic nematodes, schistosomes and roundworms (organisms responsible for untold human suffering and agricultural losses) could, in view of the conditional lethality of the ce-pcs-1-mutant phenotype, spawn new chemotherapeutic and agrochemical approaches for the many infections caused by these disease-bearing organisms.

9 Grill, E. et al. (1989) Phytochelatins, the heavymetal binding peptides of plants, are synthesized from glutathione by a specific γ-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc. Natl. Acad. Sci. U. S. A. 86, 6838–6842 10 Clemens, S. et al. (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J. 18, 3325–3333 11 Ha, S.B. et al. (1999) Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe. Plant Cell 11, 1153–1164 12 Vatamaniuk, O.K. et al. (1999) AtPCS1, a novel phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc. Natl. Acad. Sci. U. S. A. 96, 7110–7115 13 Howden, R. et al. (1995) Cadmium-sensitive mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol. 107, 1059–1066 14 Vatamaniuk, O.K. et al. (2000) Mechanism of heavy metal ion activation of phytochelatin (PC) synthase. Blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides. J. Biol. Chem. 275, 31451–31459

15 Loeffler, S. et al. (1989) Termination of the phytochelatin synthase reaction through sequestration of heavy metals by the reaction product. FEBS Lett. 258, 42–46 16 Zenk, M.H. (1996) Heavy metal detoxification in higher plants – a review. Gene 179, 21–30 17 Inzé, D. and Van Montagu, M. (1995) Oxidative stress in plants. Curr. Opin. Biotechnol. 6, 153–158 18 Vatamaniuk, O.K. et al. (2001) A new pathway for heavy metal detoxification in animals Phytochelatin synthase is required for codmium tolerance in Caenorhabditis elegans. J. Biol. Chem. 276, 20817–20820 19 Clemens, S. et al. (2001) Caenorhabditis elegans expresses a functional phytochelatin synthase. Eur. J. Biochem. 268, 3640–3643 20 Broeks, A. et al. (1996) Homologues of the human multidrug resistance genes MRP and MDR contribute to heavy metal resistance in the soil nematode Caenorhabditis elegans. EMBO J. 15, 6132–6143 21 Koga, M. et al. (2000) A Caenorhabditis elegans MAP kinase kinase, MEK-1, is involved in stress responses. EMBO J. 19, 5148–5156 22 Salt, D.E. et al. (1998) Phytoremediation Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 643–668