The secretory omega-class glutathione transferase OvGST3 from the human pathogenic parasite Onchocerca volvulus Eva Liebau 1 , Jana Ho ¨ ppner 1 , Mareike Mu ¨ hlmeister 1 , Cora Burmeister 2 , Kai Lu ¨ ersen 1 , Markus Perbandt 3 , Christel Schmetz 4 , Dietrich Bu ¨ ttner 4 and Norbert Brattig 4 1 Institute of Animal Physiology, University of Mu ¨ nster, Germany 2 Institute of Parasitology, Justus-Liebig-University, Giessen, Germany 3 Institute of Biochemistry, Center for Structural and Cell Biology, University of Lu ¨ beck, Germany 4 Bernhard Nocht Institute, Hamburg, Germany The glutathione S-transferases (GSTs) constitute a highly versatile superfamily that is thought to have evolved from a thioredoxin-like ancestor in response to the development of oxidative stress, sharing sequence and structural similarities with several stress-related proteins in a widespread range of organisms. Addition- ally, several GST-related proteins have been described, demonstrating that this ancient protein fold has been ‘recycled’ by nature for new functions, such as plant stress-induced proteins, bacterial stringent starvation Keywords glutathione S-transferase; nematode; Onchocerca; parasite Correspondence E. Liebau, Institute of Animal Physiology, University of Mu ¨ nster, Hindenburgplatz 55, Mu ¨ nster 48143, Germany Fax: +49 251 8321766 Tel: +49 251 8321710 E-mail: liebaue@uni-muenster.de Database Additional sequence data obtained in this study have been reported to GenBank. The original sequence data available under accession number AF203814 have been changed accordingly (Received 9 February 2008, revised 22 April 2008, accepted 1 May 2008) doi:10.1111/j.1742-4658.2008.06494.x Onchocerciasis or river blindness, caused by the filarial nematode Oncho- cerca volvulus, is the second leading cause of blindness due to infectious diseases. The protective role of the omega-class glutathione transferase 3 from O. volvulus (OvGST3) against intracellular and environmental reactive oxygen species has been described previously. In the present study, we con- tinue our investigation of the highly stress-responsive OvGST3. Alternative splicing of two exons and one intron retention generates five different tran- script isoforms that possess a spliced leader at their 5¢-end, indicating that the mechanism of mature mRNA production involves alternative-, cis- and trans-splicing processes. Interestingly, the first two exons of the ovgst3 gene encode a signal peptide before sequence identity to other omega-class gluta- thione transferases begins. Only the recombinant expression of the isoform that encodes the longest deduced amino acid sequence (OvGST3 ⁄ 5) was successful, with the purified enzyme displaying modest thiol oxidoreductase activity. Significant IgG1 and IgG4 responses against recombinantly expressed OvGST3 ⁄ 5 were detected in sera from patients with the general- ized as well as the chronic hyperreactive form of onchocerciasis, indicating exposure of the secreted protein to the human host’s immune system and its immunogenicity. Immunohistological localization studies performed at light and electron microscopy levels support the extracellular localization of the protein. Intensive labeling of the OvGST3 was observed in the egg shell at the morula stage of the embryo, indicating extremely defined, stage-specific expression for a short transient period only. Abbreviations CDNB, 1-chloro-2,4-dinitrobenzene; GSH, reduced glutathione; GST, glutathione S-transferase; mf, microfilaria; Ni-NTA, nickel–nitrilotriacetic acid; SL, spliced leader. 3438 FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS proteins, yeast nitrogen metabolism regulator URE2, the c-subunit of the elongation factor 1 or even ion channels [1]. A prominent catalytic activity of the GSTs is the conjugation of reduced glutathione (GSH) to numer- ous electrophilic substrates, usually promoting their inactivation, degradation and excretion. The family is characterized by a broad range of substrate specificity with low affinity K m values. This lower catalytic effi- ciency has probably been an integral part of the evolu- tion of GSTs as detoxifiers of a wide range of endogenous and environmental chemicals. Moreover, a growing number of nondetoxification functions have now been attributed to GSTs, essentially making them multifunctional enzymes, devoted to various aspects of cell defense. They participate in the catabolism of aro- matic amino acids, the synthesis of eicosanoids, the binding and transport of potentially toxic nonsubstrate molecules (ligands), the clearance of oxidative stress products and they can physically interact with kinases involved in signal transduction [2–5]. Evidence of func- tional flexibility can be found within one particular GST-class, as exemplified by the sigma-class, where members fulfill structural functions (e.g. S-crystallins) [6] or participate in prostaglandin synthesis [7]. Inter- estingly, comparative studies of free-living and para- sitic nematodes demonstrated prostaglandin synthesis activity only in sigma-class GSTs of parasites [8]. Whereas the model nematode Caenorhabditis elegans only has cytosolic sigma-class GSTs, the filarial para- site Onchocerca volvulus has the secreted form OvGST1 that acts as a prostaglandin D 2 synthase directly at the parasite–host interface, making an interception of the local immune response appear to be feasible [9,10]. Divergent preferences of ligands, such as hemin, have also been observed within the same GST-class of free- living and parasitic nematodes, with this function appearing to be an adaptation to parasitism or, specifi- cally, to blood feeding [11]. Similarly, kinetic and structural data obtained from the sole GST from the malarial parasite Plasmodium falciparum indicate that the enzyme optimized its binding property with the parasitotoxic hemin rather than its catalytic efficiency towards electrophilic compounds, possibly responding to specific evolutionary pressures [12,13]. Distinct from the prototypical tyrosine or serine resi- dues characteristic of other GST-classes, the omega- class has a cysteine residue in the active site that can form a mixed disulfide bond with GSH. It is therefore not surprising that the omega-class GSTs have a dis- tinct substrate profile, most notably GSH-dependent thiol-transferase and dehydroascorbate reductase activ- ity, reflecting their structural similarity to glutaredoxins [14]. Recently, their participation in the multistep bio- transformation of inorganic arsenic has been demon- strated and variations in the human omega-1 genes have been found that modify the age-at-onset of Alz- heimer and Parkinson diseases [15]. Other described functions of the omega-class GSTs include a modula- tion of ryanodine receptor calcium release channels [16], a participation in the post-translational processing of interleukin-1b in monocytes [17] and synthesis of an important intermediate in drosopterin biosynthesis [18]. A role of omega-class GSTs in the oxidative stress response has been shown [19,20], including studies of the omega-class GST from the human pathogenic fil- arial worm O. volvulus (OvGST3) [21,22]. In the pres- ent study, we continue our investigation of the OvGST3. Gene analysis identified an additional exon at the 5¢-end that encodes the first part of a signal peptide. Alternative splicing of two exons and one intron retention results in five different transcripts that have the spliced-leader (SL1) trans-spliced to their 5¢-end. To analyze the capacity of the secretory protein to stimulate host immune responses, the antibody responses of onchocerciasis patients against the recom- binant OvGST3 were studied. Immunohistological localization by light and electron microscopy demonstrates an intensive staining of the egg shell at the morula stage of the embryo, indicating defined expression for a short transient period only. Results and Discussion Genomic structure and alternative splicing of the OvGST3 The gene of the ovgst3 was isolated by screening an O. volvulus lambda Fix II genomic library. In addition to the previously described ovgst3 gene structure [22], 438 bp of 5¢-upstream region and one new exon, encoding the first part of a signal peptide, were identi- fied. The gene now consists of 2117 bp composed of eight exons and seven introns (Fig. 1). The nucleotide sequence at the splice junctions is consistent with the canonical GT-AG rule. The cDNA sequence confirms the intron–exon boundaries predicted from the geno- mic sequence. Interestingly, we discovered one cDNA where exon 5 was absent. Using 5¢ RACE, additional cDNA clones were obtained and a total of five differ- ent types of mRNA variants were detected. These were generated by exon skipping (‘alternative splice region’) (Fig. 1) and one intron retention (intron 5), with the potential to produce five different proteins (OvGST3 ⁄ 1–OvGST3 ⁄ 5). The three isoforms OvGST3 ⁄ 1to OvGST3 ⁄ 3 identified by Kampko ¨ tter et al. [22] have a E. Liebau et al. Omega-class glutathione transferase from O. volvulus FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS 3439 novel 5¢-exon previously not described. The isoform OvGST3 ⁄ 5 encodes the longest deduced amino acid sequence and most likely is the ancestral form because it is thought that, in multi-intron genes, constitutive splicing predates exon skipping [23]. Retention of intron 5 results in the inclusion of a premature termi- nation codon exon, potentially producing the truncated OvGST3 ⁄ 1. The isoforms OvGST3 ⁄ 2, OvGST3 ⁄ 3 and OvGST3 ⁄ 4 either do not include cassette exons 4 or 5 or both, respectively (Fig. 1). Up to now, scarce infor- mation about alternative splicing in GST genes is available; however, this mechanism is probably more prevalent than previously assumed. Unlike the situation observed for the ovgst3, spliced transcripts of other described GSTs appear to share the same N-terminus involved in glutathione binding, whereas splicing occurs in the C-terminal domain, conferring variation in substrate specificity and expanding the substrate range of the enzyme [24]. The most comprehensive structural and functional information about alternative spliced variants comes from the insect delta-class GSTs in the Anopheline mosquitoes. All alternative transcripts share a common NH 2 -terminal domain (exon 2), which is spliced to one of several alternative exons encoding variable COOH- termini to yield mature transcripts. The resulting alter- natively spliced products share high amino acid sequence identity but possess different catalytic efficiencies and substrate specificities. Splicing is an efficient means of expanding substrate diversity recog- nized by GSTs with a minimal increase in gene dupli- cation [25,26]. Recently, it has been shown that individual GST-isoforms from insects can differentially interact with components of the c-Jun N-terminal kinase pathway and their role as positive or negative regulators of signalling through this pathway is suggested [27]. Alternative splicing is a powerful mechanism generat- ing multiple forms of mRNA from a single gene and thereby expanding the diversity of expressed transcripts. The system either produces nonfunctional truncated proteins or proteins with altered regulation, distribution or physiological function. In an alternative mode, alter- native splicing can also function as an on ⁄ off switch by producing mRNA in which translation is suppressed due to the presence of a premature termination codon, such as the one observed in the OvGST3 ⁄ 1-mRNA. Blotting of O. volvulus homogenate followed by immun- odetection with affinity-purified anti-OvGST3 serum revealed a faint band of around 18 kDa only after pro- longed staining and it is not clear whether this protein is the OvGST3 ⁄ 1 (predicted molecular mass without signal peptide = 17.4 kDa), the OvGST3⁄ 3(19.9kDa), a proteolytic product or even a nonspecific cross- reacting antigen (data not shown). Therefore, it remains uncertain whether the OvGST3 ⁄ 1-transcript is AAAA AAAA AAAA AAAA AAAA OvGST3/5 OvGST3/1 OvGST3/2 OvGST3/4 OvGST3/3 E1 E1 E1 E1 E1 E2 E2 E2 E2 E2 E3 E3 E3 E3 E3 E4 E4 E4 E4 E5 E5 E5 E5 E6 E6 E6 E6 E7 E7 E7 E7 E8 E8 E8 E8 E1 E2 E3 E6 E7 E8 SLA SLB SLA SLA SLA SLA SLB * stop 23 bp 77 bp 133 bp 97 bp 123 bp 115 bp 158 bp 72 bp 372 bp 217 bp 97 bp 216 bp 63 bp 133 bp 221 bp 122 bp 438 bp Alternative splice region Fig. 1. Schematic diagram of alternative splicing in the ovgst3 gene. Schematics illustrate the exon and intron organization of the ovgst3 gene, the location of the ‘alternative splice region’ (exon 4 and 5) and five different isoforms. Exons are shown as numbered boxes. The five different transcripts (OvGST3 ⁄ 1–OvGST3 ⁄ 5) obtained, possess the spliced leader sequence at their 5¢ end, indicating that the mechanism of mature mRNA production involves both cis- and trans-splicing processes. SLA indicates the acceptor site located 119 nucleotides from the start codon ATG, SLB is an alternative acceptor site located 30 nucleotides downstream of the acceptor site SLA. Retention of intron 5 intro- duces a stop codon (marked by the asterisk) in the ORF, leading to a premature termination of translation. Omega-class glutathione transferase from O. volvulus E. Liebau et al. 3440 FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS translated into a truncated protein or is a candidate for nonsense-mediated mRNA decay. In addition to its role in eliminating faulty transcripts and thereby pre- venting the accumulation of truncated and potentially toxic protein fragments, nonsense-mediated mRNA decay also has a role in controlling gene expression and is implicated in several essential physiological pro- cesses [28–30]. Protein sequence analysis An alignment with representative sequences from dif- ferent GST classes (data not shown) was used to generate a phylogenetic tree, clearly grouping the OvGST3 into the omega-class (Fig. 2A). The OvGST3 ⁄ 5 was aligned with the omega-class GST1 from human (GSTO1) (AF212303) demonstrating approximately 30% identity. Residues that contribute to the binding of GSH, as described for the human omega-class GST, are either conserved or conserva- tively replaced. Another distinguishing feature is the active site cysteine (Cys33). The first two exons of the ovgst3 gene encode a signal peptide before sequence identity to other omega-class GSTs begins. The pre- dicted cleavage site and the start of the mature protein lies between amino acid residues Ala20 and Ile21 (Fig. 2B). Even though the human GSTO1 does not have a signal peptide, the enzyme was recently found in spu- tum supernatant, whereas intracellular markers were negative. This demonstrates that GSTO1 is excreted into airway secretions, where its role in the mainte- nance of GSH homeostasis in the extracellular space is postulated [31]. To obtain a clearer picture of the potential effects of splicing on protein structure, a model of the OvGST3 ⁄ 5 was generated based on the structure of the human omega-class GSTO1 (Fig. 3). In general, the principal isoform OvGST3 ⁄ 5 consists of two domains that are linked by a loop between helices a3 and a4. The N-terminal thioredoxin-like domain har- bours the glutathione-binding (G)-site. The G-site is formed by helix a2, by residues connecting helix a2 and strand b3 and by a segment connecting strand b4 to helix a3. The C-terminal domain is largely a-helical and consists of five a-helices that are connected by a variety of loops. Based on the full-length model, we have deduced models of the observed splice variants (Fig. 3A–D). Translation of the OvGST3 ⁄ 1 transcript leads to a truncated protein with questionable conformation of a4 (Fig. 3A). Whereas the N-terminal thioredoxin- like domain is maintained, the complete C-terminal domain with the exception of a4 is lost. In the iso- form OvGST3 ⁄ 2, helices a3, a4 and b4 are missing (Fig. 3B) and loss of exon 4 leads to the alternative isoform OvGST3 ⁄ 4, lacking b2, a2 and b3 (Fig. 3C). The lack of the fragment encoded by exons 4 and 5 forces the most drastic changes in structure and results in the protein product OvGST3 ⁄ 3 missing a 2, b2, b3, b4, a3 and a4 (Fig. 3D). Removing these important secondary structures would certainly affect folding and especially function because the G-site is destroyed. In general, GSTs are biologically active as homodi- mers. The interactions occurring at the intersubunit interface of the homodimers are dominated by hydro- phobic interactions between residues from domain 1 of one subunit and domain 2 of the other. Because many subunit interface residues are located in a4, a5 and b3, exon 4 and ⁄ or exon 5 deletions will break the con- served subunit interactions at the dimer interface area. Accordingly, none of the truncated splicing forms will have the ability to form intact dimers. Although it has been possible to confirm the ovgst3-splice variants at transcript level, it is impor- tant to analyze whether these splice variants are actually translated into proteins or whether isoforms with extreme deletions are misfolded and quickly degraded. To obtain evidence at the protein level, western blot analysis of homogenate of adult O. volvulus was carried out using affinity-purified anti-OvGST3 ⁄ 5 (Fig. 4B,C). Surprisingly, only one dominant isoform of approximately 30 kDa was observed, corresponding to the long isoform OvGST3 ⁄ 5. Whereas western blotting revealed signifi- cant expression of the principle splice isoform OvGST3 ⁄ 5 in adult female worms, only minor levels were detected in adult males (Fig. 4C). This result is in good agreement with immunolocalization of the OvGST3, where intensive staining is observed in the egg shell (Figs 6 and 7). The ‘alternative splice region’ of the ovgst3, com- prising both exon 4 and 5 (Figs 1 and 2b), is almost identical to exon 4 of the human omega-class GST2 (gsto2). Pronounced skipping of exon 4 is the only observed alternative splicing difference, affecting GSTO2-transcripts. Calarco et al. [32] demonstrated that GSTO2 transcripts that include or skip exon 4 have similar stabilities. However, transient expression in HeLa cells resulted in minor protein levels of the exon 4-skipped splice variant, indicating that skipping leads to expression of an unstable protein. Levels of active GSTO2 are thus determined by expression of the exon 4-containing splice variant. Interestingly, in chimpanzees, skipping of exon 4 E. Liebau et al. Omega-class glutathione transferase from O. volvulus FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS 3441 is not so pronounced, resulting in species-specific differences in the expression of the active splice vari- ant of GSTO2 [32]. Because no animal host has been identified that can be used to provide the various stages of O. volvulus in quantity (e.g. in particular, the infectious larvae can A B Fig. 2. Phylogenetic analysis of the OvGST3. (A) After CLUSTALW multiple alignment of the indicated GST proteins (data not shown), sequences were adjusted manually using BIOEDIT, version 5.0.9 [55] and phylogenetic relationships were estimated using MEGA, version 3.1 [56]. The accession numbers of the compared proteins are: Arabidopsis thaliana (A.t.ph, CAA72413), Caenorhabditis elegans (C.e.o, NP_498728; C.e.z, CAA91449), Drosophila melanogaster (D.m.d, NP_524326), Homo sapiens (H.s.a, AAB24012; H.s.m, AAA60963; H.s.o, AAF73376; H.s.pi, NP_000843; H.s.t, NM_000854; H.s.z, AAC33591), Musca domestica (M.d.d, CAA43599; M.d.s, AAA03434), Mus muscu- lus (M.m.a, AAI32577; M.m.m, P10649; M.m.o, NP_034492; M.m.t, CAA66666), Nostoc punctiforme (N.p.l, ZP_00105965), Ommastrephes sloani (O.s.s, M36938); Onchocerca volvulus (O.v.o, AAF99575; O.v.pi, P46427; O.v.s, AAG44696), Ostreococcus tauri (O.t.l, CAL49924), Petunia · hybrida (P.h.ph, CAA68993), Rattus norvegicus (R.n.pi, AAB59718). (B) Sequence alignment of OvGST3 ⁄ 5 from O. volvulus and the human omega-class hsGSTO1. Residues of the assumed active site are shown underlined and in bold. Residues that are identical are contained in black boxes and are indicated by an asterisk (*), whereas sequence similarity is indicated by a colon (:). Gaps indicated by a dash were introduced to optimize the alignment. The secondary structural elements a-helices are colored red and b-strands are in blue. Arrows indicate positions of exons and intron–exon boundaries. The putative signal peptide (italics) is based on prediction made by SIGNALP software, with the proposed cleavage site between amino acid residues Ala20 and Ile21. Omega-class glutathione transferase from O. volvulus E. Liebau et al. 3442 FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS only be obtained by dissecting infected blackflies), it is not possible to perform western blots of different developmental stages of O. volvulus. Furthermore, this inaccessibility of O. volvulus means that in vitro inves- tigations of stress-responsive genes at the protein level cannot be performed, comprehensive studies of the oxidative stress-response are unfeasible and partial purification of possibly existing low-abundant isoforms is impossible. Therefore, questions regarding possible stage- or stress-regulated OvGST3-isoform expression cannot be settled conclusively. AB CD Fig. 3. Ribbon presentation of a three dimensional model of the OvGST3 ⁄ 5. The model is based on the structure of the human omega-class GSTO1 (protein databank code 1EEM) with a-helices colored red and b-strands in blue. The N to C direction of the struc- tural elements can be deduced by the labeling of the secondary structures. The four splice isoforms (A, OvGST3 ⁄ 1; B, OvGST3 ⁄ 2; C, OvGST3 ⁄ 4; D, OvGST3 ⁄ 3) are mapped onto the OvGST3 ⁄ 5 iso- form. Deletions in the splice isoform are shown in green. It is likely that splicing will cause the structures (A–D) to fold in a substantially different fashion. 12345 12 3 45 1 - 47.5 - 32.5 - 25.0 - 16.5 23 r OvGST3 - rOvGST3/5 A B C Fig. 4. Characterization of recombinant OvGST3 ⁄ 5 and affinity puri- fication of anti-OvGST3 serum. (A) Bottom panel: Coomassie-stained SDS ⁄ PAGE [12.5% (w ⁄ v) gel]; top panel: corresponding western blot probed with affinity-purified anti-OvGST3 ⁄ 5. Supernatant- (lane 1) and pellet-fraction (lane 2) of Escherichia coli BLR DE3 containing pJC40-OvGST3 ⁄ 5. Lane 3, flow-through from the nickel-affinity chromatography after loading the E. coli supernatant, followed by NTA-purification step (lane 4) and gelfiltration (lane 5). (B) The obtained anti-OvGST3 antibody was purified by affinity chromatogra- phy using OvGST3 ⁄ 5 immobilized on CNBr-activated Sepharose 4B. Western blot of extract of E. coli overexpressing OvGST3 ⁄ 5. Lane 1, anti-OvGST3 prior to affinity purification; lanes 2–5, eluted anti- body fractions; only fractions 6 ⁄ 7 (lane 5) were used for western blot and immunolocalization experiments. (C) Immunoblot showing the abundance of OvGST3 in male and female O. volvulus homo- genate. Lanes 1 and 2, 100 lg of female and male worms, respectively; lane 3, lysate of E. coli Origami DE3 containing pJC40- OvGST3 ⁄ 5 as a control. Immunodetection was carried out using fractions 6 ⁄ 7 of the affinity-purified OvGST3-antibody. E. Liebau et al. Omega-class glutathione transferase from O. volvulus FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS 3443 Expression of the recombinant OvGST3 ⁄ 5 and substrate specificities To investigate the enzymatic characteristics of the OvGST3 ⁄ 5, the enzyme was expressed in Escherichia coli using various vectors containing different con- ventional affinity tags and fusion partners. In all host systems used, the recombinant protein accumulated intracellularly in insoluble aggregates (Fig. 4A, lane 2). The addition of 1% Triton X-100 to the lysis buffer improved the extraction of soluble rOvGST3 ⁄ 5. Due to the insolubility of the enzyme, purification of the recombinant OvGST3 ⁄ 5(rOvGST3 ⁄ 5) was difficult. Even though expression of rOvGST3 ⁄ 5 with the fusion partner maltose-binding protein resulted in enhanced yields and increased solubility, subsequent site specific proteolysis to remove the fusion partner resulted in almost immediate protein aggregation and loss of yield (data not shown). Therefore, a modified protocol for autoinduction of protein expression was used and approximately 0.4 mg of rOvGST3 ⁄ 5 was purified from 1 L liquid culture, using conventional nickel– nitrilotriacetic acid (Ni-NTA) affinity purification (Fig. 4A, lane 4). Unfortunately, due to their insolubil- ity, purification of the other recombinant OvGST3 isoforms has not been achieved under native conditions, and their biological function remains speculative. To identify catalytic activities that may reveal the biological function of the OvGST3 ⁄ 5, the substrate specificity of the recombinant enzyme with a broad range of substrates was determined. Elimination of the His-tag by factor Xa did not influence enzyme activity. The purified enzyme was able to use GSH as an elec- tron donor to reduce hydroxyethyl disulfide (57.9 ± 11.7 nmolÆmin )1 Æmg )1 ) and showed rather low GSH conjugating activity towards 1-chloro-2,4-dinitro- benzene (CDNB) (113.8 ± 22.1 nmolÆmin )1 Æmg )1 ). There was no detectable activity with the substrates dimethylarsenic acid, S-(4-nitrophenacyl)glutathione and cumene hydroperoxid (data not shown). The omega-class GST has a cysteine residue in the active site that can form a mixed disulfide bond with GSH. Therefore, conjugating reactions with GSH can only be performed if the disulfide bond is not formed or broken down in the catalytic mechanism. The low CDNB-conjugating activity observed for the OvGST3 should thus be interpreted with caution because it might also be due to the active site cysteine rapidly reacting with CDNB. The enzymatic activities observed in the present study are in contrast to the findings of Kampko ¨ tter et al. [22] who designed a recombinant protein short of seven amino acids at the N-terminus. Furthermore, Kampko ¨ tter et al. [22] dem- onstrated that the OvGST3 reacts with trans-2-none- nal, possibly indicating an involvement in the elimination of end products of lipid peroxidation. The thiol oxidoreductase activity is reminiscent of glutaredoxins and also characteristic for the omega- class, where dethiolation of specific S-glutathionylated proteins that accumulate under stress conditions has been proposed as a possible function, with the open and not particularly hydrophobic H-site being large enough to accommodate protein substrates [33]. Because the OvGST3 is dramatically up-regulated at the steady-state transcription level in response to oxi- dative stress and reacts sensitively to alterations in redox status [21,22], a role of the enzyme in reversible S-glutathionylation and glutathione-mediated redox regulation of proteins is feasible. Antibody response to the secretory OvGST3 ⁄ 5 The mechanism by which helminths down-regulate host immunity at the molecular level is the subject of intense research. Immunologists have focused on excre- tory–secretory products and surface molecules because these have the capacity to actively shape the immuno- logical environment. In the present study, we investi- gated whether the secretory OvGST3 is recognized by antibodies generated in patients infected with O. vol- vulus. We studied the reactivities of IgG1 and IgG4 by ELISA applying sera from 117 patients with onchocer- ciasis, including 77 patients with the hyporeactive gen- eralized form and 40 patients with the chronic hyperreactive form (also designated as sowda). Signifi- cantly elevated IgG1 and IgG4 titers (P < 0.001) were found on comparing the reactivitity of the patient sera with those from 20 healthy Europeans as a control (Fig. 5A). As a positive control for OvGST3, we included another O. volvulus antigen, the fatty acid- and retinol-binding protein Ov20, which is strongly immunogenic [34]. In comparison to the very high IgG1 and IgG4 reactivities with the Ov20 antigen, the responses against OvGST3 were significantly lower (P < 0.0001). With regard to the IgG1 and IgG4 reactivities in subgroups of the onchocerciasis patients, we found modest higher IgG1 titers in sera from generalized patients with high microfilaria (mf) density as well as with the hyperreactive form compared to patients with the generalized form and low mf numbers (P < 0.017 and P < 0.033, respectively) (Fig. 5B). The IgG4 titer for the patients with the generalized form and high mf density showed significantly higher reactivity (P < 0.007) compared to the hyperreactive form of onchocerciasis. Omega-class glutathione transferase from O. volvulus E. Liebau et al. 3444 FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS These results correspond to earlier observations, where high IgG1 levels to O. volvulus antigens were found predominantly in patients with high mf densities who were exposed to higher levels of filarial antigens and in patients with the chronic hyperreactive form; in the present study, IgG4 levels were lower compared to patients with a high mf load [34–36]. These findings indicate an exposure of the human immune system to the secretory OvGST3 antigen. The resulting antibody profile is characteristic of the varying forms of oncho- cerciasis that reflect different immune states. In the present study, OvGST3 was shown to be an antigen of low immunogenicity, comparable to the results obtained for other enzymatic antioxidants from O. volvulus such as the superoxide dismutase 1 (OvSOD1) or the OvGST2 [37]. Immunolocalization studies clearly show a short developmental stage-specific expression of the OvGST3 and a major localization in the egg shell. O. volvulus completes embryogenesis and the larvae hatch and leave the egg shell before leaving the maternal uterus. How- ever, uterus fluid is continuously released by female worms. Furthermore, there is a turnover in adult worm populations and proteins are exposed when the adult worm dies and degenerates. The restricted antibody response to the OvGST3 might therefore be due to the limited presence of the OvGST3 in the external environ- ment of the parasite or due to low immunogenicity. Immunohistological localization by light and electron microscopy We used immunohistochemistry to determine the stage- and tissue-specific distribution of the unusual secretory omega-class OvGST3. Using the 1 : 100 or 1 : 250 diluted yolk collected before immunization, no staining of any tissue of female or male O. volvulus was detected. The preimmune yolk did not contain any antibodies against O. volvulus. Following immuniza- tion, strong staining of the egg shells around morulae was seen. This staining was almost completely removed following absorption of the antibodies using rOvGST3. This indicated the high specificity of the antibodies for OvGST3 [38]. For further analyses, the pooled frac- tions 6 ⁄ 7 of the affinity purified antibodies were used (Fig. 4B). Strong staining was observed in the egg shells surrounding several stages of the developing embryos in the uterus of worms (Fig. 6). Oocytes in the ovary and oocytes or zygotes in the uterus were negative (Fig. 6A,B). Weak staining was first seen in young morulae (i.e. the stage where the egg shell first appears) (Fig. 6B). The staining intensity increased Fig. 5. IgG1 and IgG4 responses of patients with generalized and hyperreactive onchocerciasis to recombinantly expressed OvGST3 ⁄ 5. (A) Endpoint titers for IgG1 and IgG4 reactivities in sera from 117 patients with onchocerciasis (Ov) with OvGST3 ⁄ 5 and Ov20 compared to 20 healthy European controls (EC). Significant differences (P < 0.0001) in the titers were found for all patients groups compared to the control sera as well as between the titers for OvGST3 and Ov20 in the respective groups. (B) Comparison of the serum titers found for the patients with the generalized form of onchocerciasis and low mf density (1Mf l), high Mf density (Mf h), the hyperreactive (sowda) form (Sow) and healthy controls (EC) in response to OvGST3 ⁄ 5. The P-values for IgG1 were between 0.017 comparing patients with high and low mf densities and 0.033 comparing patients with low mf density and chronic hyperreactive onchocerciasis, respectively, indicating weak differences (P < 0.05 when corrected for multi-comparison). When comparing the IgG4 response of the generalized form showing high mf densities with the sowda form, P = 0.007. E. Liebau et al. Omega-class glutathione transferase from O. volvulus FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS 3445 with the development of the morulae as long as the shell was attached to the embryo (Figs 6C and 7A–D). The egg shells of coiled and stretched mf and those from which the mf had hatched, were distinctly but less intensively stained (Figs 6D,E and 7E,F). This staining pattern was also observed in female worms from four other species of the genus Onchocerca but not in five species belonging to other genera of the family Onchocercinae [38]. Degenerating embryos showed stronger staining of the egg shell than normal mf (Fig. 6D). This is best observed in the degenerated embryos following antifi- A B C D E G H F Fig. 6. Lightmicroscopic immunolocalization of OvGST3 within the egg shell of embryos in the uterus of O. volvulus. (A–E) Untreated patients. (A) Oocytes in the ovary are not labeled (arrow). (B) Oocytes or zygotes in the uterus are negative (arrow), whereas the egg shells of young morulae are weakly labeled (arrowheads). (C) Mature morulae show strongly labeled egg shells (arrowheads). (D) The shells of coiled microfilaria (mf) are still slightly labeled (arrow) and those of degenerating embryos are more strongly labeled (arrowheads). The mf are negative. (E) The mf are negative (arrow) but some still show well labeled shells (arrowheads). (F) Whereas degenerated morulae pres- ent strongly labeled shells (arrowheads), oocytes or zygotes are negative (arrows). Ten months after 4 weeks of doxycycline treatment. (G) Labeling of the shells of young morulae (arrow) and stronger labeling of degenerating mature morulae (arrowheads). Six weeks after suramin treatment. (H) The shells of normal coiled mf are slightly labeled and the mf are negative (arrow), whereas the degenerated stretched mf are strongly labeled (arrow heads). Typical finding 2 months after ivermectin treatment. The hypodermis and the epithelia of ovary and uterus are negative. Immunostaining using fraction 6 ⁄ 7 (diluted 1 : 20) of the purified antibody against OvGST3. Scale bar = 40 lm. Omega-class glutathione transferase from O. volvulus E. Liebau et al. 3446 FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS larial treatment with doxycycline or suramin (Figs 6F,G) or following a single dose of ivermectin, mainly causing degeneration of the stretched mf (Fig. 6H). The tissues of male and female worms were usually not, or only weakly, stained (Figs 6 and 7), and the sperms never labeled. Using light microscopy, some worms showed staining of the hypodermis, the epithe- lia of uterus and intestine and the afibrillar inner portions of the muscles [38]. Using electron micro- scopy, we did not find labeling of the morulae (Figs 7B,D) or the uterus epithelium adjacent to the egg shell, making prediction of the production site of the OvGST3 impossible. Using light microscopy, we observed distinct labeling of the outer cells of the morulae (Fig. 6C); however, because this finding is not supported by electron microscopy, it may also be an artifact. In conclusion, the immunohistological examinations showed specific labeling of the OvGST3 in the egg shell of developing embryos of O. volvulus. The staining appeared to be stronger in the shells of degenerating untreated and drug-treated embryos. The extracellular environment is highly oxidizing and, unsurprisingly, most secreted surface proteins are rich in disulfides. The maintenance of a reduced state of surface thiols requires protein disulfide oxidoreduc- tase and also GSH [39]. It is conceivable that surface thiols of the egg shell are early targets of oxidative stress. This is particularly evident for short-lived oxi- dants and those that cannot easily permeate into the cells. Because their location makes them particularly sensitive to extracellular oxidants, egg shell proteins might play a key role as sensors that signal any changes in redox state to the embryo as it moves forward to the proximal part of the uterus. In this respect, a potential A B D C F E Fig. 7. Electron microscopic localization of OvGST3 within the egg shell of embryos in the uterus of O. volvulus. (A, B) Morula with an egg shell that is well labeled (arrows in B). (C) Microfilaria with a well labeled egg shell (arrows). The mf and the epithelium of the uterus is negative. (D) Degenerated morula cell with well labeled shell (arrows). (E, F) Negative uterus epithelium and well labeled shell (F, arrows) shed by stretched mf. Immunogold labeling using fraction 6 ⁄ 7 (diluted 1 : 500) of the purified antibody against OvGST3. ba, endobacterium; mf, microfilaria; mo, morula; ut, uterus. (A–E) Scale bar = 1 lm. (F) Scale bar = 0.5 lm. E. Liebau et al. Omega-class glutathione transferase from O. volvulus FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS 3447 [...]... (2003) A dominant role for extracellular glutathione S -transferase from Onchocerca volvulus 14 15 16 17 18 19 is the production of prostaglandin D2 Infect Immun 71, 3603–3606 Perbandt M, Hoppner J, Burmeister C, Luersen K, ¨ ¨ Betzel B & Liebau E (2008) Structure of the extracellular glutathione S -transferase OvGST1 from the human pathogenic parasite Onchocerca volvulus J Mol Biol 377, 501–511 van Rossum... of the OvGST3 ⁄ 5 For illustrative purposes, a 3D model of the OvGST3 ⁄ 5 was generated based on the crystal structure of the homologous structure of human omega-class GST (protein databank code 1EEM) The modeling followed a standard stepwise procedure, starting with an alignment of the target sequence onto the template structure The software used (modeller 9v2) takes the structural features of the. . .Omega-class glutathione transferase from O volvulus E Liebau et al role of the OvGST3 in the regulation of exofacial protein function is feasible Changes in the outside environment might be caused by drug treatment with doxycycline, ivermectin and suramin, leading to degenerate embryos and a more pronounced staining due to the induction of the highly stress-responsive OvGST3 The nematode... Eindhoven, The Netherlands) Pre-immune egg yolk extract and antibodies against heat shock protein 60 were used as negative controls Omega-class glutathione transferase from O volvulus 10 11 Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG project Li 793 ⁄ 5-7) We are obliged to Dr Kwablah Awadzi for the onchocercomas from suramin-treated patients We acknowledge the technical... use glucose and, when it is exhausted, lactose can enter the cell and induce expression of the T7 polymerase Preliminary results obtained with small-scale cultures demonstrated Omega-class glutathione transferase from O volvulus the best yields of recombinant OvGST3 ⁄ 5 using the vector pJC40 and BLR DE3 E coli Briefly, a 1 mL seed culture of the transformed cells was grown overnight and transferred... Identifica¨ tion of a stress-responsive Onchocerca volvulus glutathione S -transferase (OvGST-3) by RT-PCR differential display Mol Biochem Parasitol 109, 101–110 22 Kampkotter A, Volkmann TE, de Castro SH, Leiers B, ¨ Klotz LO, Johnson TE, Link CD & Henkle-Duhrsen K ¨ (2003) Functional analysis of the glutathione S -transferase 3 from Onchocerca volvulus (Ov-GST-3): a parasite GST confers increased resistance... Pi was added at twice the volume of the yolk, followed by addition of PEG 6000 (Roth, Karlsruhe, Germany) at 3.5% and incubation FEBS Journal 275 (2008) 3438–3453 ª 2008 The Authors Journal compilation ª 2008 FEBS 3449 Omega-class glutathione transferase from O volvulus E Liebau et al of the mix for 30 min at 4 °C under rotation Following centrifugation at 21 374 g for 10 min, the supernatant was filtered... interassay variations less than 10% were accepted The results were expressed as endpoint titers in arbitrary units (U) [35] Statistical analysis was performed using the Mann–Whitney U-test for comparison of the titers obtained for the tested group of individuals and the Wilcoxon signed-rank test was applied for the comparison of the titers for the two antigens tested The Bonferroni correction was applied for... extensively cross-linked [43] Investigations of the homologous omega-class GSTO3 from C elegans are under way to analyse how the enzyme is involved in the complex modifications, folding and processing of the egg shell the generalized form of onchocercerciasis, living in endemic villages in Liberia (n = 24) or Uganda (n = 53) and from 40 patients with the chronic hyperreactive form (sowda) of onchocerciasis... and stored in liquid nitrogen Sera were collected from 77 patients with 3448 The entire protein-encoding cDNA of the OvGST3 (accession number AF203814) [19] was used to screen a lambda Fix II genomic library made from adult female worms The DNA from ten positive phages was analyzed by restriction enzyme fragmentation and Southern blot hybridization using the same screening probe Hybridizing fragments . The secretory omega-class glutathione transferase OvGST3 from the human pathogenic parasite Onchocerca volvulus Eva Liebau 1 ,. Liebau E (2008) Structure of the extracellu- lar glutathione S -transferase OvGST1 from the human pathogenic parasite Onchocerca volvulus. J Mol Biol 377,