Proteomics 2006, 6, 6263–6273 6263 DOI 10.1002/pmic.200600499 RESEARCH ARTICLE Proteomic analysis of glutathione transferases from the liver fluke parasite, Fasciola hepatica Gustavo Chemale1, Russell Morphew2, Joseph V. Moxon2, Alessandra L. Morassuti3, E. James LaCourse1, John Barrett2, David A. Johnston4 and Peter M. Brophy1 1 School of Biological Sciences, University of Liverpool, Liverpool, UK Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, UK 3 Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brasil 4 Biomedical Parasitology Division, Department of Zoology, Natural History Museum, Cromwell Road, London, UK 2 The parasite Fasciola hepatica causes major global disease of livestock, with increasing reports of human infection. Vaccine candidates with varying protection rates have been identified by pregenomic approaches. As many candidates are part of protein superfamilies, sub-proteomics offers new possibilities to systematically reveal the relative importance of individual family proteins to vaccine formulations within populations. The superfamily glutathione transferase (GST) from liver fluke has phase II detoxification and housekeeping roles, and has been shown to contain protective vaccine candidates. GST were purified from cytosolic fractions of adult flukes using glutathione- and S-hexylglutathione-agarose, separated by 2-DE, and identified by MS/MS, with the support of a liver fluke EST database. All previously described F. hepatica GST isoforms were identified in 2-DE. Amongst the isoforms mapped by 2-DE, a new GST, closely related to the Sigma class enzymes is described for the first time in the liver fluke. We also describe cDNA encoding putative Omega class GST in F. hepatica. Received: July 11, 2006 Revised: July 27, 2006 Accepted: August 12, 2006 Keywords: 2-DE / F. hepatica / Glutathione transferases / Omega / Sigma 1 Introduction The liver fluke Fasciola hepatica infects a wide variety of mammalian hosts, particularly sheep, goats and cattle, causing economic losses of over US$ 3 billion worldwide per annum through mortality, reduction in host fecundity, decrease in meat, milk and wool production and condemnation of livers. Recently, a 12-fold disease increase was recorded in European Union member states, possibly associated with Correspondence: Dr. Gustavo Chemale, School of Biological Sciences, Biosciences Building, Crown Street, University of Liverpool, Liverpool, L69 7ZB, UK E-mail: g.chemale@liverpool.ac.uk Fax: +44-151-7954408 Abbreviation: GST, glutathione transperase  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim climate change. Moreover, fasciolosis is now recognised as an emerging human disease, with 2.4 million people infected and a further 180 million at risk of infection [1]. There are no commercial vaccines for the disease, and control relies on a few classes of chemicals with toxicity and resistance issues. Possibly, a major reason for variable vaccine protection rates in field trials of defined antigens relates to a lack of understanding of the sub-proteome of candidate liver fluke protein families. The GST superfamily provides vaccine candidate for parasitic flatworms, including liver fluke [2]. Apart from their vaccine potential, sub-proteomic analysis of the major detoxification system in the liver fluke might contribute to a better understanding of drug resistance mechanisms and the host-parasite relationship. The GST superfamily (EC 2.5.1.18) includes phase II detoxification enzymes that catalyse the conjugation of the tripeptide glutathione (GSH, g-Glu-Cys-Gly) to a wide variety www.proteomics-journal.com 6264 G. Chemale et al. of non-polar electrophilic, endogenous and xenobiotic toxic compounds [3], such as products of oxidative stress, chemical carcinogens, therapeutic drugs, pesticides and herbicides [3]. GST are present in vertebrates and invertebrates, fungi, plants and bacteria as dimeric enzymes, and occur mainly in the cytosol with the exception of three membrane-bound glutathione transferases, i.e. microsomal GST I and II and leukotriene C4 synthase, all of which are found in microsomal and mitochondrial membrane of human and rat liver [4]. The family of cytosolic GST is currently subdivided into eight different and often species-independent classes, including Alpha, Mu, Pi, Omega, Sigma, Theta, Phi and Zeta classes [5]. GST are major detoxification enzymes in adult helminths, as these organisms appear to lack the important cytochrome P-450-dependent detoxification reactions [6]. In the liver fluke F. hepatica, GST account for as much as 4% of the total soluble protein, with a widespread distribution in the parasite’s tissues, especially the parenchyma [7, 8], suggesting important physiological roles for these enzymes. Five acidic/neutral cytosolic GST isoforms were previously identified from F. hepatica by a combination of GSH-affinity chromatography and chromatofocusing [9] of which none showed clear biochemical relationships to any of the mammalian GST families previously characterised. Further to this, four cDNA encoding different Mu class GST (FhGST-1, -7, -47 and -51) from F. hepatica have been previously isolated [10]. Immunocytochemical studies and protein sequencing of affinity purified GST showed that these isoforms are expressed in the adult worm [8], and confirmed heterogeneity between fluke GST isoforms. Biochemical analysis of the four recombinant GST clones revealed overlapping, but unique substrate specificities with different sensitivities to inhibitors [11]. A crystal structure of GST Fh-47 was determined that confirmed overall resemblance to the Mu class in general and to the Sj26 GST of Schistosoma japonicum in particular [12]. Molecular modelling of the other three enzymes based on this structure showed critical differences in the xenobiotic substrate-binding site, which may explain substrate/inhibitor differences between these isoenzymes as well as differences in the non-substrate-binding site. To date, only GST belonging to the Mu class have been isolated and identified from F. hepatica, with all GenBank deposited sequences grouping into several isoforms of four different GST. Besides their importance in detoxification processes, F. hepatica GST are also immunogenic in infected hosts. Several attempts to use F. hepatica GST as protective antigens in sheep and cattle have been described with varying levels of protection related to worm burden [13–15]. However, a systematic global sequence-based approach has not been undertaken to characterise the GST-ome superfamily of F. hepatica in order to identify the most appropriate GST for future vaccine formulations. For the first time we describe the characterisation of F. hepatica GST using a systematic proteomics approach coupled to an EST database. GST were purified from cytosolic  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Proteomics 2006, 6, 6263–6273 fractions of adult flukes using GSH- and S-hexylglutathione-agarose, separated by 2-DE and identified by MS/MS. Amongst the 11 isoforms mapped by 2-DE, a new GST, closely related to the Sigma class enzymes is described for the first time in the liver fluke. Moreover, we also describe cDNA encoding putative Omega class GST in F. hepatica. 2 Materials and methods 2.1 Cytosolic protein extracts Adult F. hepatica were obtained from freshly slaughtered sheep at a local abattoir. Flukes were extensively washed in PBS and protein extracts obtained by homogenisation in a glass grinder in lysis buffer, containing 50 mM Tris-Cl, pH 7.4, 0.1% Triton-X100, 5 mM DTT and a cocktail of protease inhibitors (Roche, Complete Mini, EDTA-free, 11836170001). After homogenisation, samples were sonicated with four pulses of 30 s on ice and centrifuged at 100 0006g for 1 h at 47C. Supernatants containing cytosolic proteins from adults were quantified by Bradford method (Sigma Aldrich) and stored at 2207C until needed. 2.2 GST purification Proteins were applied to a glutathione-agarose (SigmaAldrich) or an S-hexylglutathione-agarose (Sigma-Aldrich) affinity column and purified at 47C according to the manufacturer. Eluted proteins were concentrated using 10-kDa filters (Amicon Ultra, Millipore) and quantified by the Bradford method (Sigma-Aldrich). 2.3 2-DE Cytosolic protein extracts were precipitated with an equal volume of ice-cold 20% TCA in acetone w/v and washed twice in ice-cold acetone before solubilisation into sample buffer consisting of 7 M urea, 2 M thiourea, 4% w/v CHAPS, 66 mM DTT and 0.5% carrier ampholytes v/v (Biolyte 3/10, Bio-Rad). Samples were in-gel rehydrated and focused on 17-cm pH 3-10NL IPG strips (Bio-Rad) for a total of 60 000 Vh, using the Protean IEF Cell (Bio-Rad). 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 alkylating equilibration buffer (30% v/v glycerol, 6 M urea, 4% iodoacetamide). IPG strips were run in the second dimension on 20620 cm SDS-PAGE gels (12.5% acrylamide) using the Protean II xi 2-D Cell (BioRad). Gels were CBB stained (Phastgel Blue R, Amersham Biosciences) and scanned using the GS-800 calibrated densitometer (Bio-Rad). Quantitative differences between protein spots were analysed using Progenesis PG220 software, version 2006 (Nonlinear Dynamics). Spots were manually detected on gels www.proteomics-journal.com Animal Proteomics Proteomics 2006, 6, 6263–6273 and normalisation performed using total spot volume multiplied by total area. Quantitative analysis was based on average gels created from three gel replicates. Twofold differences between spots with a p ,0.05 were considered significant when average gels were compared. 2.4 Protein identification Protein spots were manually excised from the gels and in-gel digested with trypsin according to the following protocol. Gel plugs were treated in three washing steps with 180 mL of 50% ACN and 50 mM ammonium bicarbonate for 15 min, followed by 180 mL of ACN. Gel plugs were then dried by vacuum centrifugation and digested for 18–24 h at 377C using 10 mL of porcine trypsin (Proteomics grade trypsin, Sigma-Aldrich) diluted to 10 mg/mL in 25 mM NH4HCO3. After tryptic digestion, peptides were extracted in two steps with 50 mL of 50% ACN and 5% TFA for 1 h. Extracted peptides were dried and re-suspended in 10 mL of 1% formic acid. Peptides from digested protein spots were desalted for analysis using C18 ZipTips (Millipore) according to manufacturer’s protocol. Samples were manually loaded into a gold-coated nanovial (Waters, UK) and sprayed under atmospheric pressure at 800–900 V into a Q-Tof-1.5 hybrid TOF mass spectrometer (Waters). Initially, data were collected from the whole sample to provide an overview of the full range of observable peptides present in the digest, and peptides were selected individually for further MS/MS analysis. MassLynx software was used to control the mass spectrometer voltages, collect and process data, MaxEnt3 software to deconvolute multiple charged ions to singly charged species and BioLynx software to process MS/MS data (MassLynx, MaxEnt3 and Biolynx all from Waters). Peptides selected for sequencing were fragmented using CID with argon. The spectra acquired were combined and smoothed twice using the Savitzky-Golay method. Smoothed data were then subjected to MaxEnt3 (Micromass) using a minimum mass of 50 Da and maximum of 2000 Da. MS/MS data from each protein spot were analysed using Peptide Sequencing (PepSeq) in the Masslynx version 3.5 software package (Micromass) to provide fragmentation spectra and peptide sequences. For automatic peptide sequencing an intensity threshold of one was set and a fragment ion tolerance of 0.1 Da. Standard peptide modifications such as methionine oxidation and carbamidomethylation of cysteines were considered. Peptide sequences were searched separately using BLAST [16] short nearly exact matches against the GenBank database (NCBI – www.ncbi. nlm.nih.gov) and an in-house translated F. hepatica EST database (ftp://ftp.sanger.ac.uk/pub/pathogens/fasciola/ hepatica/ESTs/). Only protein entries with complete identity to that of the peptides sequenced were considered for the identification of the GST.  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 6265 2.5 Protein sequences alignment and phylogenetic tree Translated cDNA sequences from the ‘in-house’ translated EST database showing complete identity to peptides sequenced from spots 9 and 10 were aligned using ClustalW [17] and assembled as a contig, named FhepGSTs. To classify the cDNA sequences found in the library, protein sequences from different classes of GST from several species were aligned with F. hepatica GST obtained from GenBank and from the ‘in-house’ translated EST library database using ClustalW [17], and neighbour joining trees produced using BioEdit Sequence Alignment Editor version 7.0.5.2. [18]. Tree was viewed within TREEVIEW [19] to visualise the relationship between sequences and provide direction upon assigning the F. hepatica GST to an existing GST family class. Sequences obtained from the three-element signature of Omega class GST (PRINTS PR01625), were aligned with several Omega GST, including the F. hepatica sequences found in the ‘in-house’ translated EST database. Consensus sequences were constructed using the PRATT consensus sequence search tool (http://us.expasy.org/tools/pratt/) [20, 21]. Omega class GST signature sequences PR01625 were obtained from PRINTS data bank of protein family fingerprints (http://umber.sbs.man.ac.uk/ dbbrowser/sprint/). 2.6 Immunoblot S-hexylGSH-affinity purified GST samples were subjected to 2-DE, electro-transferred to NC membrane and immunoblotted with GST class antibodies to further characterise isolated GST. Mu class GST antibody was represented by the anti-Schistosoma japonicum Mu class antibody (PharmaciaBiotech 27-4577). A GST antibody to the Sigma-like GST class of the nematode helminth Haemonchus contortus recombinant GST polyclonal antibody (rHcon GST Ab) was also used [22]. Proteins were transferred according to standard procedures [23, 24] using Bio-Rad criterion blotter overnight at 47C to a NC membrane (Hybond-P, Amersham, UK). Membranes were incubated in blocking buffer, 1% skimmed-milk powder w/v in TTBS (20 mM Tris-HCl, 154 mM NaCl, 0.1 % v/v Tween 20, pH 7.2) for 2 h at 207C. After blocking, the membrane was washed three times for 5 min with TTBS before 1-h incubation at 207C with antibody diluted according to manufacturer’s instructions (1:400 for rHcon GST Ab; 1:1000 for anti-Mu antibody) in the blocking buffer. The membrane was washed as before and incubated for 1 h at 207C with the appropriate secondary antibody according to manufacturer’s instructions diluted in blocking buffer (antirabbit IgG conjugated with alkaline phosphatase, produced in goat, A3687 Sigma, UK for rHcon GST Ab, anti-goat IgG conjugated with alkaline phosphatase, produced in rabbit, A4187 Sigma for anti-Mu GST). Blots were washed as previously described and immunogenic proteins visualised with www.proteomics-journal.com 6266 G. Chemale et al. BCIP/NBT (5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium) liquid substrate system (Sigma). The development reaction was terminated by briefly washing the membranes with distilled water, and incubating for 10 min in 20 mM EDTA. 3 Results 3.1 2-DE mapping of F. hepatica GST Adult F. hepatica flukes were removed from sheep livers, and following homogenisation, 50 mg of soluble proteins were passed separately through a GSH- and an S-hexylglutathione agarose column to isolate 1.7 mg of GST protein. Purification using the GSH-agarose column yielded eight prominent proteins spots on 2-DE (Fig. 1A), and purification with the Shexylglutathione-agarose yielded ten protein spots on 2-DE (Fig. 1B). Comparison of the 2-D gels of the two purification matrices using the Progenesis PG220 gel analysis software showed highly significant quantitative differences (Fig. 1C). The greatest differences were found at the basic end of the gel, with proteins present in spots 9 and 10 being six- and tenfold, respectively, more efficiently purified using the Shexylglutathione-based matrix. Figure 1. 2-DE mapping of F. hepatica affinity purified GST. Glutathione transferases were affinity purified using a GSH (A) or S-hexylglutathione-agarose (B). Numbered protein spots were identified by MS/MS (Table 1). Strips were rehydrated with 50 mg of eluted proteins. IEF was carried out until 60 000 Vh at 207C in 17-cm IPG strips pH 3-10NL (Bio-Rad). Second dimension was carried out in 12.5 % SDS-PAGE. Gels were subsequently stained with CBB. Quantitative gel analysis was performed using Progenesis PG220, version 2006 software. The 3-D view of the GSHagarose purified GST 2-D gel (C) showing the quantitative differences per spot in number of fold compared to the S-hexylglutathione-agarose purified GST.  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Proteomics 2006, 6, 6263–6273 The identification of the GST separated by 2-DE was performed by MS/MS with the sequencing of three to eight peptides per protein spot. Peptide sequences obtained from each spot were searched against GenBank (www.ncbi.nlm. nih.gov) or an ‘in-house’ translated F. hepatica EST database (ftp://ftp.sanger.ac.uk/pub/pathogens/fasciola/hepatica/ESTs/) using BLAST algorithm [16]. Hits presenting total identity to the peptide sequences were considered for analysis. All identified protein spots were shown to be GST (Table 1). The three protein spots at the acidic end of the gel, spots 1 to 3, were shown to be the previously isolated FhGST51. Peptides corresponding to FhGST7 were identified in spot 5 and FhGST51, FhGST1 and FhGST7 were identified in spot 4, probably a result of a spot overlapping in the gels. FhGST47 was identified at spot 8, at the more basic end of the gel. We were not able to find complete identity hits in GenBank with peptides from spots 9 and 10 (Fig. 1, Table 1). However, ten translated cDNA sequences were found in the ‘in-house’ translated F. hepatica EST database with amino acid sequences showing complete identity to the peptides sequenced. One peptide found in these spots was also found in spot 7 (Fig. 2), together with peptides corresponding to FhGST1 and FhGST47. 3.2 New F. hepatica Sigma and Omega GST As many of the translated cDNA sequences from the ‘inhouse’ EST database showing complete identity to peptides sequenced from spots 9 and 10 were partial sequences, they were aligned using ClustalW [17] and assembled as a contig, named FhepGSTs (Fig. 2A). Besides these cDNA clones, which were identified in spots 9 and 10 (Fig. 1) and had not been previously deposited in the GenBank database, we also found two cDNA clones with partial similarity to some of the peptides sequenced, Fhep54b04 and Fhep49c06. To classify the GST encoded by these cDNA, they were aligned with existing classified GST from seven species-independent classes within mammals, helminths and insects, including F. hepatica sequences previously described. To visualise the relationship between sequences and provide direction upon assigning the F. hepatica GST to an existing GST family class, a phylogram tree was built from the alignment (Fig. 3). EST clones from the ‘in-house’ F. hepatica library cluster within GST classes previously unrepresented in F. hepatica. FhepGSTs clustered with GST belonging to the Sigma class, suggesting that a Sigma/ prostaglandin-D-synthase GST is present and expressed in F. hepatica. This contig shows similarities ranging from 26 to 45% to other Sigma class GST (Table 2). Highest identities were observed with S. mansoni, S. japonicum and Clonorchis sinenis Sigma GST, ranging from 42 to 45%. This Sigma GST is also supported by proteomic sequence data (Fig. 1 and Table 1). Fhep54b04 and Fhep49c06 partial cDNA clones clustered with GST belonging to the Omega class. Overall identity with other sequences was low, ranging from 9 to 32% www.proteomics-journal.com Proteomics 2006, 6, 6263–6273 Animal Proteomics 6267 Figure 2. F. hepatica Sigma GST amino acid deduced sequence from cDNA contig assembly. (A) Ten cDNA clones encoding protein sequences matching peptides sequenced from spots 9 and 10 (Fig. 1, Table 1) were assembled as a contig, named FhepGSTs, containing the complete peptide sequence of this protein. (B) MS/MS spectra resulting from the analysis of a peptide sequenced from spots 7, 9 and 10, belonging to the F. hepatica Sigma GST. (Table 2), with S. mansoni Omega GST showing the highest identity. Sequence identity for these F. hepatica sequences is lower than that typically expected to place a GST into a given class (.40%, [25]). Between classes, GST may typically be expected to share ~25% identity. This class structure is historically derived from mammalian classifications, and thus might be less applicable to invertebrate GST. It may be better to consider ‘class-specific’ domains in alignments, biochemical specificity and modelled 3-D structure, rather than overall identity, which may incorrectly exclude GST from assignment to given classes. In order to better classify F. hepatica putative Omega GST, we made a comparison of the consensus three-element signature of the Omega GST. This showed a much higher relationship of the F. hepatica Omega GST (Fig. 4A) to other members of this class, with similarity ranging from 47 to 65% similarity (Fig. 4B). Additionally, the F. hepatica sequences were found to contain the characteristic N-terminal extension of Omega GST and the highly conserved cysteine at the catalytic site, exclusive to Omega class GST that replaces the typical serine or tyrosine of all other GST classes [25].  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3.3 Immunological classification of F. hepatica GST To further characterise F. hepatica GST isolated in this work, 2-D gels containing the S-hexylglutathione-agarose purified GST were transferred to PVDF membranes and assayed with anti-S. mansoni Mu-class GST and anti-H. contortus Sigmaclass antibodies (Fig. 5). Spots containing FhGST51 and FhGST7 GST and spot 6 were recognised by the anti-Mu class antibody (Fig. 5A). As peptides belonging to FhGST1 and FhGST7 were also found in spot 6, and that this antibody did not recognise Mu-class GSTpresent in spots 7 and 8 (FhGST1 and FhGST47), the protein recognised in spot 6 is probably FhGST7 or a very similar isoform not identified yet. Surprisingly, proteins present in spots 9 and 10, identified in this work as F. hepatica Sigma GST, were not recognised by the antiSigma class antibody (Fig. 5B). Moreover, proteins in spots 6–8 were recognised by this antibody and were all identified as Muclass GST, apart from one peptide belonging to the newly described F. hepatica Sigma class GST (Table 1, Fig. 2). We also tried to identify Pi class GST in the F. hepatica affinity purified GST by immunoblot using an anti-human GST1 antibody, but no reactions were observed (data not shown). www.proteomics-journal.com 6268 G. Chemale et al. Proteomics 2006, 6, 6263–6273 Table 1. Identification of GST from F. hepatica by MS/MS. Peptide sequences from spots were used to search against GenBank or the translated EST library for the identification of the specific members of the GST superfamily. Numbers in brackets following GenBank accession numbers correspond to the peptides that share complete identity with the amino acid sequences Spot MS/MS peptidesa), b) GenBank entries E value Coverage (%) Theoretical pI/MW (Da) Class Observed pI/MW (Da) 1, 2, 3 1-ISMIEGAAMDLR 2-YLAPQCLEDFPK 3-MWSNFLGDR 4-FEEVQGDYLK 5-FNMGLDLPNLPYYIDDK 6-LTQSVAIMR, 7-LGYWK 8-LLLEYLGEEYEEHLYGR AAB28746 (1-8) AAA29141 (1-8) P30112 (1-8) 1905266A (1-8) 7e-25 41% FhGST51 5.88/25373.36 m 5.4-5.82/ 5196-25665 4 1-LTQSLAILR 2-VSMIEGAAVDLR 3-PAKLGYWK 4-ISMIEGAAMDLR 5-YLAPQCLEDFPK 6-IGFGLTCYNPK 7-YLAPQCLDDFPK P56598 (1-3) 1905266D (1-3) 8e-03 13% FhGST1 6.61/25729.55 m 5.95/26000 AAB28746 (3-5) AAA29141 (3-5) P30112 (3-5) 1905266A (3-5) 8e-04 14% FhGST51 5.88/25373.36 m P31671 (3,4,6,7) 1905266B (4,6,7) 7e-08 20% FhGST7 6.13/25327.27 m 5 1-ISMIEGAAMDLR 2-MWSDFLGDR 3-IGFGLTCYNPK 4-YLAPQCLDDFPK P31671 (1-4) 1905266B (1-4) 3e-15 20% FhGST7 6.13/25327.27 m 6.03/25582 6 1-LTQSLAILR 2-VSMIEGAAVDLR 3-PAKLGYWK 4-ISMIEGAAMDLR P56598 (1-3) 1905266D (1-3) 8e-03 13% FhGST1 6.61/25729.55 m 6.51/25554 P31671 (3-4) 1905266B (3-4) 2e-03 9% FhGST7 6.13/25327.27 m AAA29140 (1-2) P31670 (1-2) 1905266C (1-2) 2e-03 10% FhGST47 6.54/25412.38 m P56598 (3) 1905266D (3) 8e-03 5.5% FhGST1 6.61/25729.55 m FhepGSTs (4) 1e-02 5.6% local database s 7 1-MWSDFLGDR 2-ISMIEGAAMDLR 3-VSMIEGAAVDLR 4-VPLLDVTGPDGK 6.84/25360 8 1-YLAPHCLDEFPK 2-FNMGLDLPNLPYYIDDK 3-ISMIEGAAMDLR AAA29140 (1-3) P31670 (1-3) 1905266C (1-3) 7e-06 18% FhGST47 6.54/25412.38 m 7.1/25200 9, 10 1-EVYTLFR, 2-LLLTCAGVK 3-LVSESLESSGGK 4-VPLLDVTGPDGK, 5-IIGECEDLYR 6-MMGETDEEYYLIER, 7-LWYFQFR FhepGSTs (1-7) 9e-03 34% Local database 8.86/24534.41 s 8.54/25223 a) Alignment of F. hepatica GST showing peptides that differentiate between isoforms, see Supplementary Figure I. b) MS/MS spectra from peptides specific to each isoform, see Supplementary Figures 2–15. 4 Discussion The 2-DE mapping of GST can be a very useful tool, as these proteins play a major role in phase II detoxification, especially in drug and oxidative stress response. To provide a 2-DE mapping of F. hepatica GST, we purified GST from adult F. hepatica using GSH- and S-hexylGSH agarose columns. For  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim the first time, we combined purification using two affinity matrix isolation approaches, EST database searching and 2-DE array (Fig. 1B), resulting in the detection of ten predominant GST spots. This approach produced a more complete GST-ome profile compared to the classical approach of GSHagarose purification alone, with significant quantitative differences between the two methods, especially when spots 9 www.proteomics-journal.com Proteomics 2006, 6, 6263–6273 Animal Proteomics 6269 Figure 3. Classification of F. hepatica GST. Phylogenetic bootstrap tree of F. hepatica GST aligned with existing classified GST from seven species-independent classes within mammals, helminths and insects. Protein sequences from different classes of GST from seven species were aligned with F. hepatica GST obtained from GenBank and from ‘in-house’ EST library database using ClustalW [17], and neighbour joining trees produced using BioEdit Sequence Alignment Editor version 7.0.5.2. [18]. Tree was viewed within TREEVIEW [19] to visualise the relationship between sequences and provide direction upon assigning the F. hepatica GST to an existing GST family class. Accession numbers: GenBank, gb; Swiss-Prot, sp. F. hepatica (gb), Fhep, P31671, P56598, 1905266A, 1905266B, 1905266C, 1905266D, AAA29139, AAA29140, AAA29141, AAB28746, P30112,P31670. Ascaris summ (gb), asum, GST1, P46436; Caenorhabditis elegans (sp), Cele, GST1 pi, P10299; GST7, P91253; GST43 zeta, Q9N4H6; omega, P34345. Clonorchis sinensis (gb), Csin, GST28, O97096. Drosophila melanogaster (sp), Dmel, GSTT1, P20432; Haemonchus contortus (sp), Hcon, Q9NAW7. Homo sapiens (sp), Hsap, GSTM1.1, P09488; GSTA1, P08263; GSTP1, Q5TZY3; PGD2, Q6FHT9; GSTZ1.1, Q6IB17; GSTO1, P78417; GSTT1, Q5TZY2. Mus musculus (sp), Mmus, GSTM1, Q58ET5(sp); GSTA1, Q6P8Q0; GSTP1, P19157; PGD2, Q8CA80; GSTZ1, Q9WVL0; GSTO1, O09131; GSTT1, Q91X50. Schistosoma haematobium (gb), Shae, GST28, P30114. S. japonicum (gb), Sjap, mu, P08515. S. mansoni (gb), Sman, mu, P35661; GST28, P09792; omega, Q86LC0. and 10 are compared (Fig. 1C). All previously described F. hepatica GST were identified in the 2-DE gels, many of them present in more than one spot (Table 1). FhGST51 was identified in spots 1 to 3, indicating that this protein may be modified after translation or during detoxification or that very similar isoforms with slightly different pI exist. We were not able to sequence peptides belonging to other isoforms, even after determining the sequence of eight peptides from these spots, covering approximately 35% of the protein. If they are different isoforms, regions responsible for the difference in the pI observed were not covered by these peptides. Peptides belonging to different GST were found in the same protein spot, as can be seen in spots 4, 6 and 7 (Fig. 1  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim and Table 1). This might be the result of spot overlapping in the 2-DE gels, as these proteins present very similar pI, or possible modification and co-migration. Peptides corresponding to FhGST7 and FhGST47 were identified in single spots, 5 and 8, respectively. We were not able to find complete identity hits in GenBank with peptides from spots 9 and 10. However, cDNA sequences were found in the ‘in-house’ translated F. hepatica EST database matching the peptides sequenced and were used to assemble a contig (Fig. 2A). One peptide found in these spots was also found in spot 7 (Fig. 2B), together with peptides corresponding to FhGST1 and FhGST47. When aligned with GST from other organisms, FhepGSTs sequence clustered with Sigma class GST www.proteomics-journal.com 6270 G. Chemale et al. Proteomics 2006, 6, 6263–6273 Figure 4. Comparison of F. hepatica Omega GST to conserved signature motifs. Sequences obtained from the three-element signature of Omega class GST (PRINTS PR01625), were aligned with several Omega GST including the F. hepatica sequences found in the ‘In-house’ EST database (A). Consensus sequences to these three Omega domains were found to match well with the F. hepatica sequences, ranging from 47 to 65% similarity (B). Consensus sequences were constructed using the PRATT consensus sequence search tool (http://us. expasy.org/tools/pratt/) [20, 21]. Omega class GST signature sequences PR01625 were obtained from PRINTS data bank of protein family fingerprints http://umber.sbs.man.ac.uk/dbbrowser/sprint/. Accession numbers are same as in Figure 3. (Fig. 3). As well as being identified in adults, FhepGSTs was also shown to be highly expressed in F. hepatica eggs (data not shown), showing that this protein is expressed in both parasite stages. Overall identities of the F. hepatica Sigma and Omega GST were higher when compared to other trematode GST, varying from 27 to 45%. There are no clearly established and definitive criteria for placing a GST in a particular class. The extent of sequence identity from which to assign a GST to a given class varies in the literature from greater than 40% [3, 25], to greater than 60% [26]. GST with less than 30% [26] or 25% [3, 25] identity are taken to belong to separate classes. These criteria are typically met when mammalian GST are compared, but not with invertebrates enzymes. When  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Sigma GST from humans and rodents are compared, they present an overall identity of about 80% (Table 2). However, when compared to trematode GST, identity levels are much lower, presenting less than 30% of identity with S. mansoni, S. japonicum and C. sinensis Sigma GST. Identity levels are much higher when trematode Sigma GST are compared, varying from approximately 41% to 77% (Table 2), meaning that overall identity values for mammalian GST cannot be used to classify trematode GST. This was observed when we only compared signature motifs for Omega GST with F. hepatica Omega class enzymes. Overall, identity levels were low when compared to mammalian GST, (Table 2) but when compared to conserved motifs, identity levels were rose to 45%-65%, showing that trematode GST cannot www.proteomics-journal.com Animal Proteomics Proteomics 2006, 6, 6263–6273 6271 Table 2. Sequence identity matrix of F. hepatica GST from proposed Sigma and Omega classes obtained from the ‘in-house’ translated EST database compared to mammalian, helminth and insect GST from these two classes using ClustalW Sigma-class glutathione transferases Sequence Fhep GSTs Sman GST28 Sjap GST Csin GST28 Hsap PGD2 Mmus PGD2 Cele GST7 Asum GST1 Hcon GST FhepGSTs SmanGST28 SjapGST CsinGST28 HsapPGD2 MmusPGD2 CeleGST7 AsumGST1 HconGST ID 0.450 0.425 0.450 0.262 0.275 0.280 0.310 0.270 ID 0.770 0.430 0.300 0.290 0.300 0.300 0.320 ID 0.415 0.293 0.298 0.251 0.288 0.288 ID 0.273 0.264 0.240 0.282 0.259 ID 0.804 0.300 0.378 0.344 ID 0.300 0.364 0.330 ID 0.519 0.601 ID 0.475 ID Omega-class glutathione transferases Sequence Fhep 54b04 Fhep 49c06 Sman omega Hsap GST1 Mmus GST1 Cele P34345 Fhep54b04 Fhep49c06 Sman omega HsapGST1 MmusGST1 CeleP34345 ID 0.319 0.322 0.174 0.171 0.118 ID 0.278 0.096 0.093 0.093 ID 0.189 0.182 0.154 ID 0.721 0.318 ID 0.303 ID Figure 5. Immunoblot of 2-DE affinity purified F. hepatica GST. Affinity purified glutathione transferases were separated by 2-DE in 17-cm IPG strips pH 3-10NL (Bio-Rad). Second dimension was carried out in 12.5 % SDS-PAGE. Gels were subsequently transferred to PVDF membranes and incubated with anti-S. mansoni Mu class GST (A) and anti-H. contortus Sigma class GST (B) antibodies as described in Section 2. be excluded from a class when similarity is lower than 40% to mammalian GST. Additional evidence for Omega classification was found in the presence of the ‘Omega-typical’ Nterminal extension and catalytic cysteine. Other invertebrate and trematode sequences must be used for classification and conserved motifs and structures considered during analysis, so as not to exclude GST from classes based only on overall identity to mammalian GST. This method of  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim matching GST to conserved motifs within classes may be more robust than global alignment and overall identity percentages. Further characterization of F. hepatica purified GST by immunoblot analysis using antibodies raised against classspecific GST was unable to confirm the presence of the new liver fluke Sigma class GST. However, the relatively low identity shared between H. contortus Sigma GST and FhepGSTs (27%, Table 2) could be responsible for the weak recognition. However, three spots (6-8, Fig. 4B) identified as Mu GST were recognised by this antibody, suggesting that the use of classspecific polyclonal antibodies for class differentiation might be misleading. Spot 3, identified as FhGST51 was also recognised by the Sigma antibody, reflecting in part the relatively large amount of protein present in that spot, i.e. spots 1 and 2, identified as the same isoform, were not recognised. Moreover, anti-Mu class antibody raised against S. mansoni GST was not able to recognise all F. hepatica Mu class GST (Fig. 4A), even though they share more than 75% identity. Our data confirms that antibodies to generic GST classes are problematic in cross-species identifications. Highly specific monoclonal antibodies raised against GST class-specific epitopes, are probably required for confidently using immunology as a validation tool for GST classification. Sigma class GST have been strongly implicated in prostaglandin synthesis. A relationship between Sigma class GST of helminths and a GSH-dependent prostaglandin-H www.proteomics-journal.com 6272 G. Chemale et al. E-isomerase was revealed by comparison of partial sequence data [27]. This enzyme was later purified from the parasitic nematode Ascaridia galli and confirmed to be a Sigma class GST [28]. It is thought that this enzyme promotes the endogenous synthesis of prostaglandin E in multicellular parasites. The production of prostaglandins by these parasites could be an important component of their mode of subversion or suppression of host immunity [29]. The identification of this highly expressed GST in F. hepatica adults tentatively suggest that flukes could be using prostaglandin synthesis in such a way as to suppress the host’s protective immune response. Although only recently described as a new class of GST [30], Omega GST appears widespread in the animal kingdom. Human, pig, mouse, rat, C. elegans, S. mansoni, and D. melanogaster were all found, via bioinformatic analysis, to possess Omega GST [30, 31, 32, 33, 34, 35]. As sequence annotation progresses, further Omega GST continue to emerge, as for example here, in the parasitic liver fluke, F. hepatica. In humans, Omega GST have recently received much attention after their linkage to age onset of Alzheimer’s and Parkinson’s diseases [36], as well as roles in arsenic metabolism [37]. Other roles are also proposed; Human Omega GSTO1-1 has been shown to modulate ryanodine receptors, calcium channels in the endoplasmic reticulum [38] and was found to be up-regulated in radiationresistant cancer cell lines [33]. Sei et al. (1999) [39] and Xu et al. (1998) [40] propose [Ca2+] dependant immune modulation may also arise as a result of ryanodine receptor interaction in T and B lymphocytes. Newly uncovered F. hepatica cDNA belonging to Sigma and Omega class GST are currently being produced as recombinants in order to further characterise their biochemical properties and class-specific activities prior to challenge vaccinations. BBSRC and DEFRA UK for grant BB/C503638/1 to support Gustavo Chemale, and the EU (STREP DELIVER) for a grant to support James LaCourse are acknowledged. 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