Báo cáo khoa học: The secretory omega-class glutathione transferase OvGST3 from the human pathogenic parasite Onchocerca volvulus pot

OvGST3 from the human pathogenic parasiteOnchocerca volvulus Eva Liebau1, Jana Ho¨ppner1, Mareike Mu¨hlmeister1, Cora Burmeister2, Kai Lu¨ersen1, Markus Perbandt3, Christel Schmetz4, Die

OvGST3 from the human pathogenic parasite

Onchocerca volvulus

Eva Liebau1, Jana Ho¨ppner1, Mareike Mu¨hlmeister1, Cora Burmeister2, Kai Lu¨ersen1,

Markus Perbandt3, Christel Schmetz4, Dietrich Bu¨ttner4and Norbert Brattig4

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.

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 Km 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 D2synthase 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 ⁄ 1 to OvGST3⁄ 3 identified by Kampko¨tter et al [22] have a

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

NH2-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 sgenerat-ingle 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 (19.9 kDa),

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.

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 a2, 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

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.

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

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.

1 2 3 4 5

1

- 47.5

- 32.5

- 25.0

- 16.5

rOvGST3

- 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, 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.

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)1Æmg)1) and showed rather low

GSH conjugating activity towards

1-chloro-2,4-dinitro-benzene (CDNB) (113.8 ± 22.1 nmolÆmin)1Æmg)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

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.

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

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 treatmpres-ent (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.

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

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.