Characterization of an omega-class glutathione
S
-transferase
from
Schistosoma mansoni
with glutaredoxin-like
dehydroascorbate reductase and thiol transferase activities
Javier Girardini
1,
*, Alejandro Amirante
2,†
, Khalid Zemzoumi
1
and Esteban Serra
1
1
Instituto de Biologı
´
a Molecular y Celular de Rosario, IBR-CONICET, Facultad de Ciencias Bioquı
´
micas y Farmace
´
uticas, UNR;
and
2
Facultad de Odontologı
´
a, UNR, Rorario, Argentina
Glutathione S-transferases (EC 2.5.1.18) (GSTs), are a
family of multifunctional enzymes present in all living
organisms whose main function is the detoxification of

compounds including carcinogens, drugs and oxidative-
stress metabolites [1]. Several additional functions were
attributed to GSTs including the transport of hydrophobic
ligands, binding to bilirubin and carcinogens [3,4], the
isomerization of maleylacetoacetate and the regulation of
stress kinases and apoptosis [5,6].
Based on their sequence structure, catalytic activitiy,
immunogenicity, substrate specificity and sensitivity to
inhibitors, the mammalian GSTs form six evolutionary
distinct classes termed alpha, kappa, mu, pi, sigma, and zeta
[1,7,8]. A new class of the GST superfamily, designated GST
omega (GSTO) in accordance with the established human
GST nomenclature convention [9], has been recently
characterized in humans on the basis of structural data
[10]. This new enzyme (GSTO1-1) has similar functional
characteristics with previously described proteins in rats [11]
and mouse [12]. Although the mammalian GSTO has low
sequence similarity to other known GSTs, its crystallo-
graphic structure showed a GST fold composed of an
N-terminal glutathione-binding domain and a C-terminal
domain composed entirely of a-helices. In contrast, unlike
other GSTs, GSTO has an active site cysteine that is able to
form a disulfide bond with GSH and exhibits glutathione-
dependent thiol transferase (TTase) and dehydroascorbate
reductase (DHA) activities, reminiscent of thioredoxin (Trx)
and glutaredoxin (Grx) enzymes [10]. Recent studies have
shown new additional functions for human GSTO including
monomethylarsonic acid (MMA) reductase activity and the
modulation of ryanodine calcium channels [13,14]. A
particular member of the GST superfamily, designated

(Received 8 July 2002, revised 3 September 2002,
accepted 11 September 2002)
Eur. J. Biochem. 269, 5512–5521 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03254.x
At least five GST activities have been described in the
human parasite Schistosoma mansoni (SmGST-1 to
SmGST-5). SmGST-5 has been characterized as an
unstable enzyme that may be involved in the conjugation
of epoxide substrates and dichlorovos, the active form of
the anti-schistosomal drug metrifonate, but no corres-
ponding parasite gene has been cloned to date [21–23].
Two members of SmGSTs, 28-kDa Sm28GST and
26-kDa Sm26GST, have been cloned and were found to
correspond to the previously reported SmGST-1, -2, -3
and -4 isoenzymes, respectively [22,24]. Although DHA
activity was first described in plants several years ago, and
then in mammals, insects and protozoans, little is known
about nonvertebrate GSH-dependent DHA proteins at
the molecular level.
We report here the characterization of a new member
of the GST superfamily from S. mansoni. When compared
with other GSTs, S. mansoni protein showed a limited
sequence identity with omega-class GSTs. Additional
phylogenic analysis, including known GSTs classes and
S. mansoni GSTs, allowed us to place the new parasite
product among the newly identified GSTO class, and the
previously characterized Sm28GST and Sm26GST as
mu- and sigma-class, respectively. Additional evidence
placed the S. mansoni protein among the omega class of
GSTs, as the recombinant parasite protein (a) did not
have significant affinity for glutathione, but bound

lambda gt10 cDNA library by conventional methods [26].
Southern blot analysis was performed using S. mansoni
cercariae purified DNA as described [26].
Alignments and phylogenetic analysis
of
S. mansoni
GSTs
Sm26GST, Sm28GST and SmGSTO amino acid sequences
were aligned manually on the alignment provided by
L. Jermiin (see [10]). From the original alignment used by
Board et al. [10] eight not clearly class-defined sequences
were avoided. A phylogenetic tree was obtained by
maximum likelihood analysis of all the sites in the above-
mentioned alignment. The data was analyzed using the JTT-
F substitution model [27], and local bootstrap probabilities
were estimated for the internal branches using the
PROTML
program [28]. More than one analysis was performed by
using different input order of the sequences. Each analysis
involved two steps: stepwise addition and nearest neighbor
interchanges. The most likely tree was obtained by using the
test of Kishino and Hasegawa [29]. All calculations were
performed using the
MOLPHY
2.3 molecular phylogenetics
programs package [28].
Expression and purification of recombinant SmGSTO
Recombinant SmGSTO was expressed in Escherichia coli
and purified by two separate methods using either the
pQE30 vector (Qiagen) and nickel agarose affinity chroma-

GCACCTTAAACGAAATGACC-3¢; antisense primer
odTSalI) and cloned between the NdeIandSalIsitesof
the expression vector pT7-7. Protein was purified from
soluble extracts on S-hexyl glutathione agarose (Sigma) as
previously described [31]. The enzyme was eluted with 5 m
M
S-hexyl glutathione, 50 m
M
Hepes, pH 8, and dialyzed
against 50 m
M
Hepes, pH 8.0, before storage. Purification
yield approximately 500 lg of protein per milliliter of
S-hexyl glutathione agarose. In all cases, protein purity was
determined by SDS/PAGE and protein concentration was
measured by bicinchoninic acid method following manu-
facturers indications (Sigma). Antiserum against the puri-
fied protein was raised in rabbits using standard
immunization protocols.
Glutathione and S-hexyl glutathione affinity assays were
performed in batch. Briefly, 20 lL of 50% resuspended
Ó FEBS 2002 Omega-class GST from Schistosoma mansoni (Eur. J. Biochem. 269) 5513
resin was centrifuged and the supernatant was eliminated.
Ten microliters of 1 mgÆmL
)1
purified enzyme was added to
the same tube and incubated in ice with gentle agitation.
After 30 min the supernatant was recovered by centrifuga-
tion. The resin was washed four times with 250 lLof
50 m

membrane (Amersham Pharmacia). Western blot experi-
ments were carried out according to standard techniques.
Ten micrograms of DNAse I-treated total RNA from
S. mansoni miracidia, sporocyst, cercariae and adult worm
were reverse transcribed by using 100 U of SuperScript
TM
reverse transcriptase (Life Technologies) in 50 lLof
supplied reaction buffer. PCRs were performed on 1 lL
of each reverse transcription reaction and resolved by
agarose gel electrophoresis. Primers used were: GSTX1/
GSTX3 for SmGSTO and TUB3 (5¢-GAAGTGGAT
ACGAGGATAAGGTACCAG-3¢)/TUB4 (TGGAACTT
ATCGTCAACTTTTCCATCC-3¢)forS. mansoni a-tubu-
lin. SmGSTO amplification bands were quantified by using
GelPro and normalized by comparing to a-tubulin ampli-
fication products.
Enzyme assays
Enzymatic activity towards a range of substrates and
inhibitors was determined as described [32]. Thiol trans-
ferase activity was measured according to Axelsson et al.
[33] using HEDS as substrate. The reaction mixture
contained 0.2 m
M
NADPH, 0.5 m
M
GSH, 50 m
M
phos-
phate buffer, 0.5 units of glutathione reductase and an
aliquot of the protein solution. The reaction was initiated by

and used to obtain an optimized model. The WhatCheck tool
(from the
WHAT IF
package program) was used to estimate
accuracy of the structure obtained. Parameters taken into
account were: Ramachandran plot appearance Z-score, chi-
1/chi-2/Z-score, packing quality Z-score and RMS Z-scores
( />RESULTS
SmGSTO DNA and protein sequence
BLAST search of the S. mansoni EST database with the
complete sequence of Tc52 revealed a clone (EST AI975843)
with around 25% sequence identity with the GST-like
domain of the T. cruzi protein. A fragment of 549 bp was
amplified by PCR using specific primers, designed based on
the EST sequence, and cDNA from S. mansoni adult
worms. The amplified fragment was sequenced and used as
probe to screen an adult worm cDNA library. After three
rounds of hybridization, two independent clones were
purified and sequenced. The sequences obtained were
identical in both clones and corresponded to a cDNA of
934 bp including an open reading frame encoding for a 241
amino acid polypeptide, with a predicted molecular mass of
27.6 kDa (Fig. 1). Sequence identity ranging from 18 to
25% was obtained with human GST-theta, mouse p28, rat
DHA, human GSTO 1-1, as well as with several plant
DHAs and non characterized GST-like proteins. In all
cases, the most conserved amino acids were localized in the
N-terminal domain of SmGSTO. The best hit obtained
(E ¼ 8.1e
)11

Fig. 1. Nucleotide and deduced amino acid
sequences of SmGSTO 1. The codon corres-
ponding to the initial methionine is
underlined.
Fig. 2. Unrooted phylogeny showing the most likely relationship between class representative GSTs and S. mansoni GST amino acid sequences. Branch
lengths are proportional to estimates of evolutionary change. The number associated with each internal branch is the local bootstrap probability
that is an indicator of confidence. The sequences are (species name; GenBank
TM
accession number): Schistosoma omega (Schistosoma mansoni,
AF484940), nematode omega (Caenorhabditis elegans, L23651), mouse omega (Mus musculus, U80819), rat omega (Rattus rattus, AB008807),
human omega (Homo sapiens, AF212303), soybean heat-shock protein (HsPr) (Glycine max, M20363), potato GST (Solanum tuberosum, J03679),
nematode zeta (Caenorhabditis elegans, Z66560), human zeta (Homo sapiens, NM_001513), carnation zeta (Dianthus caryophyllus, M64268), mouse
theta (Mus musculus, U48419), human theta (Homo sapiens, NM_000854), blowfly delta (L. cuprina, L23126), house fly delta (Musca domestica,
X61302), fruit fly Delta (Drosophila melanogaster, X14233), Arabidopsis phi (Arabidopsis thaliana, D17672), Petunia phi (Petunia hybrida, Y07721),
mouse mu (Mus musculus, J03952), human mu (Homo sapiens, NM_000848), chicken mu (Gallus gallus, X58248), rat Pi (Rattus norvegicus,
L29427), human pi (Homo sapiens, NM_000852), rat sigma (Rattus norvegicus, D82071), human sigma (Homo sapiens, D82073), squid2 sigma
(Ommastrephens sloanei, M36938), squid1 sigma (O. sloanei, M36937), Schistosoma 28 kDa (Schistosoma mansoni, S71584), human alpha (Homo
sapiens, NM_000846), mouse alpha (Mus musculus, M73483), and chicken alpha (Gallus gallus, L15386), Schistosoma 26 kDa (Schistosoma
mansoni, M31106).
Ó FEBS 2002 Omega-class GST from Schistosoma mansoni (Eur. J. Biochem. 269) 5515
GSH binding affinity of recombinant SmGSTO
Recombinant SmGSTO was first produced as a fusion to a
histidine-tag and purified in one step using an Ni
2+
nitrilotriacetic acid resin (Fig. 4A). There are some discrep-
ancies in the literature about the ability of omega class GSTs
to bind different glutathione-coupled matrixes. To deter-
mine whether SmGSTO was able to bind glutathione
agarose, we have used the purified enzyme in batch assays.
As showed in Fig. 4B,C, His-tagged SmGSTO was not able

stages. Expression of SmGSTO was also studied at the
transcriptional level by RT-PCR using cDNA prepared
from miracidia, sporocysts, cercariae and adult worms
(Fig. 5B). A unique amplification product of the 549 bp
expected size was observed in all reactions. The intensity of
the amplified products was compared after normalization
using a-tubulin cDNA as internal control. Relative values
showed as a bar graphic determine that SmGSTO tran-
scription is higher in sporocysts and adult worms in
comparison with cercariae and miracidia. This results are
in agreement with those obtained by Western blot. Taken
together, these results suggested that SmGSTO is expressed
more in parasitic stages than in free living stages during the
S. mansoni life cycle.
Characterization of recombinant SmGSTO enzymatic
activity
Recombinant GSTO was used to assay its enzymatic
activity. Results of substrate specificity are shown in
Table 1. SmGSTO showed a negligible activity against
CDNB and ethacrynic acid and no measurable activity
against other GST substrates like 1,2-dichloro-4-nitroben-
zene (DCNB), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole;
p-nitrobenzyl chloride, vinylene thiocarbonate, t-butyl
hydroquinone and p-chloranil. In contrast, SmGSTO
showed DHA and HEDS-measured TTase activities.
Kinetic parameters were obtained for DHA and TTase
activities. SmGSTO showed a K
m
¼ 0.23 m
M

>4 m
M
for GSH in this reaction. Even though differences
in K
m
values for GSH in the two assayed reactions were
obtained, it was clear that a K
m
lower than 0.32 m
M
could
not be reached. Finally, SmGSTO showed differential
sensitivity to several GST inhibitors tested (Table 2).
Among them, it is interesting to note the inhibitory activity
of CDNB which is considered as a classical GST substrate.
Specific activity was first measured for both reactions in
standard conditions using a phosphate buffer pH 7.6 [33].
However, when the pH profile for SmGSTO activity was
carried out, optimal activities were recorded at pH 8.0 and
pH 8.6 when phosphate buffer or Tris/HCl buffer were
used, respectively (Fig. 6). A similar optimal pH profile was
recently reported for Plasmodium falciparum glutaredoxin 1
activity [37].
Construction of a homology model of SmGSTO
SmGSTO structure was built using the human GSTO 1–1
structure as template in the SWISS-MODEL modeling
Fig. 3. Southern blot analysis of SmGST. DNA from S. mansoni
cercaria (10 lg) digested with different restriction enzymes was
hybridized with the coding region of SmGST-O cDNA radiolabeled by
a random primer.

analysis of parasite extracts. Each lane contains total protein (50 lg)
from cercariae (lane 1), schistosomula (lane 2), sporocysts (lane 3), and
adult worms (lane 4) electroblotted and immunodetected by a-SmG-
STO serum. (B) RT-PCR analysis of different S. mansoni stages.
Reverse-transcribed RNA from miracidia (lane 1), sporocysts (lane 2),
cercariae (lane 3) and adult worms (lane 4) were amplified using
SmGSTO and a-tubulin specific primers as indicated in experimental
section.
Ó FEBS 2002 Omega-class GST from Schistosoma mansoni (Eur. J. Biochem. 269) 5517
referred here as SmGST omega (SmGSTO). In addition,
sequence alignment of SmGSTO with representative
sequences from the recently reported GSTO class or from
the EST data base highlights similarities, but also significant
differences. In all cases, the most conserved amino acids
were localized in the N-terminal domain of the SmGSTO
protein. However, the remaining regions of SmGSTO
presented significant divergences from the known GSTO,
at the primary structure level. Indeed, the GSTO class
represents a particular class of the GST superfamily which
possesses specific structural features, such as an active site
cysteine that is able to form a disulfide bond with GSH, a
novel domain formed by the proximity of a specific
N-terminal extension to the C-terminus and a large H-site,
as well as the ability to catalyze the GSH-dependent
reduction of dehydroascorbate [10].
We further undertook a phylogenetic protein sequence
analysis to ascertain whether the SmGSTO was a divergent
member of the GSTO class. The deduced SmGSTO protein
sequence was used in a phylogenic analysis using the
maximum-likelihood approach. Two other previously char-

than in free-living life stages, suggesting that this protein
may play a role in the survival of the parasite within the
host. An increased expression during cercariae transforma-
tion to mature adults in mammalian host was already
described as a general feature for detoxifying enzymes in
Schistosoma [41–43]. Immunohistochemical analysis of
human tissues confirmed a widespread expression of
GSTO1-1, suggesting that it has important biological
functions. Specific expression of GSTO1-1 was localized in
the nuclei and in nuclear membranes of many cell types [44].
However, no putative nuclear localization signals could be
found within GSTO1-1 or SmGSTO. Nuclear localization
Table 1. Substrate specificities of recombinant SmGSTO. Activity for
each substrate was determined in standard conditions. ND, not
detected.
Substrate
Specific activity
(lmolÆmin
)1
Æmg
)1
)
1,2-Dichloro-4-nitrobenzene ND
1-Chloro-2,4-dinitrobenzene 0.02
7-Chloro-4-nitrobenzo-2-oxa-
1,3-diazole
ND
Ethacrynic acid 0.02
p-Nitrobenzyl chloride ND
Vinylene trithiocarbonate ND

ried out using the glutathione:dehydroascorbate reductase assay.
Grafic indicates relative DHA activity measured in 100 m
M
phosphate
buffer and 100 m
M
Tris/HCl, using standard substrate concentrations.
5518 J. Girardini et al. (Eur. J. Biochem. 269) Ó FEBS 2002
of S. mansoni 28 kDa GST, which has no detectable nuclear
localization signal, was already described [45]. A cytolocali-
zation study of SmGSTO is being undertaken in our
laboratory.
Some contrasting findings were reported concerning the
ability of omega GSTs to bind matrix-linked glutathione.
Mouse p28 was reported to bind glutathione-agarose, but
human GSTO1-1 was unable to bind S-linked glutathione-
sepharose. Here, we report that SmGSTO binds S-hexyl
glutathione-agarose but not glutathione-agarose. Moreover,
S-hexyl glutathione-agarose-bound SmGSTO was not dis-
placed neither by reduced nor oxidized glutathione. These
results are in line with the high GSH K
m
value obtained for
SmGSTO and the strong inhibitory effect of S-hexyl
glutathione on the enzyme activity. The preference of
SmGSTO for more hydrophobic alkyl-bound S-hexyl
glutathione rather than glutathione could reflect some
particular characteristics of the active site of this enzyme.
As other GSTs from the omega class the parasite protein
was unable to use known GST family substrates like CDNB

with HEDS. The second group corresponds to variable
molecular mass Ôone-cysteineÕ (monothiol) glutaredoxins
with a conserved sequence C-G-F-S, such as yeast gluta-
redoxins Grx3–5. E. coli Grx2 and GSTO 1–1 were proposed
to be grouped into a third subfamily, having an N-terminal
Grx-like domain and the helical C-terminal domain and the
general structure reminiscent to the GST superfamily of
proteins. Sequence similarity and predicted structure show
Fig. 7. Model showing SmGSTO structure.
Structure was built based on homology
modeling using human GSTO1-1 as template
in the SWISS-MODEL modeling environ-
ment. (A) General chain fold view of human
GSTO 1–1 and SmGSTO. (B) Scheme illus-
trating position of GSH contacting residues
determined for human GST 1–1 and modeled
for SmGSTO.
Ó FEBS 2002 Omega-class GST from Schistosoma mansoni (Eur. J. Biochem. 269) 5519
that SmGSTO belongs to this last group. At this point, it
should be noted that the questions as to whether E. coli Grx-
2 is a GST or if GST-O are glutaredoxins is not completely
solved. When active cysteine-containing tetramers were
sought in these proteins, a striking sequence divergence was
observed. E. coli Grx2 contains a Trx1-like two-cysteine
sequence, C-P-Y-C; GSTO 1–1 has a one-cysteine C-P-F-A
sequence; and SmGSTO contains C-P-Y-V, similar to the
Trx1-like sequence but with only one cysteine. Sequence
comparison at the active site level and HEDS-measured
SmGSTO TTase activity strongly suggest that SmGSTO
could participate in glutathionylation and reduction of

The authors wish to thank Dr Lars Jermiin for GST sequences
alignments and for His help with Molphy 2.3 utilization, Dr Luis
Esteban for His help with Linux operative system installation and
utilization and Dr Eleonora Garcı
´
aVe
´
scovi for her critical reading
of the manuscript. This research was supported by Fundacion
Antorchas, Third World Academy of Sciences and the Research
Program of the UNR. ECS is member of the National Research
Council (CONICET, Argentina) and JEG is Fellow of the same
institution.
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