Proteomics and in Silico

Article pubs.acs.org/jpr

Proteomics and in Silico Approaches To Extend Understanding of the Glutathione Transferase Superfamily of the Tropical Liver Fluke Fasciola gigantica Russell M. Morphew,*,† Neil Eccleston,‡ Toby J. Wilkinson,† John McGarry,‡ Samirah Perally,† Mark Prescott,§ Deborah Ward,§ Diana Williams,‡ Steve Paterson,§ M. Raman,∥ G. Ravikumar,∥ M. Khalid Saifullah,⊥ S. M. Abbas Abidi,⊥ Paul McVeigh,# Aaron G. Maule,# Peter M. Brophy,† and E. James LaCourse¶ †

Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, SY23 2DA, U.K. Faculty of Veterinary Science, University of Liverpool, L69 7ZJ, U.K. § School of Biological Sciences, University of Liverpool, L69 7ZB, U.K. ∥ Tamil Nadu Veterinary and Animal Sciences University, Chennai 600-051, India ⊥ Aligarh Muslim University, India # School of Biological Sciences, Queen’s University Belfast, Belfast, Northern Ireland, U.K. ¶ Liverpool School of Tropical Medicine, L3 5QA, U.K. ‡

S Supporting Information *

ABSTRACT: Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member KEYWORDS: Fasciola gigantica, glutathione transferase, Sigma, Omega, Zeta, Mu

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INTRODUCTION Fasciolosis is a foodborne zoonotic disease affecting grazing animals and humans worldwide. The causative agents of fasciolosis are the liver flukes Fasciola hepatica (typically found in temperate regions) and Fasciola gigantica (typically found in tropical regions), which have distinct and overlapping distributions. Liver fluke cause global economic losses of over US$ 3 billion per annum to livestock through mortality; reduction in host fecundity; susceptibility to other infections; decrease in milk, meat, and wool production; condemnation of livers; and in many resource-poor settings, loss of performance in draft animals.1 Fasciolosis is also a re-emerging human disease with estimates of between 2.4 and 17 million people infected worldwide.2 Indeed, current policy within the World © 2012 American Chemical Society

Health Organisation for this re-emerging disease has paved the way for fasciolosis to be added to the preventative chemotherapy concept.3 Current methods of control of fasciolosis are dominated by chemotherapy, with triclabendazole (TCBZ) as the “drug of choice” for both humans and livestock due to its broad effectiveness against juvenile and adult fluke stages. However, fluke resistance to TCBZ is increasingly reported,4−6 raising much concern in light of the absence of any new flukicidal drugs as effective as TCBZ becoming commercially available in the near future. Furthermore, despite significant research into Received: July 17, 2012 Published: November 20, 2012 5876

dx.doi.org/10.1021/pr300654w | J. Proteome Res. 2012, 11, 5876−5889

Journal of Proteome Research

Article

In this work we have taken a global view of the GST superfamily from the tropical liver fluke F. gigantica and have describe for the first time the identification of a Sigma class GST designated as FgGST-S1. FgGST-S1 has been cloned and expressed in a recombinant and active form. The potential role of this FgGST-S1 in the establishment and maintenance of infection and parasite survival is considered along with the possibilities for this protein as an anthelmintic drug target or vaccine candidate. In addition, we have identified from an in silico analysis an Omega class GST from F. gigantica, which has been subsequently cloned (FgGST-O1), a novel Mu class isoform, and for the first time in trematodes, a Zeta class GST. The global characterization of Fasciola GSTs will support the understanding of xenobiotic and internal toxin responses, potential drug resistance mechanisms, vaccine candidature, and the host−parasite relationship.

potential mode of TCBZ action and detoxification, current understanding of the mechanisms of resistance to TCBZ is limited, severely hindering efforts at the molecular and biochemical level to detect and combat TCBZ resistance and preserve drug efficacy.7 In the developing world the cost of short-term drug regimes and sporadic availability excludes the use of TCBZ as a control option. Research therefore has also concentrated on the search for potential vaccines against fasciolosis as an alternative to chemotherapeutic control methods. However, although several vaccination studies have shown varying levels of protection in cattle, goats, and sheep, none has yet resulted in the commercial availability of a suitable vaccine. Of particular interest within both drug resistance studies and vaccine trials are the glutathione transferases (GSTs) (EC 2.5.1.18). This family of ubiquitous multifunctional enzymes are involved in a range of systems for a variety of parasites including xenobiotic detoxification8−10 and immunomodulation11,12 and have shown promise as vaccine candidates.13−16 GSTs are possibly the major detoxification enzymes in adult helminths, since these organisms have an apparent lack of, or reduced expression of, the important phase I cytochrome P-450 dependent detoxification components.17,18 To this end, GSTs account for as much as 4% of the total soluble protein in F. hepatica, with a widespread distribution in parasite tissues, and are present in excretory/secretory products following in vitro culture, suggesting important physiological roles for these enzymes in this organism.12,19,20 Specific interest in GSTs in terms of a vaccine for fluke has also stemmed from the promise shown in trials and phase II clinical status of the Sigma class GST-based vaccine candidate against the related fluke parasites of humans, the Schistosoma sp.21 Several attempts to use Fasciola sp. Mu class GSTs as protective antigens in sheep and cattle have been described with varying levels of protection related to worm burden.16,22−25 These data were encouraging and highlighted Fasciola sp. GSTs as viable vaccine candidates. However, these studies were likely conducted predominately with Mu class GSTs for two main reasons: native liver fluke Sigma class GST appears not to bind in any significant quantities when in the presence of the overwhelming quantity of native Mu class GST to glutathione-affinity matrix, and recombinant vaccine candidates have all been of the Mu class to date. In contrast, the Sigma GST vaccine candidate from schistosomes was initially identified via a non-biochemical immunology route in the 1980s, and it has been assumed that Fasciola sp. expressed only Mu type GSTs until a study in F. hepatica in 2006.26 Previous sequence and structural comparisons of F. hepatica Mu class GSTs have clearly highlighted distinct differences to the schistosome GST vaccines of the Sigma class. The study by LaCourse et al.,12 using a recombinant protein, looked at the biochemical and immunological properties of the Sigma class GST in F. hepatica. In addition, this study is the first study to use Sigma class GST as a vaccine candidate against F. hepatica, in this case against a particularly aggressive strain of F. hepatica. While no reduction in worm burden was recorded, a significant reduction in the associated pathology was observed suggesting Sigma class GST from Fasciola is a viable vaccine target for inclusion into multivalent vaccines.12 Therefore, experiments are required to determine if the Sigma class GST is present in F. gigantica and, as suggested previously,27 investigate the GST protein superfamily as a whole before pressing on with future vaccine trials.

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MATERIALS AND METHODS

Source of Fasciola Species Material

Adult Fasciola gigantica samples were collected from local abattoirs in both India and Egypt, and adult F. hepatica samples were collected from a local abattoir in mid-Wales, U.K. Indian F. gigantica were used for all cloning and expression work, whereas Egyptian F. gigantica were used for all native purification and proteomic analysis. Fasciola Species Verification, Total RNA Extraction and cDNA Synthesis

For species verification the method of McGarry et al.28 was adopted. Briefly, approximately 50 mg tissue were removed from each of the frozen fluke (including F. hepatica and F. gigantica confirmed species as positive controls), and genomic DNA was extracted using a DNeasy kit (QIAGEN, Crawley, U.K.). The extracted DNA was treated with RNAase (100 mg/ mL), split into aliquots, and stored at 22 °C until use. PCR species verification was performed using two primer sets from RAPD-derived sequences28 to amplify a non-coding region of genomic DNA. Primer set 1, forward primer FhepF (5′-GCG GCC AAA TAT GAG TCA-3′) and reverse primer FhepR (5′CTG GAG ATT CCG GTT ACC AA-3′). Primer set 2, forward primer FgigF (5′-GTT CAG GTG ACA AGC CAA3′) and reverse primer FgigR (5′-ATC ACA CCG TGA AGC AGA-3′). PCR thermocycler conditions used were as described previously.28 Total RNA was isolated using RNeasy (Qiagen) blood and tissue procedure according to the manurfacturer’s instructions using 25 mg of frozen F. gigantica and subsequently treated with DNase I (Ambion Inc.) to remove contaminating genomic DNA. RNA quality and quantity from each sample was determined by spectrophotometry at 260 nm. Total RNA from the F. gigantica was used as template to synthesize cDNA by reverse transcription using SuperScript III (Invitrogen) according to the manufacturer’s instructions. Transcriptome Searches

Sequences representing known GST classes were used to tBLASTn the F. gigantica transcriptome of Young et al.,29 which is available to search at http://bioinfosecond.vet.unimelb.edu. au/wblast2.html and an F. hepatica transcriptome database (EBI-ENA archive ERP000012: an initial characterization of the F. hepatica transcriptome using 454-FLX sequencing). Sequences used were taken from Genbank: Pi class, P09211; Alpha class, P08263; Theta class, P30711; Sigma class, 5877



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bursts of 1 min with intervals of 1 min on ice. The lysed sample was centrifuged at 21,000 × g for 30 min at 4 °C. The supernatant was quantified as previously mentioned. Both supernatants, F. gigantica and E. coli (containing rFgGST-S1), were passed through 0.45 μm syringe filters prior to enzyme purification.

ABI79450; Mu class (Fasciola 26, 27, 28, and 29 Isoforms), P30112, P31670, P31671, and P56598; Omega class, P78417; Zeta class, O43708. All sequences were aligned using ClustalW through BioEdit Version 7.0.5.3 (10/28/05).30 RACE PCR of FgGST-S1, Expression of rFgGST-S1, and Cloning of FgGST-O1

Enzyme Purification and Activity Assays

Rapid Amplification of cDNA ends (RACE) PCR was performed to obtain the full coding sequence of the F. gigantica Sigma class GST. cDNA for 5′ and 3′ RACE was synthesized by according to the manurfacturer’s specifications (GeneRacer Kit, Invitrogen). A nested PCR amplification of F. gigantica Sigma class GST was performed using a specific primer for 5′ RACE PCR (5′ TCG ATT CTT GAT AAC GTC TAA GTT TCC 3′) and a specific primer for 3′ RACE PCR (5′ GAC AAT TCA AAA TGA TGG GTG AAA CG 3′). RACE PCRs for FgGST-S1 were performed as follows: 2 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 68 °C, followed by a final extension of 10 min at 68 °C. The PCR products were separated on 1% agarose gel and then gelpurified with the Purelink Gel Extraction Kit (Invitrogen). The purified PCR products were ligated into the pCR4-TOPO vector (Invitrogen) and transformed into competent E. coli TOP10. Positive transformants were selected by ampicilin resistance. The positive clones were sequenced in house. By aligning the sequences of the 3′ and 5′ RACE products, a fulllength cDNA sequence of FgGST-S1 was deduced and obtained through RT-PCR using specific primers (forward primer 5′ AAA ATA GCG CAC ACG AAT CA 3′ and reverse primer 5′ ACC ACA AGT CGG CGA ATA AC 3′). FgGST-S1 was then amplified via PCR using the following primer pair: rFgGST-S1 forward primer, 5′ CAT ATG GAC AAA CAG CAT TAC AAG TTG TGG 3′; rFhGST-S1 reverse primer, 5′ GCG GCC GCG AAC GGA GTT TTT GCA C 3′. Restriction enzyme sites (in bold type and underlined) for Nde I (forward primer) and Not I (reverse primer) were included so that the entire ORF could be directionally cloned into the pET23a (Novagen) vector. Recombinant protein was produced in E. coli BL21(DE3) cells (Novagen). Omega class GSTs were cloned using primers designed on F. hepatica Omega class GST contigs Fhep54b04 and Fhep49c0626 (forward primer 5′ ACA ATT GTG AAT CAC AAG CA 3′ and reverse primer 5′ GCT GGC TTC GAA TTG GTT G 3′). Omega class GST sequences were amplified using PCR. F. gigantica and F. hepatica sequences were then cloned into the pGEM-T easy vector (Promega) according to manufacturer’s instructions, screened and sequenced in-house.

Enzymes were purified according to the method of Chemale et al.26 Briefly, native F. gigantica cytosolic protein batches were applied to a S-hexylglutathione-agarose (S-hexyl-GSH agarose, Sigma-Aldrich, U.K.) affinity column and E. coli lysate containing rFgGST-S1 was applied to a glutathione-agarose (GSH agarose, Sigma-Aldrich, U.K.) affinity column. GSTs in both samples were purified at 4 °C according to the manufacturer’s instructions. Eluted proteins were concentrated using 10 kDa filters (Amicon Ultra, Millipore) and quantified using Bradford Reagent31 as previously described. This process was repeated for all biological replicates of F. gigantica cytosolic proteins, five in total. GST enzyme activity was determined spectrophotometrically by assessing the change in absorbance at 340 nm brought about by the conjugation of reduced glutathione (GSH) with 1chloror-2,4-dinitrobenzene (CDNB). The purified Sigma class GST was assayed for GST activity in 1 mL volumes under conditions detailed according to the method of Habig et al.32 (i.e., 1 mM CDNB, 1 mM GSH, pH 6.5, 25 °C). All assays were performed upon a Cary 50 Bio UV−vis spectrophotometer (Varian, U.K.). Protein Preparation and 2DE

Cytosolic protein extracts prepared previously were precipitated with an equal volume of ice-cold 20% TCA in acetone (w/v) and washed twice in ice-cold acetone before solubilization into isoelectric focusing buffer (IEF buffer; 8 M urea, 2% w/v CHAPS, 33 mM DTT, and 0.5% carrier ampholytes v/v Biolyte 3/10). Purified GST enzymes were diluted in IEF buffer (as previous) at 1.2× concentration. For purified enzymes, 10−50 μg of each biological replicate sample batch from pooled F. gigantica (10 μg for Western blot; 50 μg for Coomassie stain) was actively in-gel rehydrated for 16 h and isoelectrically focused on 17 cm pH 3−10L IPG strips to 50,000 V h, on a Protean IEF Cell (Bio-Rad). For cytosolic samples 500−1000 μg of each replicate sample (500 μg for Western blot; 1000 μg for Coomassie stain) and focused as with the purified enzymes. After focusing, strips were equilibrated for 15 min in reducing equilibration buffer (30% v/v glycerol, 6 M urea, 1% DTT) followed by 15 min in alkylating equilibration buffer (30% v/v glycerol, 6 M urea, 4% iodoacetamide). IPG strips were run upon SDS-PAGE (12.5% acrylamide) using the Protean II xi 2D Cell (BioRad). Gels were either Coomassie blue stained (Phastgel Blue R, Amersham Biosciences) and scanned on a GS-800 calibrated densitometer (BioRad) or transferred to nitrocellulose membrane for Western blot according to the methods of Towbin et al.33 Quantitative differences between 2DE protein spots were analyzed using Progenesis PG220, software version 2006 (Nonlinear Dynamics Ltd.) using five biological replicates.

Cytosol Preparations

Adult F. gigantica somatic extracts were obtained by homogenization of batches of 4 frozen fluke at 4 °C in a glass−glass homogenizer in lysis buffer, containing 20 mM KHPO4, pH 7.0, 0.1% Triton-X 100 and a cocktail of protease inhibitors (Roche, Complete-Mini, EDTA-free). Homogenates were centrifuged at 100,000 × g for 1 h at 4 °C. Supernatants were considered as the cytosolic fraction and were quantified using Bradford Reagent (Sigma-Alridch, U.K.) according to manufacturer’s instructions based upon the adapted method of Bradford31 before storage at −20 °C until needed. E. coli preparations containing rFgGST-S1 were suspended in lysis buffer (containing 5 mM MgCl, 400 mM NaCl, and 20 mM sodium phosphate pH 7.4) and lysed via a freeze/thaw method, freezing in liquid nitrogen followed by thawing at 42 °C three times. The E. coli sample was then ultrasonicated for 3

GST Class Assignment via Immunoblotting

Native F. gigantica S-hexyl-GSH-affinity purified GST samples and F. gigantica cytosolic samples were subjected to standard SDS-PAGE and 2DE, as described previously, electro-transferred to membranes and Western blotted with a polyclonal 5878



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Figure 1. Fasciola species verification. Using the method of McGarry et al.28 with F. hepatica specific primers (A) and F. hepatica and F. gigantica specific primers (B), all 20 fluke samples are identified as F. gigantica. Lanes 1−20 show DNA from F. gigantica. Lanes Fh and Fg are positive controls for F. hepatica and F. gigantica (previously verified by McGarry et al.28), respectively. Lane (-ve) is a negative control without DNA template.

Figure 2. Representative 2DE protein arrays of F. gigantica GSTs purified from somatic extracts. Fifteen micrograms of S-hexyl-GSH agarose binding proteins were profiled on a 12.5% polyacrylamide gel via two-dimensional electrophoresis (2DE) and stained with Coomassie blue. Proteins were isoelectric focused on 17 cm pH 3−10 nonlinear immobilized pH gradient (IPG) strips. Numbered protein spots correspond to putative protein identifications found in Table 1.

antibody raised in rabbits to the recombinant F. hepatica Sigma GST.12 Membranes were also probed with a polyclonal Mu class GST antibody represented by the anti-Schistosoma japonicum GST26 Mu class antibody (Pharmacia-Biotech 274577). Proteins were transferred according to standard procedures33,34 using the methods described in Chemale et al.26 Primary antibodies (1°Ab) were diluted at 1:30,000 for rFhGST-S1 and at 1:1000 for Mu class anti-S. japonicum GST26 Ab. The appropriate secondary antibodies (2°Ab) were diluted as per manufacturer’s instructed dilution at 1:30,000 (anti-rabbit IgG conjugated with alkaline phosphatase, produced in goat, A3687 Sigma-Aldrich, U.K.). Western blots were developed and immunogenic proteins visualized with BCIP/NBT (5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium) liquid substrate system (Sigma-Aldrich, U.K.).

Search parameters were as follows: enzyme set at trypsin with one missed cleavage allowed; fixed modification set for carbamidomethylation with variable modification considered for oxidation of methionine; monoisotopic masses with unrestricted protein masses were considered with peptide and fragment mass tolerances at ±1.2 and ±0.6 Da for an ESIQUAD-TOF instrument. Protein identifications resulting from MASCOT ions scores greater than 52 were considered as showing significant identity or extensive homology (p < 0.05) to the predicted identification displayed (www.matrixscience. com). De novo sequencing of peptides from proteins identified as Sigma GSTs was also performed. These peptides were interpreted via BioLynx semiautomated peptide sequencing tool (MassLynx v 3.5). Raw spectra were combined, smoothed, subtracted, and centered as described by Moxon et al.36 and deconvoluted using MaxEnt 3 software (MassLynx v 3.5). Precursor ion tolerance was set to 0.5 Da, fragment mass tolerance was set to 0.1 Da, and the intensity threshold was set at 1%. Amino acid modifications considered were carboxyamidomethylcysteine, cysteine acrylamide, and methionine sulphoxide, with trypsin specified as the enzyme used for protein digestion; two missed cleavages were permitted. The minimum mass standard deviation was set at 0.025 Da and the sequence display threshold (% Prob) set to 1. Sequences derived from de novo sequencing were used to BLAST using BioEdit (Version 7.0.5.3)30 against an “in-house” database constructed from 6260 (858,763 residues) F. hepatica EST sequences downloaded and translated from the Sanger Institute (ftp://ftp.sanger.ac.uk/pub/pathogens/fasciola/ hepatica/ESTs/). The resulting matched F. hepatica ESTs

Protein Identification via Mass Spectrometry and BLAST

Protein spots were manually excised from the gels and in-gel digested with trypsin according to the method of Chemale et al.35 Tandem mass spectrometry (MS/MS) was performed according to the method of Moxon et al.36 followed by data processing for database searching. Briefly, Sequest compatible (.dta) file peak mass lists for each spectrum were exported, and spectra common to each 2-DE spot were merged into a single MASCOT generic format (.mgf) file using the online Peak List Conversion Utility available at www.proteomecommons.org.37 Merged files were submitted to a MASCOT MS/MS ions search within a locally installed MASCOT server (www. matrixscience.com) to search the NCBI GenBank nonredundant protein database (NCBI http://www.ncbi.nlm.nih. gov/nr release April 2011 183.0 containing 135,440,924 sequences; 126,551,501,141 residues). 5879



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Table 1. Protein Identification of F. gigantica Glutathione Transferases Using MASCOTa spot no.

NCBI accession no.

mascot Score

protein

organism

% coverage

unique peptides

GST class

1

Q24JY1 P31671 AAD23997 P11598 AAD23997 AAA29139 P31671 P30112 AAD23997 P31671 P30112 AAD23997 P31671 P30112 AAD23997 P30112 P31671 AAD23997 P31671 P30112 AAD23997 P31671 AAD23997 P31671 P30112 P56598 P30112 AAD23997 P30112 AAD23997 P30112 P31670 AB179450 AB179450 AB179450 AB179450

220 114 108 218 141 96 96 62 207 191 168 314 178 149 278 172 129 104 89 52 178 103 385 194 172 214 55 109 58 109 90 90 200 370 256 264

60S RIbosomal Protein L23a GST28 Mu Class Glutathione Transferase Glutathione Transferase Protein Disulfide-lsomerase A3 Precursor Glutathione Transferase Glutathione Transferase GST28 Mu Class Glutathione Transferase GST26 Mu Class Glutathione Transferase Glutathione Transferase GST28 Mu class Glutathione Transferase GST26 Mu class Glutathione Transferase Glutathione Transferase GST28 Mu class Glutathione Transferase GST26 Mu class Glutathione Transferase Glutathione Transferase GST26 Mu class Glutathione Transferase GST28 Mu class Glutathione Transferase Glutathione Transferase GST28 Mu class Glutathione Transferase GST26 Mu class Glutathione Transferase Glutathione Transferase GST28 Mu class Glutathione Transferase Glutathione Transferase GST28 Mu class Glutathione Transferase GST26 Mu class Glutathione Transferase GST29 Mu class Glutathione Transferase GST26 Mu class Glutathione Transferase Glutathione Transferase GST26 Mu class Glutathione Transferase Glutathione Transferase GST26 Mu class Glutathione Transferase GST27 Mu class Glutathione Transferase Glutathione Transferase (Sigma class) Glutathione Transferase (Sigma class) Glutathione Transferase (Sigma class) Glutathione Transferase (Sigma class)

60S taurus F. hepatica F. gigantica Rattus norvegicus F. gigantica F. hepatica F. hepatica F. hepatica F. gigantica F. hepatica F. hepatica F. gigantica F. hepatica F. hepatica F. gigantica F. hepatica F. hepatica F. gigantica F. hepatica F. hepatica F. gigantica F. hepatica F. gigantica F. hepatica F. hepatica F. hepatica F. hepatica F. gigantica F. hepatica F. gigantica F. hepatica F. hepatica F. hepatica F. hepatica F. hepatica F. hepatica

20.5 9.6 15 10.3 9.0 10.0 9.6 9.6 19.3 19.3 19.3 34 21.6 29.4 26.0 21.6 13.8 16.0 9.6 9.6 15.0 9.6 34.0 21.6 21.6 16.7 11.9 9.0 4.0 9.0 9.0 9.6 20.4 32.2 30.3 26.0

4 2 3 4 2 2 2 2 4 4 4 6 4 5 5 4 3 3 2 2 3 2 6 4 4 4 2 2 1 2 2 2 4 6 5 4

Mu

2

3

4

5

6

7 8

9 10 11

12 13 14 15

Mu

Mu

Mu

Mu

Mu

Mu Mu

Mu Mu Mu

σ σ σ σ

a

Spectra from mass spectrometry were subjected to MS/MS ion searches using MASCOT (Matrix Science). Significant hits, at P = 5%, have a MASCOT score of 52 or greater. All reported accession numbers are from GenBank.

et al.28 In this study, all 20 individuals used to purify GSTs were confirmed as F. gigantica rather than the related temperate fluke, F. hepatica (Figure 1). Post species confirmation, all F. gigantica fluke were homogenized and passed through a Shexyl-GSH agarose column to isolate F. gigantica GST protein. Following purification S-hexyl-GSH agarose samples were subjected to 2DE proteomic profiling. Purification using a Shexyl-GSH agarose column yielded 14 protein spots on 2DE profiles (Figure 2). As with previous studies on Fasciola GSTs using S-hexylGSH agarose purification columns, proteins (presumed GSTs) were found at the basic end of the gel, with proteins spots 12, 13, and 14 (Figure 2) well represented in the GST subproteome. All protein spots consistently present on the 2DE arrays were then extracted and identified via tandem mass spectrometry. All spots subjected to MS/MS protein identification contained proteins identified as GSTs from Fasciola species (Table 1). Only two protein spots contained additional proteins that were not identified as GSTs, namely, spot 1 containing a 60S Ribosomal Protein L23a and spot 2 containing Protein Disulfide-Isomerase A3 Precursor protein.

were subjected to BLAST analysis against the NCBI nonredundant database. Phylogenetics and Epitope Prediction

To construct a phylogenetic tree an alignment of all GST sequences was exported into Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.38 Analysis was performed using a neighbor-joining method, 1000-replicate, bootstrapped tree. The amino acid data were corrected for a gamma distribution (level set at 1.0) and with a Poisson correction. Sequences from F. hepatica and F. gigantica encoding the Sigma class GST were also subjected to epitope prediction using a Kolaskar and Tongaonkar Antigenicity prediction method,39 available at http://tools.immuneepitope.org/tools/ bcell/iedb_input.

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RESULTS

Species Confirmation and GST 2DE Profiling

Prior to GST purification, F. gigantica flukes were first confirmed as F. gigantica according to the method of McGarry 5880



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Table 2. De Novo Sequencing of the Novel Sigma Class GSTa de novo peptide sequence TDEEYYUER LVSESLESSGGK VPVLDVTGPDGK YQESMAIAR

matched peptide sequence TDEEYYUER LVSESLESSGGK VPLLDVTGPDGK YQESMAIAR

% identity

EST e value

EST

accession no.

100

0.089

Fhep27aO5.qlk

ABI79450

100 91 100

0.063 0.013 6.7

Fhep27aO5.qlk Fhep27aO5.qlk Fhep27aO5.qlk

ABI79450 ABI79450 ABI79450

protein Glutathione Transferase (Sigma class) Glutathione Transferase (Sigma class) Glutathione Transferase (Sigma class) Glutathione Transferase (Sigma class)

organism

BLAST e value

F.

2.00 × 10−120

hepatica F. hepatica F. hepatica F. hepatica

2.00 × 10−120 2.00 × 10−120 2.00 × 10−120

a

De novo sequences from spots 12−14 were subjected to BLAST analysis against an in house F. hepatica EST database. Matching EST sequences were then subjected to BLAST analysis themselves against the NCBI non-redundant protein database.

Figure 3. Evidence for sequence variations between F. hepatica and F. gigantica Sigma GST. MS/MS spectra from the analysis of a 2+ peptide m/z 589.69 (sequence VPV LDV TGP DGK) sequenced from spots 12−15, belonging to a the novel Sigma GST from F. gigantica, FgGST-S1. Sequencing was performed automated using MassLynx v 3.5.

characterized Sigma class GST from F. hepatica (Table 2 and Supplementary Figures S1 and S2). The remaining de novo sequenced peptide showed a single amino acid substitution (underlined in bold) at position 55: VPV LDV TGP DGK, a conservative switch from a leucine to a valine (Figure 3).

In total, 14 protein spots from S-hexyl-GSH agarose purification contained GSTs, identified as representatives from both Mu (11 in total) and Sigma class (3 in total) GSTs. Nearly all sequences matched to F. gigantica despite F. hepatica Mu class GST sequence deposits available in the public databases vastly outnumbering those of F. gigantica. In all cases, identification was significant at p = 0.05. All of the Mu class GST identifications, with one exception, matched to the solitary F. gigantica database entry AAD23997 (Genbank) and to one or more F. hepatica entries. Of the F. hepatica hits, the most common were that of GST 28 (alternatively named GST-7) and GST26 (alternatively named GST-51) identified in all 11 Mu class hits. Only 2 spots had the F. gigantica sequence (AAD23997) combined with a single F. hepatica hit; spot 7 matching GST28 (GST-7) and spot 10 matching GST26 (GST-51). GSTs 29 (alternatively named GST-1) and 27 (alternatively named GST-47) were only identified in spots 9 and 11 respectively. All Sigma class GST identifications matched the sequenced FhGST-S1, accession number DQ974116. To look for sequence variations between the identified Sigma class GST from F. gigantica and the related Sigma class GST from F. hepatica, four peptides were de novo sequenced. All four peptides de novo sequenced matched to a Sigma class GST EST sequence with three matching 100% to the previously

Immunological Classification

To further characterize the F. gigantica GST isolated in this work, 2DE gels containing the S-hexyl-GSH agarose purified GST were transferred to PVDF membranes and assayed with anti-S. japonicum Mu class GST and anti-F. hepatica Sigma class GST antibodies (Figure 4). Probing for Mu class GSTs revealed spots 1−11, all identified as Mu class GST, having reactivity. In addition spot 13, identified as a Sigma class GST, showed reactivity, albeit at a significantly lower level (Figure 4B). When probing the 2DE profile for Sigma class GST (Figure 4C) spots 12−14 were identified consistently as having reactivity. On occasion, spot 15 (Figure 4C) would be purified using Hexyl-GSH affinity chromatography and was also identified as a Sigma GST both immunologically and through tandem mass spectrometry (Table 1). This spot was never identified using Western blotting of cytosolic sample and therefore most likely reflects a product of degradation during the purification process. No Mu class GSTs were recognized using this antibody. 5881



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performed. After transfer and antibody binding, the only antibody recognition was seen toward the basic end of the gel where the purified Sigma GST was seen previously (Figure 4D and E). No other recognition was seen using anti-F. hepatica Sigma class GST antibodies. Transcriptome Analysis, Cloning, and RACE PCR

Searching the F. gigantica transcriptome database with the Genbank F. hepatica Sigma class GST entry ABI79450 identified 3 matching contigs (Figure 5). None of the matching contigs provided the full 211-amino-acid sequence (contig 14158, 11 amino acids; contig 27045, 30 amino acids; contig 24647, 72 amino acids). However, the largest contig, contig 24647, did span the mass spectrometry identified amino acid substitution at position 55. In this case, the amino acid at position 55 was leucine, matching ABI79450 from F. hepatica. The full-length cDNA encoding the Sigma class GST (FgGST-S1) from F. gigantica was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The cDNA contained a 636-bp open reading frame (ORF) coding for 211 amino acids with a predicted theoretical isoelectric point (pI) of 8.85 and average molecular weight of 24595.47 Da (Figure 5). In addition, a 34-bp 5′ UTR and a 40-bp 3′ UTR were identified. In comparison with the published FhGST-S1, FgGST-S1 showed 22 nucleotide sequence variations within the coding region. Of these 22 nucleotide changes, 4 were show to be non-synonymous producing amino acid variations at positions 6, 109, 121, and 195. In line with the published F. gigantica transcriptome data, the amino acid at position 55 was a leucine, yet positions 195 and 209 showed single amino acid polymorphisms (SAAPs). The complete cDNA sequence of the F. gigantica Sigma class GST gene was deposited in GenBank under accession number JX157879. Phylogenetics showed FgGST-S1 clustered with FhGST-S1 and additional Sigma class GST sequences as noted previously12 into a trematode specific clade (Figure 6). In comparison with the Schistosome Sigma

Figure 4. Representative 2DE Western blots of F. gigantica global and S-hexyl-GSH purified proteins using anti-GST antibodies. Proteins were isoelectric focused on 17 cm pH 3−10 nonlinear immobilized pH gradient (IPG) strips, run on 12.5% polyacrylamide gels and transferred onto PVDF membranes for antibody binding. GST class specific antibodies (AB) and F. gigantica protein samples were probed are as follows: (A) 2DE gel of S-hexyl-GSH-binding proteins; (B) antiMu class GST AB against S-hexyl-GSH-binding proteins; (C) antiSigma class GST AB against S-hexyl-GSH-binding proteins; (D) 2DE gel of the global F. gigantica proteome; (E) anti-Sigma class GST AB against the global F. gigantica proteome. The Sigma class GST identifications have been numbered for clarity.

As this was the first identification of the Sigma class GST in F. gigantica, a Western blot using anti-F. hepatica Sigma class antibodies probing a global somatic profile of F. gigantica was

Figure 5. Multiple alignment of the Fasciola Sigma GST protein sequences. Fasciola Sigma GST sequences were aligned using ClustalW and used the F. hepatica sequence available from Genbank (DQ974116), the newly cloned F. gigantica sequence (JX157879), and the matching contigs from the transcriptome project of Young et al.29 (contig 24647, 27045, and 14158). Regions of sequence boxed in black represent identical sequence between the two cloned and sequenced Fasciola sequences and the F. gigantica transcriptome data. Boxed sequence indicates peptides sequenced during de novo mass spectrometry, four sequenced peptides in total. Residues indicated by a solid black arrow highlight amino acid variations between the two species of Fasciolid. The remaining arrow, unfilled, indicates a SAAP specific to F. gigantica. RC, Reverse complement. 5882



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Figure 6. Phylogenetic analysis of the soluble cytosolic GST superfamily. Neighbor-joining phylogenetic tree constructed using amino acid sequences through MEGA v 4.0 with 1000 bootstrapped support and a Poisson correction. All reported accession numbers are from Genbank. Where sequences were identified in silico, only contig numbers are reported. Those from F. gigantica were taken from the study of Young et al.,29 and those from F. hepatica were taken from transcripts produced by the University of Liverpool (EBI-ENA archive ERP000012: An initial characterization of the F. hepatica transcriptome using 454-FLX sequencing).

variations were observed (Figure 7A). Both sequences were deposited in GenBank (accession numbers: FgGST-O1, JX1157881; FhGST-O1, JX1157880). As with the Sigma class GSTs the Omega class representatives cluster into a trematode clade in an overall Omega class clade (Figure 6). Two sequences were also identified showing significant identity to Zeta class GSTs and using phylogenetics clustered with additional Zeta class GSTs (Figure 6). The two transcriptome databases revealed full length contigs for Zeta class GSTs from both studied Fasciolids (Fgig contig21372 and Fhep contig1132); a 642-bp ORF encoding 213 amino acids

class GSTs, FgGST-S1 shares 44% sequence identity with Sm28 of S. mansoni (P09792) and 43.6% sequence identity with Sh28 from S. hematobium (P30114). This is an increase of around 25% when compared with previous Mu class GSTs where sequence identity with Sm28 and Sh28 varies from 17.4% to 19.5%. Using the transcriptome data to identify alternative GST classes in Fasciola revealed contigs matching an Omega class GST. The full sequences from both F. gigantica and F. hepatica were then cloned and yielded a 723-bp ORF encoding a 240amino-acid protein. Between both species 12 amino acid 5883



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Figure 7. Multiple alignment of the Fasciola Omega and Zeta class GST protein sequences. All GST sequences were aligned using ClustalW. (A) Multiple alignment of the newly cloned/sequenced F. gigantica and F. hepatica Omega class GSTs with those of genome sequenced organisms. Underlined are the proline-rich residues in the Omega class characteristic N-terminal extension. The catalytic cysteine residue characteristic of Omega class GSTs is indicated with *. Residues indicated with an arrow relate to the identified Omega class GST motifs identified by Chemale et al.26 (B) Multiple alignment of F. gigantica and F. hepatica in silico identified Zeta class GSTs with those of genome sequenced organisms. Underlined are the characteristic Zeta class N-terminal motif, SSCX(W/H)RVRIAL,52 and C-terminal motif, LLVLEAFQVSHPCR.50

contig 10310) matching known Mu class GSTs from Schistosoma species (Figure 6).

with 10 amino acid variations between species (Figure 7B). The separation here between Zeta class GSTs does not show as strong a relationship as those of the Omega class (Figure 6). The last matches to known GST classes were from the well documented Mu class GSTs. Searching the F. gigantica transcriptome revealed partial sequences representing Mu class GSTs 27 and 29 (contig 21494 and contig 15733, respectively). In addition, a third Mu class GST isoform was identified in both Fasciola species (Fgig contig 27593 and Fhep

Recombinant GST Production, Activity Assay, and Bioinformatics

Full length sequenced, recombinant F. gigantica Sigma class GST (rFgGST-S1) was shown to be purified to a high level from transformed E. coli cytosol following expression. A total of 8.97 mg of rFgGST-S1 was purified from a total of 100 mL of 5884



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Figure 8. Expression and purification of recombinant FgGST-S1. (A) ESI mass spectrum of the GSH-affinity purified rFgGST-S1 showing the MW of rFgGST-S1 at 25875.96 ± 0.80 Da (Peak B) and 1, 2, and 3 conjugated GSH molecules (peaks A, C, and D, respectively). (B) SDS-PAGE gel of the expression and purification of rFgGST-S1. Lanes 1 and 7: Molecular weight markers. Lane 2: E. coli total insoluble protein. Lane 3: E. coli total cytosolic protein. Lane 4: Flowthrough collected after passing through the column. Lane 5: Wash with equilibration buffer. Lane 6: GSH-affinity purified recombinant rFgGST-S1 protein. Run on 12.5% SDS-PAGE and Coomassie blue stained.

between both species, spanned amino acids 99−114 in F. hepatica (IGE CED LYR EVY TIFR) and 99−105 in F. gigantica (IGE CEDL), significantly shorter as a result of the amino acid variation at position 109. The identified F. gigantica specific SAAPs at positions 55, 195, and 209 did not impact epitope recognition either individually or in combination.

E. coli lysate using a 1 mL GSH-agarose affinity column. The purity was assessed by the presence of a single band upon SDSPAGE at the estimated size and dominating peaks in an ESI trace at the calculated theoretical masses for the protein sequence (Figure 8). The ESI trace revealed four peaks corresponding to the rFgGST-S1 which related to the native protein minus the N-terminal methionine (Figure 8A Peak B), plus one GSH conjugation (Figure 8A Peak A), 2 GSH molecules conjugated (Figure 8A Peak C), and 3 GSH molecules (Figure 8A Peak D). Analyzing the purified rFgGST-S1 by 2D SDS-PAGE revealed a single protein resolving into 3 major protein spots (Figure 9A, a). In addition, at least 10 higher molecular weight proteins (ranging from 44 kDa to the 97 kDa marker) with the same pI as the resolved rFgGST-S1 were seen (Figure 9A, b). Finally, a selection of small molecular weight proteins were also visualized (Figure 9A, c). Western blotting of the 2DE profile with an anti-rFhGST-S1 antibody12 confirmed all resolved protein spots as rFgGST-S1 (Figure 9B) as did mass spectrometry (Supplementary Table S1). No recognition was seen probing the 3 spots with an anti-Mu class antibody (Figure 9C). rFgGST-S1 was produced as an active protein, displaying significant enzymic activity toward the model GST substrate 1chloro-2,4-dinitrobenzene (CDNB). The specific activity of rFgGST-S1 was recorded at 3846.75 ± 167.52 nmol min−1 mg−1 using 5 biological replicates. Application of a Kolaskar and Tongaonkar Antigenicity prediction method39 revealed both FgGST-S1 and FhGST-S1 to have 10 predicted epitopes (data not shown). All epitopes with one exception matched between both species 100%. The one exception predicted the epitope to encompass an amino acid substitution at position 109. This epitope, which varied

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DISCUSSION High resolution 2DE proteomics coupled with transcriptomics is a powerful tool to identify members of protein superfamilies,27 including the GSTs.26 Here, we initially, used PCR to confirm flukes under study as F. gigantica prior to the purification of the expressed GST pool by S-hexyl-GSH agarose columns. The choice of using the less well-known S-hexyl-GSH agarose matrix as opposed to the usual GSH column step was taken as previous work has highlighted the efficiency of S-hexylGSH agarose columns to purify a greater range of expressed GSTs in the related F. hepatica.26 Thus, this GST isolation strategy has identified the well characterized Mu class GSTs and, for the first time in F. gigantica, a Sigma class GST protein mirroring the temperate liver fluke F. hepatica.26 In F. hepatica there are 4 clear isoforms of Mu class GST, namely, GSTs 26, 27, 28, and 29 (or 51, 47, 7, and 1, respectively). Unfortunately, there are only two deposits currently in the public databases that have been made for F. gigantica. Both entries, AAD23997 and ACH88355, match one another 100% and correspond to GST isoform 26 of F. hepatica. However, from our results it appears as if all 4 representatives of the Mu class GSTs found in F. hepatica are present in F. gigantica. Searching the public databases using MASCOT hit all four of the F. hepatica Mu class isoforms with 5885



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hepatica sequence (DQ974116) and F. gigantica transcriptome sequences. The Sigma class GSTs in the closely related schistosomes share a further 25% amino acid similarity with the F. gigantica and the F. hepatica Sigma GSTs over the Fasciola Mu class GSTs. The schistosome GSTs have shown significant promise as vaccine candidates, having now entered clinical trials.21 Given the importance of Sigma class GST in Fasciola biology as an immune modulator,12 that all previous GST vaccination trials against fascioliasis used Mu class GSTs, and in view of this similarity to the schistosome GSTs, the F. gigantica and F. hepatica Sigma GSTs warrant immediate immunological investigations. The complete coding sequence of the Sigma class GST of F. gigantica was deduced using RACE PCR. The Sigma class GSTs of F. gigantica and F. hepatica were found to share over 98% similarity with only 4 amino acid variations. The sequence similarity of the two Fasciola Sigma GSTs suggests the functions described for the F. hepatica Sigma class GST12 may also occur in the F. gigantica orthologue. However, the presence of one of the four amino acid variations may cause changes in the predicted biological function. Our de novo peptide sequencing (F. gigantica sourced from Egypt) identified an amino acid substitution at amino acid 55 in the mature protein (L55V) not observed in the predicted F. gigantica transcriptome sequences (sourced from Thailand) or the RACE PCR acquired FgGST-S1 (sourced from India). In addition, two other amino acid variations between F. gigantica sequences (between fluke from India and Thailand) were seen (positions 195 and 209) indicating at least 3 sites for SAAPs. SAAPs such as these have been identified previously in protein superfamilies within the Fasciolids,27 and the evidence presented here points toward geographical SAAPs in the F. gigantica Sigma class GST protein, FgGST-S1. Searching the F. hepatica EST database revealed none of the F. gigantica variants suggesting that these 3 SAAPs could be F. gigantica specific, and their impact upon the functionality of the protein will need further clarification. However, it appears that all 3 SAAPs may be of low minor allele frequencies suggesting that FgGST-S1 may have limited plasticity to host selection pressure. In addition, it seems as if there is a single gene encoding FgGSTS1 making this a stable protein to target with immunotherapy. Looking at the identified SAAPs at positions 55, 195, and 209 in relation to the G-site residues41 shows the SAAP at position 55 to lie within the active site and directly adjacent to residues 52−54 all involved in GSH binding. However, it appears no adverse affect to GSH binding activity is incurred as this particular variant was only identified in GSH affinity-purified proteins. Both residues at 195 and 209 lie away from the active site and would appear not to affect enzyme activity. FgGST-S1 and FhGST-S1 share close sequence homology to one another and toward the schistosome Sigma class GSTs. Should the similarity of the liver fluke Sigma GSTs extend to biochemical activity the potential for therapy could be greatly enhanced. FgGST-S1 may also function in the mediation of host immune responses through the production of prostaglandins through prostaglandin synthase activity as with FhGSTS1.12 The liver fluke transcription data revealed Omega class GST present in both F. gigantica and F. hepatica (also found previously26), both of which were subsequently cloned in this study. Omega class GSTs appear to be, via mainly bioinformatic analyses, ubiquitous in the animal kingdom.42 Omega GSTs, including those of F. gigantica and F. hepatica, have an active

Figure 9. 2DE Western blots of rFgGST-S1 using anti-GST antibodies. rFgGST-S1 was isoelectric focused on 7 cm pH 3−10 linear immobilized pH gradient (IPG) strips, run on 12.5% polyacrylamide gels and transferred onto PVDF membranes for antibody binding. GST class specific antibodies (AB) are as follows: (A) 2DE gel of rFgGST-S1, 10 μg Coomassie blue stained; (B) antiSigma class GST AB against rFgGST-S1, 2 μg; (C) anti-Mu class GST AB against rFgGST-S1, 2 μg. Corresponding regions of the 2DE gel profile and Western blots are circled and designated a, b, and c.

GSTs 26 and 28 the most well represented, mirroring work in F. hepatica.26 Only two proteins from S-hexyl glutathione based isolation were classified as non-GST liver fluke proteins. We note the possibility of host-derived proteins with GSH affinity40 being present in the gut content of the fluke following feeding on blood and bile, although this would be at a significantly lower level than the overwhelmingly greater quantity of fluke proteins. Non-GST contamination is a more likely occurrence when purifying with S-hexyl-GSH agarose due to the hydrophobic alkyl side chain attached to the GSH molecule binding nonspecifically to partially exposed hydrophobic areas of proteins during the purification method (Supplementary Figure S3). Thus the presence of these two proteins is of no real significance. Purifying native Sigma class GST from the tropical liver fluke provides the first confirmation that this is a functional protein, as only small fragments were identified through a recent transcriptome study.29 Importantly, Sigma class GST confirmation was also supported via mass spectrometry and antibody recognition. This previously undiscovered Sigma class GST in F. gigantica appears to be highly similar to the recently discovered Sigma class GST of F. hepatica26 showing a solitary amino acid change (T209S) between the published F. 5886



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immune modulating Sigma class GST in both Fasciola species of interest, it is perhaps more important, in light of the success of the Schistosome Sigma class GST progressing to clinical trials, to further investigate the immunological properties of Fasciola Sigma GST as a vaccine candidate rather than pursuing Mu class representatives as in previous trials. Furthermore, variance of only 4 amino acids between the two Sigma class GST sequences suggests a very similar immunological response to both would be observed. Therefore, the potential to produce a vaccine that could target both F. gigantica and F. hepatica equally well would be of significant benefit to the world’s agricultural economy and to human populations in endemic areas. Furthermore, the similarity between Fasciola and Schistosoma Sigma class GST, while still needing further investigation, lends itself to the possibility of producing a single cross protecting vaccine (observed with fatty acid binding proteins51) that would be effective against at least two parasites of veterinary and human importance.

site cysteine residue and can catalyze a range of thiol transferase and reduction reactions.43 Roles of Omega class GSTs have been proposed in drug resistance,44,45 yet the response seen may actually represent a more general cellular response to oxidative stress.43,46 In C. elegans, Omega class GST has been localized exclusively to the gut.46 It is therefore possible that FgGST-O1 and FhGST-O1may function in the fluke gut to provide a detoxification role combating oxidative stress from the products of blood digestion. Additional roles in oxidative stress response may be associated with ascorbate, an important antioxidant. Omega class GSTs display dehydroascorbate reductase activity and may be involved in the maintenance of sufficient ascorbate levels,43 linking the Omega GSTs with the detoxification roles assigned to typical GSTs. Continuing analysis of the recently published transcriptome29 provided confirmation that Mu class GST isoforms of F. gigantica mirror those of F. hepatica. Partial sequences were identified representing GSTs 27 and 29 but not that of GSTs 26 and 28. However, GST 26 is represented by the two sequences deposited in the public domain, leaving only GST 28 unconfirmed. In addition, when searching transcripts using known Mu class GSTs, a fifth Mu class GST was identified in both F. gigantica and F. hepatica. This fifth Mu class GST is more closely related to the schistosome Mu class GSTs than to the four current Fasciola Mu class GSTs. This new Mu class GST appears to be a more ancient Mu isoform and suggests common functions in both schistosomes and fasciolids. Unfortunately, the sequences from the two transcript runs were not included in the database used for MS/MS identification and so would not have been identified during our proteomics study. Finally, in silico analysis also revealed, for the first time, a Zeta class GST in Fasciola species. Zeta class GSTs, like Omega class, were first identified via sequence alignment techniques using the human EST database47 after perhaps eluding discovery in humans and other organisms, due to their poor capacity to bind GSH-affinity matrices and low/no activity with the model substrate CDNB. Zeta class GST members are highly conserved across eukaryotes;47 such high level of conservation suggests a functional importance of the Zeta class GST and perhaps the inability of other enzymes to accommodate its loss of function. Regarding function, Zeta class GSTs are found to transform dichloroacetic acid (DCA) to glyoxylic acid48 and maleylacetoacetate (MAA) to fumarylacetoacetate. 49 Due to its significantly higher catalytic activity, the isomerization of MAA to fumarylacetoacetate is proposed as the major reaction in vivo for this enzyme. MAA isomerization is an important step in the tyrosine degradation pathway, although non-enzymatic isomerization of MAA to fumarylacetoacetate may be sufficient to bypass the loss of a Zeta class GST50 in the presence of low levels of tyrosine or phenylalanine.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Table S1 and Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +44 (0)1970 621511. Fax: +44 (0)1970 622350. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors acknowledge the BBSRC (BBC503638 and BBH0092561) and the EU DeLiver project (FOOD-CT2005-023025) for support.

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REFERENCES

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CONCLUSIONS Delineating the soluble GST superfamily in a parasite provides insight into drug metabolism and potential resistance routes and host−parasite relationships. Understanding the functionality of the newly identified GST representatives is the next step, with reverse genomics approaches likely to reveal key insights into how these enzymes function in vivo; importantly, both Sigma and Omega class Fasciola GSTs are amenable to RNA interference (data not shown). Having identified the 5887



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