Enzyme Catalysis and Regulation:

Platyhelminth Mitochondrial and Cytosolic

Redox Homeostasis Is Controlled by a
Single Thioredoxin Glutathione Reductase
and Dependent on Selenium and
Glutathione

Mariana Bonilla, Ana Denicola, Sergey V.

Novoselov, Anton A. Turanov, Anna Protasio,
Darwin Izmendi, Vadim N. Gladyshev and
Gustavo Salinas

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J. Biol. Chem. 2008, 283:17898-17907.
doi: 10.1074/jbc.M710609200 originally published online April 11, 2008

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http://www.jbc.org/content/suppl/2008/04/14/M710609200.DC1.html

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 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 26, pp. 17898 –17907, June 27, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Platyhelminth Mitochondrial and Cytosolic Redox

Homeostasis Is Controlled by a Single Thioredoxin
Glutathione Reductase and Dependent on Selenium
and Glutathione*□ S

Received for publication, December 31, 2007, and in revised form, March 27, 2008 Published, JBC Papers in Press, April 11, 2008, DOI 10.1074/jbc.M710609200
Mariana Bonilla‡, Ana Denicola§, Sergey V. Novoselov¶, Anton A. Turanov¶, Anna Protasio‡, Darwin Izmendi‡,
Vadim N. Gladyshev¶, and Gustavo Salinas‡1
From the ‡Cátedra de Inmunologı́a, Facultad de Quı́mica-Facultad de Ciencias, Instituto de Higiene, Universidad de la República,
Avda. A. Navarro 3051, Piso 2, Montevideo 11600, Uruguay, the §Laboratorio de Fisicoquı́mica Biológica, Facultad de Ciencias,
Universidad de la República, Montevideo 11400, Uruguay, and the ¶Department of Biochemistry and Redox Biology Center,
University of Nebraska, Lincoln, Nebraska 68588

Platyhelminth parasites are a major health problem in develop- The control of parasitic infections, which are a major cause of
ing countries. In contrast to their mammalian hosts, platyhelminth disability, mortality, and economic losses in many developing

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thiol-disulfide redox homeostasis relies on linked thioredoxin-glu- countries, remains as one of the most important challenges for
tathione systems, which are fully dependent on thioredoxin-gluta- medicine in the 21st century (1). In the case of the phylum
thione reductase (TGR), a promising drug target. TGR is a platyhelminthes (flatworms), which include the causative
homodimeric enzyme comprising a glutaredoxin domain and thi- agents of schistosomiasis (bilharzia) and hydatid disease, phar-
oredoxin reductase (TR) domains with a C-terminal redox center macotherapy with praziquantel has met great success in the
containing selenocysteine (Sec). In this study, we demonstrate the treatment of infection. However, drug resistance is a serious
existence of functional linked thioredoxin-glutathione systems in issue as it has been the case for other antiparasitic drugs (2). In
the cytosolic and mitochondrial compartments of Echinococcus the case of platyhelminths, this may have severe consequences,
granulosus, the platyhelminth responsible for hydatid disease. The because praziquantel is the only drug that is readily available for
glutathione reductase (GR) activity of TGR exhibited hysteretic large scale treatment of these infections (3). Thus, the need for
behavior regulated by the [GSSG]/[GSH] ratio. This behavior was new drugs and/or vaccines is of great importance. In recent
associated with glutathionylation by GSSG and abolished by deglu- years, evidence has accrued that the selenocysteine (Sec)2-con-
tathionylation. The Km and kcat values for mitochondrial and cyto- taining enzyme thioredoxin glutathione reductase (TGR) is
solic thioredoxins (9.5 ␮M and 131 sⴚ1, 34 ␮M and 197 sⴚ1, respec- essential for platyhelminth parasites and has emerged as a
tively) were higher than those reported for mammalian TRs. rational target for chemotherapy and/or immunotherapy
Analysis of TGR mutants revealed that the glutaredoxin domain is (4 – 8). In most organisms, including the mammalian hosts of
required for the GR activity but did not affect the TR activity. In platyhelminths, cellular redox homeostasis, antioxidant
contrast, both GR and TR activities were dependent on the Sec- defenses, and supply of reducing equivalents to several targets
containing redox center. The activity loss caused by the Sec-to-Cys and essential enzymes rely on two major pathways: the gluta-
mutation could be partially compensated by a Cys-to-Sec mutation thione (GSH) and the thioredoxin (Trx) systems, which have
of the neighboring residue, indicating that Sec can support cataly- overlapping and differential targets and functions (9, 10). In
sis at this alternative position. Consistent with the essential role of contrast, platyhelminth parasites lack conventional thiore-
TGR in redox control, 2.5 ␮M auranofin, a known TGR inhibitor, doxin reductase (TR) and glutathione reductase (GR), and
killed larval worms in vitro. These studies establish the seleni- hence conventional Trx and GSH systems (4, 6, 7). Instead, they
um- and glutathione-dependent regulation of cytosolic and rely exclusively on linked thioredoxin-glutathione systems,
mitochondrial redox homeostasis through a single TGR enzyme with TGR being the key enzyme that provides reducing equiv-
in platyhelminths. alents to both pathways. Another feature of the linked systems
in platyhelminths is that cytosolic and mitochondrial TGR derive
from a single gene and have identical sequence, once the leader
peptide of the mitochondrial variant is removed (5). In the mam-
* This work was supported, in whole or in part, by National Institutes of Health
Grant GM065204 (to V. N. G.). This work was also supported by Fogarty malian hosts, different thioredoxin reductase isozymes function in
International Research Collaboration Award-NIH Grant TW006959 (to the cytosol and the mitochondria (11, 12), TGR expression is
V. N. G.) and by the Uruguayan Research Council (Grants PDT 29-171 and largely restricted to testis (13), and GR exists as a distinct gene (14).
PDT 63-105 to G. S.). The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
2
marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to The abbreviations used are: Sec, selenocysteine; DMEM, Dulbecco’s modified
indicate this fact. Eagle’s medium; DTNB, 5,5⬘-dithiobis(2-dinitrobenzoic acid); DTT, dithiothrei-
□S
The on-line version of this article (available at http://www.jbc.org) contains tol; EGFP, enhanced green fluorescent protein; GR, glutathione reductase; Grx,
supplemental Figs. S1 and S2 and Table S1. glutaredoxin; GSSG, glutathione (oxidized form); TGR, thioredoxin glutathione
1
To whom correspondence should be addressed: Tel./Fax: 5982-487-4320; reductase; TR, thioredoxin reductase; Trx, thioredoxin; SECIS, selenocysteine
E-mail: [email protected]. insertion sequence; mtTrx, mitochondrial Trx; cTrx, cytosolic Trx.

17898 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 26 • JUNE 27, 2008
 Linked Thioredoxin-Glutathione Systems
In sum, the dissimilar arrangements of redox pathways as com- (ATCC) were cultured in DMEM supplemented with 10% fetal
pared with their hosts, the lack of back-up systems, and the fact bovine serum in the presence of 100 units/ml penicillin and 50
that parasitic organisms are subjected not only to the endogenous units/ml nystatin. Transfections were carried out in 35-mm glass
oxidative stress, but also to the oxidative challenge imposed by the bottom culture dishes using Lipofectamine 2000 (Invitrogen),
host’s immune system, provide a strong rationale to target platy- according to the manufacturer’s instructions. Transfection mix
helminth TGRs. Recent studies support this idea: inhibition of was prepared using 3 ␮g of plasmid DNA (mitochondrial Trx
TGR expression by RNA interference caused death of the platy- construct, mitochondrial TGR construct, or pEGFP-N2, used
helminth parasite Schistosoma mansoni, and auranofin, a potent as a control) and 6 ␮l of Lipofectamine per dish. Transfections
inhibitor of TGR and Sec-containing TRs (15), caused a partial were carried out in Opti-MEM (Invitrogen) for 8 h. The transfec-
cure in experimental Schistosoma infection (8). tion medium was replaced with a DMEM culture medium con-
TGR possesses a fusion of conventional TR domains with a taining MitoTracker Red CM-H2XRos (Molecular Probes), a
glutaredoxin (Grx) domain (13, 16). TGR, like GR and TR, is a marker of the mitochondrial compartment; cells were incubated
homodimer, with monomers oriented in a head-to-tail manner. for 30 min and then washed twice with DMEM. Transiently trans-
Based on biochemical data, the current model of the mecha- fected cells were detected by confocal microscopy (Bio-Rad,
nism of reaction for TGR proposes that electrons flow from MRC1024ES laser scanning microscope).
NADPH to FAD, to the C156XXXXC redox center (numeration
according to Echinococcus granulosus TGR), to the C-terminal Cloning, Expression, and Purification of Recombinant TGR
GC595UG (U is Sec) redox center of the second subunit, and and Its Mutants
finally to the C31XXC redox center of the Grx domain of the Different constructs were made for expression of wild-type

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first subunit. The fully reduced enzyme can reduce either oxi- TGR (TGRGCUG) (where U is Sec, and GCUG is the C-terminal
dized Trx using the C-terminal active site GCUG, or GSSG tetrapeptide) and the following mutants: Sec596 to Cys
through the CXXC redox center of the Grx domain (13, 17). (TGRGCCG), Sec596 to stop (TGRGC*), Sec596 to Cys, and
Recently, a crystallographic structure of an S. mansoni C-ter- Cys595 to Sec (TGRGUCG), as well as a mutant lacking the entire
minally truncated TGR (GCstop) has been solved. Based on the Grx domain of TGR (TRGCUG). In all cases mRNA from trizoled
residual GR activity of the mutant, the authors proposed an E. granulosus protoscoleces (larval worms) was used as a tem-
alternative view in which GSSG could be reduced directly by plate for reverse transcription and PCR, using ThermoScript
the CXXXXC redox center of TR domains (18). reverse transcriptase (Invitrogen) and Pfu (Fermentas), respec-
In the current study, we have characterized the linked thiore- tively. Forward and reverse gene-specific primers were derived
doxin-glutathione system of the platyhelminth E. granulosus, from the previously published TGR sequence (5). In the case of
the causative agent of hydatid disease. We demonstrated the mutant TGRs, the reverse primers were modified appropri-
occurrence of functional linked systems in both cytosol and ately. For Sec-encoding constructs (TGRGCUG, TGRGUCG, and
mitochondria. The analysis of activities of TGR mutants TRGCUG), further engineering of the reverse primers was
revealed that the Grx domain is required for the GR activity, but needed to specify Sec, because the UGASec codon requires
does not affect the TR activity; in contrast, both Trx- and recoding by an Sec insertion sequence (SECIS) element present
glutathione-dependent activities require selenocysteine (Sec) in the selenoprotein mRNAs. Thus, for these constructs, the
residue. Our results also indicate that [GSSG]/[GSH] ratio reg- reverse gene-specific primer contained, at the 5⬘-end, the
ulates TGR activities and strongly suggest that glutathionyla- SECIS element of Escherichia coli formate dehydrogenase H at
tion/deglutathionylation is involved in this regulation. In addi- a 10-nucleotide distance from the UGASec codon (sequences of
tion, we show that larval worms are killed by very low primers for every construct are detailed in supplemental Table
concentrations of auranofin, a TGR inhibitor, and discuss our S1). This strategy with a bacterial-type SECIS has been previ-
results in light of the current models that have been put forward ously used for C-terminal Sec incorporation in E. coli (19). The
to explain the GR activity of TGR. amplified products were first cloned into pGEM-T-easy (Pro-
mega), and the construct sequences were verified prior to sub-
EXPERIMENTAL PROCEDURES sequent subcloning into pET28a (Novagen). Constructs were
used to transform E. coli BL21(DE3) cells, or, in the case of
Cloning of Mitochondrial Isoforms of Trx and TGR as selenoprotein constructs, BL21(DE3) cells previously trans-
N-terminal Fusions to EGFP formed with pSUABC, a plasmid that supports high level
To analyze the subcellular localization of the putative mito- expression of genes involved in Sec synthesis and decoding
chondrial variants of Trx and TGR, constructs were generated (selA, selB, and selC) (19). Expression of recombinant proteins
using pEGFP-N2 (Clontech). In the case of Trx, the sequence was carried out following the protocol described in a previous
was retrieved from Partigen (cluster EGC03292), and the entire study (20), which has been optimized for expression of seleno-
coding region, including the leader peptide, was cloned as an proteins. Essentially, induction of recombinant proteins was
in-frame fusion to EGFP. In the case of TGR the N-terminal carried out with 100 ␮M isopropyl 1-thio-␤-D-galactopyrano-
fragment of mitochondrial TGR, containing the leader peptide side at late exponential phase (A600 ⫽ 2.4), during 24 h at 24 °C.
followed by the Grx domain of TGR, was cloned as an EGFP Recombinant clones were grown in modified LB media accord-
fusion. In both cases, a Kozak consensus sequence was included ing to a previous study (21), supplemented with 0.1 g/liter cys-
in the forward primer for initiation of translation at the first teine and 0.37 g/liter methionine (22), in the presence of kana-
AUG codon. For transient expression, mouse NIH 3T3 cells mycin (50 ␮g/ml), and chloramphenicol (33 ␮g/ml); the latter

JUNE 27, 2008 • VOLUME 283 • NUMBER 26 JOURNAL OF BIOLOGICAL CHEMISTRY 17899
Linked Thioredoxin-Glutathione Systems
was used only in the case of bacterial cultures harboring the reducing conditions, and by size exclusion chromatography on
TGRGCUG, TGRGUCG, and TRGCUG constructs. At the time of a Superdex 75 column (GE Healthcare).
induction the culture was supplemented with 5 ␮M sodium sel-
enite, 20 ␮g/ml riboflavin, 20 ␮g/ml pyridoxine, and 20 ␮g/ml Metabolic Labeling of Selenoproteins
niacin according to a previous study (21). For recombinant To label cells with 75Se, E. coli cells carrying the different
TGRs that did not contain Sec (TGRGCCG and TGRGC*) the constructs were grown at 37 °C until A600 reached 0.4, and the
same protocol was followed, except that the plasmid pSUABC culture was supplemented with ⬃50 ␮Ci 75Se (as freshly neu-
was not used. The bacterial cultures were centrifuged, and the tralized sodium selenite, specific activity of 1000 Ci/mmol,
pellets were resuspended in modified nickel-nitrilotriacetic Research Reactor Facility, University of Missouri, Columbia,
acid lysis buffer (300 mM NaCl, 50 mM sodium phosphate, 20 MO). After an additional 30 min, isopropyl 1-thio-␤-D-galacto-
mM imidazole, pH 7.2) containing 1 mM phenylmethylsulfonyl pyranoside was added to each cell culture at a final concentra-
fluoride and 1 mg/ml lysozyme, and sonicated (10 pulses of 1 tion of 100 ␮M. After 3 h of induction at 37 °C, cells were col-
min with 1-min pauses). The lysates were centrifuged for 1 h at lected, washed, and lysed by boiling in SDS-PAGE sample
30,000 ⫻ g, and supernatants were applied to a nickel-nitrilo- buffer containing 50 mM 1,4-dithiothreitol (DTT). Cell lysates
triacetic acid column (Qiagen), washed with 300 mM NaCl, 50 were then subjected to SDS-PAGE followed by transfer of pro-
mM sodium phosphate, 30 mM imidazole, pH 7.2, and eluted teins onto a polyvinylidene difluoride membrane. 75Se signal
with 250 mM imidazole. The protein-containing fractions were was visualized with a phosphorimaging device (Fuji).
applied to PD10 desalting columns (GE Healthcare) using
Enzymatic Assays
phosphate-buffered saline, 150 mM potassium chloride, 50 mM

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sodium phosphate, pH 7.2. Fractions containing the recombi- Insulin Reduction Assay for Trx Activity—The efficient reduc-
nant proteins were stored at ⫺70 °C before use. Total protein tion of two interchain disulfides of insulin catalyzed by Trx in the
concentration and FAD content were determined spectropho- presence of DTT was used as a measure of Trx activity, according
tometrically at 280 (⑀ ⫽ 54.24 mM⫺1 cm⫺1) and 460 nm (⑀ ⫽ to a previous study (24). The reaction was followed by the increase
11.3 mM⫺1 cm⫺1), respectively. The selenium content of sel- in absorbance at 650 nm due to the precipitation of free insulin
enoproteins was determined by atomic absorption using a B-chain. The 0.8-ml reaction mixtures contained 0.33 mM DTT,
Plasma Emission Spectrometer (Jarrell-Ash 965 ICP) in Chem- 130 ␮M insulin, and 2 mM EDTA in 100 mM potassium phosphate
ical Analysis Laboratory, University of Georgia. The purity of buffer, pH 7.0. Runs with DTT alone were performed as controls.
the recombinant proteins was analyzed by running 10% SDS- DTNB Reduction Assay for TR Activity—The reduction of
PAGE gels, under reducing conditions, and by size-exclusion 5,5⬘-dithiobis (2-dinitrobenzoic acid) (DTNB) with concomi-
chromatography on a Superose 12 column (GE Healthcare). tant NADPH oxidation was determined by the increase in
absorbance at 412 nm due to formation of 5⬘-thionitrobenzoic
acid at 25 °C (25). The 0.8-ml reaction mixtures contained 0.2
Cloning, Expression, and Purification of E. granulosus
mM NADPH, 5 mM DTNB, and 10 mM EDTA in 100 mM potas-
Recombinant Mitochondrial and Cytosolic Trx Forms
sium phosphate buffer, pH 7.0.
mRNAs encoding cytosolic and mitochondrial E. granulosus Insulin Reduction Assay for TR Activity—The Trx-coupled
Trxs were amplified by reverse transcription-PCR from total assay of TR activity takes advantage of the NADPH-dependent
larval worm mRNA as described above. Specific forward and reduction of Trx by TR, which is followed by the decrease in
reverse primers for cytosolic and the predicted mature mito- absorbance at 340 nm; in this assay, excess of insulin is used as an
chondrial Trx were derived from previously published electron sink to maintain a constant concentration of oxidized Trx
sequences (23) and from Partigene (cluster EGC03292), respec- (25). The 0.8-ml reaction mixtures contained 0.2 mM NADPH, 1
tively. The amplified products were first cloned into pGEM-T- mM EDTA, 0.5 mg/ml insulin, and E. granulosus cytosolic or mito-
easy (Promega), sequenced and subsequently subcloned into chondrial Trx (concentrations ranged from 0 to 80 ␮M and from 0
pET28a (Novagen) using appropriate restriction enzymes. to 140 ␮M, respectively), in 50 mM potassium phosphate buffer, pH
Constructs were used to transform E. coli BL21(DE3) host cells. 7.0. The kinetic parameters of TGR with its physiological sub-
Expression of recombinant proteins was carried out following strates, cytosolic and mitochondrial Trx, were determined from
the standard protocol for expression of recombinant proteins. Michaelis-Menten plots of vo (derived from time-course experi-
Essentially, recombinant clones were grown on LB in the pres- ments) against substrate concentration.
ence of kanamycin, and induction of recombinant proteins was GR Assay—The GR activity was assayed as NADPH-depend-
carried out with 100 ␮M isopropyl 1-thio-␤-D-galactopyrano- ent reduction of oxidized glutathione (GSSG), which is fol-
side at early exponential phase (A600 ⫽ 0.5), for 3 h at 37 °C. The lowed as the decrease in absorbance at 340 nm (26). The 0.8-ml
bacterial cultures were centrifuged, and the recombinant pro- reaction mixture contained 0.125 mM NADPH, 1 mM GSSG, 1
teins were purified and desalted as described above for TGR, mM EDTA in 100 mM potassium phosphate buffer, pH 7.0.
except that all buffers had pH 7.8. Fractions containing the All enzymatic assays were carried out in a Cary 50 (Varian)
recombinant proteins were stored at ⫺70 °C prior to use. Pro- spectrophotometer at 25 °C. Analyses of the kinetic data were
tein concentration was determined spectrophotometrically at performed using ORIGIN software (OriginLab).
280 nm (⑀ ⫽ 7.6 and 6.1 mM⫺1 cm⫺1 for cytosolic and mito- Mass Spectrometry Analysis—Wild-type TGR samples (10
chondrial Trx, respectively). The purity of the recombinant nM concentration) were incubated with GSSG in the presence
proteins was analyzed by running 15% SDS-PAGE gels under or absence of 0.125 mM NADPH at molar ratios under which

17900 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 26 • JUNE 27, 2008
 Linked Thioredoxin-Glutathione Systems
hysteresis was or was not observed (1 mM or 30 ␮M GSSG, In Vitro Culture of Larval Worms—50,000 protoscoleces,
respectively) in 100 mM potassium phosphate buffer, pH 7.0, obtained from asceptical punction of a single hydatid cyst from
containing 1 mM EDTA, and immediately passed through a bovine lung, were washed several times with phosphate-buff-
PD10 desalting column (GE Healthcare). Protein-containing ered saline and then incubated at 37 °C, 5% CO2, in DMEM
fractions were digested with trypsin and subjected to analysis supplemented with antibiotics and 20 mM HEPES, pH 7.4. Cul-
by matrix-assisted laser desorption ionization time-of-flight tured protoscoleces were treated with 1, 2.5, 5, and 10 ␮M
mass spectrometry (4800 Analyzer, Applied Biosystems). The auranofin or with the vehicle (DMSO), in the presence or
mass spectrometry analysis was carried out at the Institut Pas- absence of 100 ␮M hydrogen peroxide. Protoscoleces were
teur, Montevideo. observed under the microscope, and viability was assessed by
exclusion of the vital dye eosin.

RESULTS
Mitochondrial Localization of Trx and TGR Forms—Se-
quence analyses suggested the occurrence of both cytosolic and
mitochondrial forms of TGR and Trx, with TGR forms gener-
ated from a single gene, and two genes for Trx forms. The
results of transient expression in mammalian NIH 3T3 cells of
the predicted mitochondrial forms of Trx and TGR are shown
in Fig. 1. Both EGFP fusion proteins co-localized with Mito-

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Tracker, indicating that the signal peptides of these proteins
direct the fusions to the mitochondrial compartment. No obvi-
ous staining of the cytosol or other subcellular compartments
was observed. TGR has been previously shown to be present in
the mitochondrial subcellular fraction of a larval worm aqueous
extract (5); however, the mitochondrial location of Trx was pre-
viously limited to in silico predictions in platyhelminths (7).
TGR Can Provide Electrons to Both Cytosolic and Mitochon-
drial Trx Forms—Prior to determining the enzymatic parame-
ters of TGR with its physiological substrates, the quality of
every recombinant protein was assessed in several ways. First,
FIGURE 1. Subcellular localization of GFP-fused mitochondrial TGR and the purity of TGR and its mutants, and of cytosolic and mito-
mtTrx. NIH3T3 cells were transiently transfected either with the non-recom- chondrial Trx forms, was determined by SDS-PAGE under
binant pEGFP vector (A) or with the pEGFP-derived constructs carrying mito-
chondrial TGR (B) and mtTrx (C) N-terminally fused to GFP. Images were
reducing conditions (Fig. 2A) and by size exclusion chromatog-
obtained at 8 h post-transfection using an I-confocal microscope. A set of raphy (data not shown). In the case of selenoproteins, Sec incor-
three panels is shown for each construct. Left panels show green fluorescence poration was evaluated by metabolic labeling of the bacterial
corresponding to transiently expressed GFP fusion proteins. Center panels
show the red fluorescence of the mitochondrial dye (MitoTracker). The right cultures with 75Se. The results are shown in Fig. 2 (B and C);
panels show merged images from left and center panels. specific labeling at the expected molecular weight was observed

FIGURE 2. Analysis of recombinant proteins. A, SDS-PAGE analysis of purified recombinant proteins. After purification on a nickel-nitrilotriacetic acid column
and desalting, recombinant proteins were run on a 12.5% polyacrylamide gel. 1 ␮g of each recombinant protein was loaded on the gel. Lanes 1–5, TGRGCUG,
TRGCUG, TGRGUCG, TGRGCCG, TGRGC*, respectively; lane 6, molecular weight markers; lane 7, mtTrx; and lane 8, cTrx. The positions of molecular weight marker are
indicated on the right. B and C. 75Se incorporation into recombinant TGRs. BL21(DE3) cells expressing TGRGCUG, TGRGUCG, TGRGCCG, and TGRGC* were induced
with 100 ␮M isopropyl 1-thio-␤-D-galactopyranoside for 3 h at 37 °C. 50 ␮Ci of 75Se were added to 10-ml cultures 30 min before induction. Total cell protein
samples were resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. B, Coomassie Blue staining of the polyvinylidene difluoride
membrane. C, 75Se detection by phosphorimaging device analysis. Lanes 1– 4, 10 ␮l of total cell protein samples from cells expressing recombinant TGRGCUG,
TGRGUCG, TGRGCCG, and TGRGC*, respectively; lane 5, molecular weight marker. FDH-O, formate dehydrogenase O (110 kDa), the single selenoprotein expressed
by E. coli under aerobic conditions. The bands around 66 kDa are indicated by an asterisk on lanes 1 and 2 on the right panel and correspond to 75Se-labeled
Sec-containing recombinant TGRs. Lower molecular mass bands on these lanes probably correspond to secondary initiation or degradation products of these
Sec-containing recombinants. The absence of bands on lanes 3 and 4 indicates no unspecific selenium incorporation was detected. The positions of molecular
weight markers are indicated on the right.

JUNE 27, 2008 • VOLUME 283 • NUMBER 26 JOURNAL OF BIOLOGICAL CHEMISTRY 17901
Linked Thioredoxin-Glutathione Systems
TABLE 1
Kinetic parameters of wild-type TGR and its mutants
Apparent kinetic parameters for mtTrx and cTrx (physiological TGR substrates)
were obtained by varying the mtTrx and cTrx concentrations at a constant and
saturating concentration of NADPH and constant enzyme concentrations. Appar-
ent kcat values for DTNB were determined from the slope of the initial velocities (v0)
versus enzyme concentration plots (Fig. 4). The enzyme concentrations used for kcat
calculations for selenoproteins (TGRGCUG, TRGCUG, and TGRGUCG) considered the
actual concentrations of active enzymes (i.e. their values were corrected according
to their selenium content).
Parameter Substrate TGRGCUG TRGCUG TGRGUCG TGRGCCG
Apparent Km mtTrx 9.5 ⫾ 0.5 13.0 ⫾ 0.6 12.0 ⫾ 0.6 14.3 ⫾ 0.6
(␮M) cTrx 34 ⫾ 2
Apparent kcat mtTrx 131 ⫾ 2 80 ⫾ 2 6.4 ⫾ 0.2 0.530 ⫾ 0.007
(s⫺1) cTrx 197 ⫾ 3
DTNB 118 ⫾ 3 60 ⫾ 3 6.0 ⫾ 0.5 0.63 ⫾ 0.03
Apparent kcat/Km mtTrx 13.8 ⫾ 0.9 6.1 ⫾ 0.4 0.27 ⫾ 0.03 0.037 ⫾ 0.002
(␮M⫺1s⫺1) cTrx 5.8 ⫾ 0.4

FIGURE 3. Kinetic parameters of TGRGCUG with mtTrx and cTrx. Apparent

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Km and kcat of TGRGCUG with mtTrx and cTrx were obtained using the Trx-
coupled assay. The initial reaction velocities (v0) at different Trx concentra-
tions were measured at a constant and saturating NADPH concentration (200
␮M) and a constant enzyme concentration (0.5 nM). Plots of v0 versus substrate
concentration for mtTrx and cTrx are shown. The data were fitted to the
Michaelis-Menten equation using Origin 7.5 software. Apparent Km and vmax
were obtained from these fittings and apparent kcat was calculated from
apparent vmax. Apparent Km, kcat, and kcat/Km values are indicated. The
enzyme (TGRGCUG) concentration used for kcat calculation was corrected
according to its selenium content.

exclusively in the bacterial lysates expressing selenoproteins,

indicating that full-length selenoproteins were synthesized. In
addition, the selenium content of recombinant selenoproteins
was determined. Sec incorporation was close to 10% in all FIGURE 4. TR activity of TGR mutants. The TR activities of wild-type and
recombinant selenoproteins (9.2% for wild-type TGRGCUG, mutant TGRs were compared using the DTNB assay. The assay was carried out
at constant and saturating concentrations of DTNB and NADPH (5 mM and
7.4% for inverted TGRGUCG, and 8.7% for TRGCUG). Taken 200 ␮M, respectively) and different concentrations of each enzyme. The plots
together, all these data indicated that the strategy was success- of initial velocities (v0) versus enzyme concentration are shown. The selenoen-
ful to produce full-length TGR in a bacterial host, although zyme (TGRGCUG, TRGCUG, and TGRGUCG) concentrations used for kcat calcula-
tions were corrected according to their selenium contents.
higher percentages of Sec incorporation (up to 50%) have been
previously reported using this methodology for other seleno-
proteins (19). Because only a fraction of the TGR molecules out the Grx domain), Sec596 to stop mutant (TGRGC*), Sec596 to
incorporate Sec (due to prevalent termination of translation at Cys mutant (TGRGCCG), and Cys595 to Sec and Sec596 to Cys
Sec UGA codons), active protein concentrations of selenopro- double mutant (TGRGUCG). Analysis of TR activity with the
teins were corrected according to their selenium content. DTNB assay (shown in Fig. 4 and summarized in Table 1)
The activities of recombinant TGR and Trx were initially revealed that TRGCUG and wild-type TGR have similar kcat.
assessed independently of each other, using the DTNB and TGRGC* had negligible activity even at 500 nM enzyme concen-
insulin reduction assays (see “Experimental Procedures”), tration (data not shown). TGRGCCG had a kcat more than two
respectively. Both recombinant enzymes displayed activity in orders of magnitude lower than that of wild-type TGR. The
these independent assays (data not shown). Then, using the double mutant with Sec and Cys at inverted positions,
insulin coupled assay, the kinetic parameters of TGR with its TGRGUCG, had a kcat one order of magnitude higher than that of
physiological substrates, cytosolic and mitochondrial Trx, were the Sec596 to Cys mutant. We next evaluated the kinetic param-
determined from Michaelis-Menten plots of vo against sub- eters of the mutants with mitochondrial Trx. The results are
strate concentration (Fig. 3). Km and kcat values were of the summarized in Table 1. The catalytic efficiency (kcat/Km) of the
same order for both Trxs (Fig. 3, Table 1). The catalytic effi- Sec to Cys mutant was 2.5 orders of magnitude lower than that
ciency of TGR was (13.8 ⫾ 0.9) ⫻ 106, and (5.8 ⫾ 0.4) ⫻ 106 M⫺1 of the wild-type TGR, whereas the double mutant was approx-
s⫺1 for mitochondrial and cytosolic Trx, respectively. imately one order of magnitude higher than the Cys mutant
Sec but Not the Grx Domain Is Essential for TR Activity—To (Table 1). Both TR assays indicated that, although the Grx
assess the role of the Grx domain and of Sec at the GCUG domain did not affect the TR activity, the C-terminal Sec resi-
C-terminal redox center of TGR in the catalysis, we generated a due was essential for this activity. Interestingly, a Sec residue at
set of TGR forms: wild-type TGR (TGRGCUG), TRGCUG (with- the resolving position of the C-terminal redox center could par-

17902 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 26 • JUNE 27, 2008
 Linked Thioredoxin-Glutathione Systems
cantly change once the enzyme was
fully active (see Fig. 5, B and C). We
next assayed whether Trx can
relieve GR hysteresis and found that
20 ␮M Trx, without preincubation,
abolished the hysteretic behavior
(Fig. 5D).
GR Activity Is Reversibly Regu-
lated by Glutathionylation/Deglu-
tathionylation—Rendón et al. (6)
have postulated that TGR must pos-
sess two GSH-binding sites, one of
them regulatory, with high affinity
for reduced GSH. We examined
binding of E. granulosus TGR to a
glutathione matrix and found that
neither oxidized nor NADPH-re-
duced TGR associated with GSH-
agarose. Because the [GSSG]/[GSH]

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ratio controls the activity of the
enzyme, we reasoned that TGR may
be glutathionylated by GSSG at high
concentrations and deglutathiony-
lated by GSH. Thus, we incubated
10 nM TGR with NADPH and 1 mM
GSSG for 1 min (conditions at
which the enzyme is still under hys-
FIGURE 5. Hysteretic behavior of GR activity of TGR. Full-time courses obtained using different assay condi- teresis, see Fig. 5B) and for 10 min
tions are shown. In all cases the reactions were started by the addition of TGRGCUG at the indicated final
concentrations. A, effect of enzyme concentration. Assays were performed at varying TGRGCUG concentrations (the enzyme is no longer under hys-
and constant NADPH and GSSG concentrations (100 ␮M and 1 mM, respectively). B, effect of GSSG concentra- teresis, see Fig. 5B). We then sub-
tion. GSSG concentration was varied at constant NADPH and enzyme concentrations (100 ␮M and 10 nM,
respectively). Note that at 31 and 62 ␮M GSSG reactions come to their end at higher A340, i.e. before depleting jected the enzymes to tryptic digest
NADPH, because GSSG becomes the limiting reagent. C, effect of GSH concentration. GSH was included at and mass spectrometry analysis.
various concentrations while maintaining constant GSSG, NADPH, and enzyme concentrations (1 mM, 125 ␮M, The results indicated that TGR is
and 10 nM, respectively). D, effect of Trx concentration. cTrx was added at different concentrations to reaction
mixtures containing 1 mM GSSG, 125 ␮M NADPH, and 10 nM TGRGCUG. The TGRGCUG concentration considered glutathionylated at two Cys resi-
was corrected according to its selenium content. dues: Cys88 and Cys354 after 1-min
incubation with 1 mM GSSG and
NADPH (supplemental Fig. S1).
tially compensate for the loss of activity due to Sec to Cys Absence of NADPH did not prevent glutathionylation. In con-
mutation. trast, after 10-min incubation with 1 mM GSSG and NADPH,
GR Activity Exhibits Hysteretic Behavior That Is Dependent TGR was found to be deglutathionylated (supplemental Fig.
on the Ratio of Oxidized and Reduced Glutathione—A hyster- S1). In similar experiments, no glutathionylation was found
etic behavior (i.e. the existence of a lag time before catalysis when 10 nM TGR was incubated for 1 min with 30 ␮M GSSG
takes place) of the GR activity of TGR was previously reported (a concentration under which there is no hysteresis, see Fig.
for the TGR of Taenia crassiceps (another platyhelminth para- 5B) with or without NADPH. In all cases, neither the Cys
site) (6). This behavior has not been reported for E. granulosus, residues belonging to the Grx active site nor those of the
S. mansoni, or mammalian TGR (5, 13, 27). We observed that E. CXXXXC catalytic redox center of TGR were detected as
granulosus TGR exhibited hysteretic behavior for its GR activ- glutathionylated peptides; instead, they were detected to be
ity, which became evident at TGR concentrations below 15 nM forming disulfides. Some Cys-containing peptides, including
(Fig. 5A) or high GSSG concentrations (Fig. 5B). Although the the Sec-containing peptide could not be detected. Alto-
results were analogous to those described for T. crassiceps TGR, gether the results indicated that the enzyme is glutathiony-
the E. granulosus TGR exhibited less marked hysteresis (i.e. lated at high GSSG concentrations and suggest that it
lower lag times for similar experimental conditions). Rendón et becomes deglutathionylated once the enzyme is active.
al. (6) described that preincubation of TGR with GSH abolishes We then tested whether the TR activity was preserved in
the hysteretic behavior of the enzyme. We found that preincu- TGR at conditions at which the GR activity was under hystere-
bation was not needed to relieve GR hysteresis of E. granulosus sis. Because it is not possible to measure the TR activity of TGR
TGR (Fig. 5C). Although the lag time correlated directly with in the presence of GSSG, we incubated 10 nM TGR with 1 mM
the concentration of GSSG, and inversely with the concentra- GSSG for 1 min (conditions under which there is GR hysteresis
tion of GSH, the maximal slope of the curves did not signifi- and glutathionylation) and applied the glutathionylated TGR to

JUNE 27, 2008 • VOLUME 283 • NUMBER 26 JOURNAL OF BIOLOGICAL CHEMISTRY 17903
Linked Thioredoxin-Glutathione Systems

FIGURE 6. TR activity of TGRGCUG and TRGCUG analyzed at the hysteresis conditions for GR activity. A, the TR activities of untreated and glutathionylated
TGRGCUG were compared using the Trx-coupled assay. B, the GR activities of untreated and glutathionylated TGRGCUG were compared at 100 ␮M GSSG. In both
A and B the enzyme preparations were assayed at 1 nM TGR concentration and 150 ␮M NADPH. It should be noted that, to calculate the volume of enzyme
preparation that ought to be used in the assay, glutathionylated TGR was assumed to be 2-fold diluted following desalting. This approximation could explain
the slightly smaller slopes observed for this enzyme in both assays, as compared with the untreated one. C, the TR activity of TRGCUG was evaluated using the
Trx-coupled assay both in the absence and presence of high concentration (1 mM) GSSG. The enzyme was assayed at 1 nM final concentration and 150 ␮M
NADPH. The selenoenzyme (TGRGCUG and TRGCUG) concentrations considered were corrected according to their selenium contents.

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FIGURE 7. GR activity of TGR mutants. Time courses obtained for the GR activity of wild-type TGR and its mutants are shown. In all cases the reaction was
started by the addition of the enzymes at the indicated final concentrations. A, comparison of the GR activity of TGRGCUG, TGRGCCG, TGRGC*, and TRGCUG at 31 ␮M
GSSG and 125 ␮M NADPH. B, the GR activity of TGRGUCG at different enzyme concentrations was evaluated at 31 ␮M GSSG and 125 ␮M NADPH. C, the effect of
GSH addition on the hysteretic behavior of TGRGUCG was studied by including GSH at 1 mM in a reaction mixture containing 1 mM GSSG, 125 ␮M NADPH, and
25 nM TGRGUCG. The enzyme concentrations for selenoproteins (TGRGCUG, TRGCUG, and TGRGUCG) were corrected according to their selenium contents.

a PD10 desalting column to remove GSSG. We measured the tration, but also on GSSG and GSH concentrations, we eval-
TR activity of this treated TGR by both the insulin-coupled uated the activity of these mutants at high enzyme concen-
(Fig. 6A) and DTNB assays (data not shown) finding no hyster- trations (200 nM) and different concentrations of GSSG and
etic behavior. In contrast, the GR activity of the treated TGR in the presence of GSH. None of these enzymes exhibited
was under hysteresis at low GSSG concentrations (condition significant activity at low GSSG concentrations (30 ␮M) (Fig.
under which there is no hysteresis if the enzyme was not pre- 7A), and addition of 1 mM GSH did not affect the enzymatic
treated) (Fig. 6B). In addition, we examined whether Grx activity (data not shown). Altogether, these results indicated
domain contributed to glutathionylation. For this purpose, we that the C-terminal and Grx redox centers are essential for
evaluated glutathionylation and TR activity of TRGCUG at high GR activity. Interestingly, and similar to what was observed
[GSSG]. We found that the TR activity was unaffected at high for the TR activity, the double mutant TGRGUCG exhibited
[GSSG] using the Trx-coupled assay (Fig. 6C); however, the significant GR activity (Fig. 7B). We then studied the effect of
peptide containing Cys354 was not detected in TRGCUG (either GSH addition on the GR activity of the double mutant. At
native or glutathionylated). high concentration of GSSG (1 mM), the addition of 1 mM
Both the Grx Domain and the Sec Residue Are Essential for GSH abolished hysteresis (Fig. 7C). Finally, we examined
GR Activity—We further investigated whether the GR activity whether a combination of functional TR domains (TRGCUG)
was affected by the Grx domain and the Sec residue at the with functional Grx domain (TGRGC*) displays GR activity.
C-terminal redox center. TRGCUG, TGRGCCG, and TGRGC* did No activity was observed at high concentrations of both pro-
not display significant activity at the standard conditions of the teins, indicating that electron transfer between these two
assay (i.e. 1 mM GSSG), even at high concentrations of enzymes proteins is not efficient. This also rules out the possibility
(200 nM). Because the phenomenon of hysteresis observed for that the ⬃10-fold Grx excess with respect to functional TR
the GR activity is dependent not only on the enzyme concen- domains present in TGRGCUG and TGRGUCG (due to the

17904 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 26 • JUNE 27, 2008
 Linked Thioredoxin-Glutathione Systems
and/or vaccine development (8). In
this study, we provide further evi-
dence that validates TGR as a key
platyhelminth molecule to target.
We have previously shown that
cytosolic and mitochondrial vari-
ants of TGR from E. granulosus
derive from a single gene and have
the same amino acid sequence, once
FIGURE 8. Auranofin effect on E. granulosus larval worms. Protoscoleces were incubated in vitro at 37 °C, 5% the leader peptide of the mitochon-
CO2 in DMEM with 10 ␮M auranofin, a TGR inhibitor, or its vehicle (DMSO) as a control. A, control protoscoleces
after 30 h of culture. B, treated protoscoleces after 12 h of culture (all protoscoleces were dead, note the drial isoform is removed. We now
disorganization of the parenchyma and the loss of the hooks or the entire crown of hooks). C, treated proto- show, by transient expression in
scoleces after 30 h of culture. The scale bar on C corresponds to 100 ␮m. eukaryotic cells that the mitochon-
drial variant of TGR and a putative
mitochondrial Trx from E. granulosus localize to the mitochon-
drial compartment when assessed by confocal microscopy. No
experimental evidence for mitochondrial co-localization of
platyhelminth TGR and Trx had been previously reported. We
thus also proved that TGR provides electrons to E. granulosus

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cytosolic and mitochondrial Trxs as well as to GSSG, indicating
the existence of TGR-dependent functional thiol systems in
cytosol and mitochondria. Furthermore, TGR functions
equally well with both Trx isozymes, with a catalytic efficiency
of 107 M⫺1 s⫺1, similar to the values reported for other TGRs
and TRs. The Km and kcat values for Trxs are higher than those
reported for mammalian TRs (⬃3 ␮M and 40 to 60 s⫺1, respec-
tively) (15, 28, 29). However, Km values of the TGRs of the
phylogenetically closest organisms to E. granulosus (T. crassi-
ceps and S. mansoni) are also higher than those for mammalian
FIGURE 9. Hysteretic behavior and glutathionylation of TGR. At high GSSG
concentrations, the GR activity of TGR exhibited hysteretic behavior (filled
TRs. The reported kcat values for these TGRs are lower than the
squares); TGR was found to be glutathionylated under hysteresis and deglu- reported herein for E. granulosus TGR, but it should be noted
tathionylated once hysteresis was abolished. At low GSSG concentrations, that no corrections by protein selenium content have been
the GR activity did not exhibit hysteretic behavior (open squares), and the
enzyme was not glutathionylated. The figure also shows that hysteretic done in those cases (6, 8).
behavior is favored by high [GSSG]/[GSH] ratios or low enzyme concentra- During characterization of the GR activity of E. granulosus
tions and is relieved at low [GSSG]/[GSH] ratios or high enzyme TGR we observed that at low concentrations the enzyme exhib-
concentrations.
ited hysteretic behavior. Furthermore, we showed that the
prevalent truncated forms present in these preparations) can [GSSG]/[GSH] ratio controlled the activity of the enzyme: the
affect the GR activity of these selenoproteins. lag time correlated directly with GSSG concentration and
Auranofin, a TGR Inhibitor, Kills Larval Worms at Low inversely with GSH concentration. Hysteretic behavior has
Concentrations—Because glutathione and thioredoxin activi- been associated with changes in conformation and/or oli-
ties depend on TGR in both compartments, mitochondria and gomerization in response to substrates, products, or modifiers
cytosol, we evaluated, in animal culture, the effects of auranofin (30). We did not detect changes in the oligomerization state of
on protoscoleces (larval worms). All protoscoleces were dead TGR upon incubation of the enzyme with high GSSG concen-
12 h after addition of 10 ␮M auranofin. The disruption of pro- trations by size exclusion chromatography (data not shown).
toscoleces tegument integrity was the first microscopic sign of Our results strongly indicated that the observed hysteretic phe-
the drug effect; latter changes included disorganization of the nomenon correlated to the glutathionylation state of TGR as
protoscolex parenchyma (Fig. 8). At 5 ␮M auranofin, 20% of summarized in Fig. 9. Further evidence that the hysteretic
protoscoleces were dead at 12 h, and 100% at 30 h. At 2.5 ␮M behavior of TGR was due to glutathionylation derived from the
auranofin, all protoscoleces were dead after 48 h, whereas at 1 fact that, once GSSG was removed using a desalting column,
␮M auranofin, 90% of protoscoleces survived, but development the glutathionylated enzyme was hysteretic even at low GSSG
toward cyst was severely compromised. We then evaluated the concentrations. Of the two Cys residues found to be glutathio-
effect of auranofin on protoscoleces subjected to oxidative nylated, Cys88 is present in vertebrate TGRs and in Grx,
stress (100 ␮M hydrogen peroxide). A lower percentage of pro- whereas Cys354 is present only in E. granulosus TGR (supple-
toscoleces, only 60%, survived at 1 ␮M auranofin. mental Fig. S2). Interestingly, Cys88 is close to the mobile linker
of Grx-TR domains (100 –105), and Cys354 is located close to a
DISCUSSION solvent accessible mobile loop (359 –363) (18). Glutaredoxins
Previous studies have shown that TGR is an essential enzyme have been shown to catalyze deglutathionylation of target pro-
in platyhelminth parasites and an attractive target for drug teins (31–33). Thus, an additional issue relates to whether

JUNE 27, 2008 • VOLUME 283 • NUMBER 26 JOURNAL OF BIOLOGICAL CHEMISTRY 17905
Linked Thioredoxin-Glutathione Systems
deglutathionylation is autocatalyzed by TGR. We observed mutant) (38). Our results suggest that Sec not only provides
deglutathionylation of TGR by GSH in the absence of NADPH a catalytic advantage over Cys at the nucleophilic position,
(data not shown). This suggests that deglutathionylation under but a Cys to Sec mutation at the resolving position can also
these conditions can occur, but catalysis by the Grx domain enhance enzymatic activity compared with the correspond-
cannot be ruled out. Because TGR catalyzes numerous thiol- ing Cys form. The fact that Sec is superior to Cys at the
(selenol)-based reactions, and at the same time its activity is resolving position of the redox active center may also apply
controlled by the [GSSG]/[GSH] ratio, it constitutes an inter- to other selenoproteins and the corresponding thiol oxi-
esting model to study enzymatic regulation by glutathionyla- doreductases. It would be interesting to explore this possi-
tion/deglutathionylation, as well as substrate inhibition and bility in the context of engineering Cys-containing enzymes
product activation phenomena. for enhanced catalysis.
The fact that hysteresis is found at high GSSG concentrations Regarding the role of the Grx domain in TGR functions, the
raises the question of whether this phenomenon takes place in comparison of wild-type TGR and TR revealed that the Grx
vivo. It should be noted that parasites are under oxidative stress domain does not positively or negatively affect the TR activity
mainly due to the host’s immune response. This generates not of the enzyme, indicating that the Grx domain neither assists
only GSSG, but also thiyl radicals, sulfenic acids, and S-nitrosy- nor hinders the TR activity of TGR. In contrast, the TR module
lated glutathione and protein intermediates, which have been of TGR has no GR activity, and thus both the Grx and TR
proposed as glutathione donors and acceptors for protein- domains are needed for this activity.
SSG formation, respectively (34). Glutathionylation of pro- Our data clearly indicate that efficient GSSG reduction
teins has been proposed to regulate diverse intracellular sig- requires both the C-terminal Sec-containing redox center

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naling pathways, particularly in mammalian cells under and the N-terminal Grx domain, in agreement with the
oxidative stress (9, 31, 34) and to provide a protective mecha- model originally proposed for mammalian TGR (13), and
nism for the damaging effects of oxidative agents (34, 35). Our later supported by characterization of the enzyme (17). This
results indicated that TGR activity is preserved for reduction of model put forward that electrons can flow from the C-ter-
Trx during oxidative stress; under these conditions TGR would minal GCUG redox center directly to Trx, or to the N-ter-
be inactivated for the GR function, whereas the TR function minal CXXC redox center of the Grx domain and finally to
would remain unaffected. GSSG. This implies that a conformational switch must exist
To gain further insights into the catalytic mechanism of to allow either Trx or the “in built” Grx domain to receive
TR and GR activities of TGR, we generated a set of con- electrons from the C-terminal redox center. An alternative
structs designed to study the roles of its C-terminal redox electron pathway for GSSG reduction has recently been sug-
center and the N-terminal Grx domain. Consistent with the gested based on the crystallographic structure of a C-termi-
results obtained for mammalian TGR (17), we found that the nally truncated S. mansoni TGR. To account for the residual
Sec residue of TGR is essential for both activities. The Sec to GR activity of the truncated enzyme, the authors proposed
stop mutant (a truncated mutant at the penultimate amino that GSSG could be reduced directly by the CXXXXC redox
acid) has negligible GR and TR activities. Furthermore, a Sec to center of TR domains (18). The fact, that auranofin, an inhib-
Cys mutation also results in a remarkable loss of TR activity and itor of Sec-containing TRs and TGRs, inhibits not only TR
almost complete loss of GR activity. These results indicated but also GR activities of S. mansoni TGR, suggests that the
that in E. granulosus the overall redox homeostasis is con- proposed alternative electron pathway is marginal and is
trolled by TGR and is dependent on selenium. A striking unlikely to be physiologically relevant (8, 27).
observation was that the additional mutation at the C-termi- Finally, we performed in vitro studies on E. granulosus larval
nal redox center (TGRGUCG) partially compensated for the worms to evaluate the effects of auranofin. Concentrations as
loss of activity caused by the single Sec to Cys mutation low as 2.5 ␮M of auranofin kill all larval worms within 48 h, and
(TGRGCCG). This recovery of activity was observed with all lower concentrations severely hampered in vitro development
substrates studied: DTNB, thioredoxin, and GSSG. Seleno- of larval worms toward cyst. The effect of the drug on larval
protein redox active sites are characterized by the presence worms challenged with hydrogen peroxide was even more
of Sec at the nucleophilic (attacking) position, and a Cys marked. Low concentrations of auranofin have also been
residue is usually observed at the resolving position of the reported to rapidly kill juvenile and adult S. mansoni in culture.
catalytic mechanism (17, 36 –38). The higher nucleophilicity Mammalian cells, however, are able to survive higher concen-
and low pKa of the selenol group of Sec (39) are thought to trations of auranofin (8).
confer Sec a catalytic advantage over Cys at the attacking Data available indicate that in platyhelminth parasites the
position. In addition, recent evidence supports the model biochemical scenario of thiol-disulfide redox homeostasis
that Sec is the leaving group during reduction of the C ter- differs greatly from that of their mammalian hosts. In platy-
minus during the catalytic cycle (38). No natural selenopro- helminths, the overall redox homeostasis is controlled by
tein with Sec at the resolving position has been described TGR and is dependent on selenium. The substitution of con-
(40), and it is assumed that Sec would not confer a significant ventional Trx and GSH systems by linked systems makes
advantage to the catalytic efficiency when present at this TGR (the molecular link for Trx and glutathione-dependent
position. Furthermore, a semisynthetic mammalian TR with functions) a target molecule for drug or vaccine develop-
an inverted C-terminal active site resulted in 100-fold ment. In this study, we demonstrated the existence of func-
decrease in catalytic activity (comparable to the Sec to Cys tional linked systems in both mitochondria and the cytosol

17906 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 26 • JUNE 27, 2008
 Linked Thioredoxin-Glutathione Systems
that depends on a single TGR. Thus, targeting this enzyme 16. Sun, Q.-A., Yalin, W., Zappacosta, F., Jeang, K.-T., Lee, B. J., Hatfield, D. L.,
can be safely expected to compromise the overall cellular and Gladyshev, V. N. (1999) J. Biol. Chem. 274, 24522–24530
17. Sun, Q. A., Su, D., Novoselov, S. V., Carlson, B. A., Hatfield, D. L., and
redox homeostasis in these organisms.
Gladyshev, V. N. (2005) Biochemistry 44, 14528 –14537
18. Angelucci, F., Miele, A. E., Boumis, G., Dimastrogiovanni, D., Brunori, M.,
Acknowledgments—We greatly appreciate the gift of pSUABC from
and Bellelli, A. (2008) Proteins, in press
Dr. Elias Arnér (Karolinska Institutet). We also thank Lic. Bruno 19. Arnér, E. S., Sarioglu, H., Lottspeich, F., Holmgren, A., and Bock, A. (1999)
Manta for technical assistance and Dr. Beatriz Alvarez, Dr. Alvaro J. Mol. Biol. 292, 1003–1016
Dı́az, and Dr. Cecilia Fernández for helpful discussions. We also 20. Rengby, O., Johansson, L., Carlson, L. A., Serini, E., Vlamis-Gardikas, A.,
thank the Institut Pasteur, Montevideo (Unidad Tecnológica de Karsnas, P., and Arnér, E. S. (2004) Appl. Environ. Microbiol. 70,
Bioquı́mica y Proteómica Analı́ticas), for technical assistance with 5159 –5167
mass spectrometry. 21. Bar-Noy, S., Gorlatov, S. N., and Stadtman, T. C. (2001) Free Radic. Biol.
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