From the Cátedra de Inmunología,
Facultad de Química/Ciencias, Universidad de la
República, Avenida Alfredo Navarro 3051, Piso 2, CP 11.600, Montevideo, Uruguay and ¶ Sección Bioquímica,
Facultad de Ciencias, Universidad de la República, Iguá
4225, CP 11.500, Montevideo, Uruguay
Received for publication, September 10, 2002
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ABSTRACT |
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Thioredoxin and glutathione systems are
the major thiol-dependent redox systems in animal cells.
They transfer via the reversible oxidoreduction of thiols the reducing
equivalents of NADPH to numerous substrates and substrate reductases
and constitute major defenses against oxidative stress. In this study,
we cloned from the helminth parasite Echinococcus
granulosus two trans-spliced mRNA variants that encode
thioredoxin glutathione reductases (TGR). These variants code for
mitochondrial and cytosolic selenocysteine-containing isoforms that
possess identical glutaredoxin (Grx) and thioredoxin reductase (TR)
domains and differ exclusively in their N termini. Western blot
analysis of subcellular fractions with specific anti-TGR antibodies
showed that TGR is present in both compartments. The biochemical
characterization of the native purified TGR suggests that the Grx and
TR domains of the enzyme can function either coupled or independently
of each other, because the Grx domain can accept electrons from either
TR domains or the glutathione system and the TR domains can transfer
electrons to either the fused Grx domain or to E. granulosus thioredoxin.
The reversible thiol-disulfide reaction is a central theme in
biology. On the one hand, this redox exchange is an efficient mechanism
of electron transport. On the other hand, this reaction is a molecular
device used by nature as a switch to control protein function and
localization through the redox state of critical thiol groups (1).
The central role in thiol-disulfide exchange in animal cells is played
by the thioredoxin (Trx)1 and
glutathione (GSH) systems. Both systems have overlapping and distinct
properties and targets but function in a similar way. They transfer via
the reversible oxidoreduction of thiols the reducing equivalents of
NADPH to numerous substrates and substrate reductases (2). They
maintain the cellular redox homeostasis and constitute major defenses
against oxidative stress, acting directly by reducing oxidized
compounds and proteins or providing reducing equivalents to the
hydrogen peroxide reductases present in animal cells, namely
thioredoxin peroxidase and glutathione peroxidase (3). Both systems
provide electrons to ribonucleotide reductase and control vital
cellular processes such as transcription and signal transduction
through the redox regulation of kinases, phosphatases, and
transcription factors (4-6).
The animal thioredoxin system comprises the thioredoxin reductase (TR)
and Trx, whereas the glutathione system consists of glutathione
reductase (GR), glutathione (GSH), and glutaredoxin (Grx). Trx and Grx
are thiol-disulfide oxidoreductases that transfer electrons to various
substrates and substrates reductases. GSH, another thiol-based
reductant, recycles Grx to its reduced state and reduces other protein
and non-protein substrates. TR and GR are pyridine nucleotide-disulfide
oxidoreductases that transfer the reducing equivalents of NADPH to
oxidized Trx and other substrates and to GSH, respectively. Mammalian
and Caenorhabditis elegans TR possess C-terminal electron
transfer centers containing redox active cysteine and selenocysteine
(Sec) residues (7-10). The selenol group of Sec is a strong
nucleophile (11) that confers a profound reductive capacity to the
enzyme. The C-terminal redox center is not present in GR. The
equivalent redox link in the GSH system is thought to be provided by
GSH (10).
In mammals, both systems are present in mitochondria and the cytosol.
This is because of the existence of genes encoding mitochondrial and
cytosolic variants for the proteins from both systems and also because
of alternative splicing of single genes (12-17). Recently, mitochondrial and cytosolic variants of Drosophila
TR have also been described to be derived from alternative splicing of
a single gene (18).
Recently, a more complex machine exhibiting specificity for both
thioredoxin and glutathione systems has been characterized in mammals
(19). This enzyme termed thioredoxin glutathione reductase (TGR) is a
selenoprotein oxidoreductase that possesses thioredoxin reductase,
glutathione reductase, and glutaredoxin activities, achieving this
broad substrate specificity by a fusion of TR and Grx domains. This
enzyme has been reported to be present in the microsomal fraction of
testis cells from mice. A multifunctional TGR has also been reported in
Schistosoma mansoni, a platyhelminth organism (20),
indicating that this fusion domain has been evolutionarily conserved.
In this article, we report the cloning and characterization of a
thioredoxin glutathione reductase of the larval stage of Echinococcus granulosus, a cestode parasite that
dwells in the liver and lungs of human and cattle hosts. The results
indicate that the enzyme possesses glutathione reductase, thioredoxin
reductase, and glutaredoxin activities and suggest that there is domain
communication between the C-terminal Cys-Sec redox center and the
N-terminal glutaredoxin domain. In addition, we provide conclusive
evidence that mitochondrial and cytosolic variants of TGR are generated from a single TGR gene.
Isolation of Full-length cDNAs Encoding E. granulosus
TGR--
Fertile hydatid cysts (larval stage of E. granulosus, the larva is a fluid-filled cyst bounded by the
hydatid cyst wall; the larval worm or protoscoleces are found within
the cyst) were collected from the lungs of naturally infected cattle in
Uruguay. The hydatid cyst fluid and protoscoleces were aseptically
aspirated with a vacuum pump. Once settled by gravity, the
protoscoleces were extensively washed in phosphate-buffered saline
(PBS) to remove dead protoscolex debris. They were observed under a
light microscope for viability (flame cell and vital dye exclusion).
Total RNA was isolated from freshly isolated E. granulosus
protoscoleces using TriZOL reagent (Invitrogen) and reverse-transcribed at 50 °C using an oligo(dT) primer and Superscript reverse
transcriptase (Invitrogen) following the manufacturer's instructions.
The cDNA obtained was subsequently used as template for PCR
reactions following standard techniques. Sense and antisense
oligonucleotides were designed from the mammalian and C. elegans thioredoxin reductase-conserved amino acid regions
VNVGCIPKKLMH and PNAGEVTQG, respectively. The sense oligonucleotide,
5'-GTGAATGTIGGITGYATYCCTAARAARYTNATGCA-3', was used in polymerase chain
reactions in combination with antisense oligonucleotides R1
(5'-TTGDATVACYTCICCNGCGTT-3') or R2 (5'-TTGDATVACYTCICCNGCATT-3'). A
PCR product was obtained with the sense oligonucleotide and R1. This
product was isolated (GeneClean, BIO101), cloned into pGEM-T-Easy
(Promega), and sequenced using AmpliTaq FS according to manufacturers'
and standard protocols on a ABI Prism 377 DNA sequencer, and found to
encode the central fragment of a thioredoxin reductase.
The sequence information obtained was used to clone the full-length
cDNA. The 3'-end of the gene was PCR-amplified using a gene-specific sense oligonucleotide (5'-CGACAACCGCGTGGTAGGA-3') in
combination with oligo(dT). The 5'-ends of the cDNA were also obtained by reverse transcription-PCR using the gene-specific antisense primer 5'-GGATTTGGCATCCTCCATGTAGTG-3' and the sense oligonucleotide 5'-CACCGTTAATCGGTCCTTACC-3' derived from the sequence of the 36 nucleotide splice leader (SL) sequence present at the 5'-end
of some E. granulosus mRNAs (21). The PCR products
obtained were isolated, cloned and sequenced as described above. Since the amplification of the 5'-end of the cDNA yielded two distinct but sequence-related PCR products, subsequent PCRs were carried out to
confirm the sequences of the two 5'-end cDNA products. These PCRs
were performed using (i) the SL oligonucleotide and two different
gene-specific antisense primers and (ii) two different sense
oligonucleotides corresponding to the junctions of the SL exon (present
in both cDNAs) with the differential downstream nucleotides present
in each of the two cDNAs in combination with a gene-specific
antisense oligonucleotide (see Fig. 4 for details of cDNA
sequences). In all of the cases, sequence analysis was performed using
the following programs: (i) Blast 2.0 (www.ncbi.nlm.nih.gov/BLAST) for searching homologies in
GenBankTM and the echinobase, a recent EST data base of
E. granulosus (22) (www.nematodes.org/Lopho/LophDB.php); (ii) SignalP and
PSORT for detection of localization signals; and (iii) Mfold to
identify secondary mRNA structures
(www.bioinfo.rpi.edu/nzukerm/seganal/).
Partial Characterization of EgTGR Gene--
Genomic E. granulosus DNA was isolated from protoscoleces as described
previously (23). This template was subsequently used in PCR reactions
to isolate the 5'-end of EgTGR gene using the forward
oligonucleotide 5'-ATGTTTGGCTGTCATTGTCT-3' and the reverse primer
5'-GGATTCTTACGAAGCATTTCAACCTG-3'.
Expression and Purification of the Recombinant EgTGR Central
Fragment and Recombinant EgTrx--
The central fragment of the
EgTGR gene corresponding to the initial cDNA fragment
isolated was cloned in the appropriate reading frame into
BamHI/HindIII sites of pET28a expression vector
(Novagen) to produce the His-tagged recombinant fragment. The
Escherichia coli BL21(DE3) strain was transformed
with the construct, and single bacterial colonies were inoculated into
LB medium supplemented with 50 µg ml Antiserum against the EgTGR--
A rabbit polyclonal serum
reactive with EgTGR was obtained by intramuscular immunization with the
purified recombinant fragment of EgTGR. The immunization protocol
consisted of a priming immunization with 300 µg of EgTGR in Freund's
complete adjuvant (Sigma) and two subsequent immunizations on days 30 and 45 with 150 µg of EgTGR in Freund's incomplete adjuvant (Sigma).
On day 55, the rabbit was bled and the reactivity of the serum-analyzed
by Western blot against the recombinant fragment of EgTGR and total
protoscolex extract (see below).
Purification of E. granulosus TGR from
Protoscoleces--
Protoscoleces were homogenized under liquid
nitrogen in PBS containing 1 mM phenylmethylsulfonyl
fluoride, 10 mM EDTA, 5 µg ml Subcellular Fractionation of E. granulosus
Protoscoleces--
0.5 ml of gravity-decanted protoscoleces were
suspended in 2.5 ml of ice-cold 10 mM Tris, pH 7.6, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride,
10 mM EDTA, 5 µg ml Western Blot--
Total protoscolex extract and subcellular
fractions were probed with the anti-EgTGR serum and with a monoclonal
antibody that recognizes the cytosolic parasite protein P-29 (24).
Extracts were diluted in 2× loading buffer (62.5 mM Tris,
pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) mercaptoethanol,
0.02% (v/v) bromphenol blue), heated for 5 min at 100 °C, and
centrifuged at 10,000 × g for 10 min. Supernatants
were separated on 10% (w/v) gels and transferred onto nitrocellulose
membranes. The nitrocellulose membranes were incubated with blocking
buffer (PBS, 1% Tween 20) and subsequently incubated with the rabbit
anti-EgTGR serum diluted 1:500 in PBS, 0.1% Tween 20, or with
the monoclonal antibody followed by a 2-h incubation with the
corresponding secondary antibody coupled to alkaline phosphatase.
Protein bands were visualized by revealing the alkaline phosphatase
activity using 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue
tetrazolium as substrates.
Enzyme Assays--
Thioredoxin reductase, glutathione reductase,
and glutaredoxin activities were measured in the native purified TGR.
Thioredoxin reductase activity was assayed by the DTNB reduction assay
(25) and the insulin reduction assay (26). In the DTNB reduction assay,
the purified enzyme was added to a reaction mixture containing 100 mM potassium phosphate, pH 7.0, 10 mM EDTA, 0.2 mg ml
GR activity was assayed as the NADPH-dependent reduction of
GSSG determined as the decrease in absorbance at 340 nm at 25 °C in
a reaction mixture containing 100 mM potassium phosphate, pH 7.0, 10 mM EDTA, 200 µM NADPH, and 1 mM GSSG. One unit of enzyme was defined as the oxidation of
1 µM/min NADPH/min at 25 °C (27).
The glutaredoxin assay was performed as described previously (28). A
reaction mixture containing 1 mM GSH (Sigma), 2 mM EDTA, 0.1 mg ml
Thioredoxin reductase, GR, and Grx assays were carried out in the
absence and presence of 10 nM auronofin, a gold compound known to selectively inhibit the redox active selenol group of the
enzyme (29). At the concentration used, the activity of control yeast
glutathione reductase was not affected. For assays in presence of
auronofin, the purified enzyme was preincubated with NADPH. All of the
assays were carried out in triplicates.
In addition, thioredoxin reductase and citrate synthase activities were
determined in the subcellular fractions of E. granulosus protoscoleces. Citrate synthase activity was determined according to
Darley-Usmar et al. (30).
Cloning of a cDNA (EgTGR) Coding for a Pyridine
Nucleotide-disulfide Oxidoreductase Containing Selenocysteine from
E. granulosus--
Degenerate primers derived from consensus
C. elegans and mammalian TR amino acid sequences
were successfully used to clone the central fragment of a
E. granulosus thioredoxin reductase cDNA by
PCR. This information allowed the cloning of two full-length cDNAs
encoding TGR variants that differ in their N-terminal sequences but
encode identical Grx and TR domains. The full-length cDNAs sequences have been deposited in GenBankTM. The deduced
amino acid sequences are shown in Fig. 1.
Specific features derived from the sequence analysis are presented and discussed below.
The analysis of the deduced amino acid sequence of the cloned cDNAs
indicates the presence of characteristic thioredoxin reductase domains,
namely the FAD- and NADPH-binding domains and interface domain as well
as the conserved active site sequence CVNVGC and the additional
C-terminally located redox active center. This latter electron transfer
center displays the molecular signature characteristic of selenium
thioredoxin reductases containing the amino acid SEC (U in one-letter
code) within the conserved motif GCUG (Fig. 1). The Sec amino acid
co-translationally incorporated to the protein synthesis is encoded by
an in-frame UGA codon and decoded as a UGASec codon by a
selenocysteine incorporation signal (SECIS) element present in the
3'-untranslated region of Echincoccus cDNA,
180 bp downstream of the UGASec codon (Fig.
2). An UAG stop codon is present in EgTGR
cDNAs that are two codons downstream from the UGASec
codon.
The SECIS element present in EgTGR mRNA possesses the
characteristic stem loop structure (31-33) containing the functional core with the AUGA sequence in the 5'-arm and the GA in the 3'-arm of
the stem and the unpaired AAN bases in the apical loop, both elements
spaced by a 11-bp helix. Furthermore, the location of all of the
structural features (helix I, internal loop, core, helix II, and apical
loop) of the stem loop is well conserved with respect to vertebrate
SECIS elements (see Fig. 2). It has recently been proposed that UG-,
UC-, and CA-rich sequences around the UGASec codon of
mRNAs of mammalian selenoproteins may also participate in decoding
the UGASec codon. Although the location of these regions
may vary between mRNAs encoding selenoproteins, they adopt a
stem-loop secondary structure (34). Putative UC-, UG-, and CA-decoding
regions surrounding the UGASec could be identified in EgTGR
cDNA. Moreover, the sequence around this codon may fold as a
stem-loop-like structure (data not shown).
E. granulosus TGR Possesses TR and Glutaredoxin Domains That
Confers the Enzyme the Capacity to Shuttle Electrons to Targets of Both
GSH and Trx Systems--
In addition to the TR domains, the EgTR
cDNAs encode an N-terminal Grx domain. This domain possesses the
structural features required for Grx activity: the redox center
CXXC characteristic of glutaredoxin, a GSH binding motif,
and the hydrophobic surface area (Fig. 1). A similar domain fusion has
recently been described for a mouse thioredoxin reductase (19). The
enzyme was renamed by the authors as TGR, because the isolated enzyme
possessed not only TR activity but also glutathione reductase and
glutaredoxin activity. More recently, a TGR cDNA encoding a TGR has
been isolated from the trematode parasite S. mansoni
and the enzyme was characterized displaying similar biochemical
characteristics to the mouse TGR (20). Similar to the Schistosome TGR
and unlike the mammalian TGR, the glutaredoxin domain of EgTGR
belongs to the two Cys glutaredoxin subfamilies.
The native purified TGR was purified from protoscolex extracts by
sequential salt precipitation, ion-exchange chromatography, and
affinity chromatography on an ADP-agarose column. After the affinity
chromatography, the preparation contained no visible bands other than a
66-kDa protein on SDS-PAGE (Fig. 3). The
native enzyme possessed TR activity toward both the general substrate DTNB and recombinant E. granulosus thioredoxin
(Table I). NADPH-reduced TR was
completely inhibited by near stochiometric concentration of auronofin,
giving further confirmatory evidence that EgTGR is a selenoenzyme.
Interestingly, no bands in the range of 52-57 kDa typical of isolated
vertebrate TRs were observed in the purified active fraction,
suggesting that most of the TR activity in the protoscolex extract
corresponds to the isolated 66-kDa TGR.
Glutathione reductase assays were also performed with the purified
E. granulosus TGR. The results indicate that the enzyme is
also capable of reducing oxidized glutathione (Table I) with a specific
activity similar to mouse TGR (19). The enzyme activity was inhibited
by auronofin, indicating that the Cys-Sec redox center participates in
the mechanism of electron transfer to oxidized glutathione (Table
I).
Glutaredoxin assays were performed as described previously (28) in a
reaction mixture containing GR and GSH; GSH but not GR; and neither GR
nor GSH, all of them with or without auronofin. The results are
summarized in Table I. The Grx activity in the presence of GR and
glutathione was 1.7 units min
Taken together, all of these results indicate that both Grx and TR
domains encoded by the EgTGR cDNA are functionally active in the
native enzyme. This domain fusion in EgTGR makes it possible to
transfer electrons from NADPH toward the downstream targets of both
thioredoxin and glutathione systems.
Alternative mRNAs Arising from Trans-splicing Code for
Mitochondrial and Cytosolic Variants of TGR--
Trans-splicing
involving a splice leader (SL) mini exon that is added to the
5'-ends of pre-mRNAs has been recently reported to occur in ~20%
of Echinococcus transcripts (21). Although the function of
trans-splicing in these organisms is still unknown, it has already been
used to construct Echinococcus cDNA libraries from
trans-spliced mRNAs (22). The 5'-end of the EgTGR gene was cloned by a PCR splice leader strategy using an oligonucleotide derived from the E. granulosus SL exon and gene-specific
reverse primers (see "Experimental Procedures"). Two PCR bands of
similar intensities differing in 85 nucleotides were amplified from
protoscolex cDNA (Fig.
4A). The PCR products were
isolated, cloned, and sequenced. The nucleotide sequence revealed that
both cDNAs contained the entire splice leader exon. However, the
longer cDNA contained an 85-bp sequence immediately downstream of
the SL exon, absent in the shorter cDNA. The downstream 3'-end
sequence that follows the extra 85-bp sequence in the longer cDNA
and the SL sequence in the shorter cDNA was identical in both
cDNAs and encodes the N-terminal Grx domain and C-terminal
thioredoxin reductase domains of the TGR (Fig. 4A).
The possibility that the two PCR bands were derived from a PCR artifact
was ruled out by performing different PCR reactions with the SL forward
oligonucleotide and different gene-specific primers. In every case, two
bands of equal intensity and differing in 85 bp were obtained
(data not shown). In addition, PCR were performed using as forward
primers the joining sequences of the SL exon with the two downstream
bases of each cDNA. in either case, a single band of the expected
size was obtained (data not shown). These independent PCR products were
cloned and sequenced and found to be identical to the originally
isolated cDNAs. In all of the cases, the PCR results were
consistent not only respect to their sizes and sequences but also with
regard to band intensity, suggesting that both mRNAs are produced
in similar amounts.
The results indicate that both mRNAs contain the trans-spliced
leader exon and are derived from a single TGR gene, because the coding sequence for the Grx and TR domains is identical in both
cDNAs. Two plausible models for generation of the mature trans-spliced mRNAs from the TGR gene are depicted in
Fig. 4, B and C. Both of them predict that the
longer cDNA contains an extra exon not present in the shorter
cDNA and the existence of an intron between the first and second
exons of EgTGR gene. The model depicted in Fig.
4B involves a single primary transcript that is
alternatively trans-spliced immediately upstream of a first exon,
(route a) and immediately upstream of a second exon (route b). The former pre-mRNA then would be cis-spliced
joining the first and second exons (route a). An alternative
explanation for the results would be the existence of two alternative
primary transcripts from the TGR gene (Fig. 4C),
each of them trans-spliced at the 5'-end. In this latter model, the
longer primary transcript would contain exons I and II and an intron
between them, whereas the shorter primary transcript would not contain
the first exon. This model implies the existence of a transcriptional
initiation site within the intron.
Whether trans-splicing occurs on a single primary transcript or on two
alternative transcripts, an intron should be located between the first
and second exons so that there is an acceptor site for
cis/trans-splicing. Thus, we examined the 5'-end genomic organization
of EgTGR gene (see "Experimental Procedures") and found
that the gene encoding EgTGR possesses a first exon that corresponds to
the cDNA portion present in the longer cDNA and absent in the
shorter cDNA followed by an intron and a downstream exon (Fig.
4C). The intron contains a GT-donor and an AG-acceptor site
consistent with the models (Fig. 4D). In addition, an
examination of the intron sequence revealed the presence of a TATA
consensus sequence 80 nucleotides upstream of TGR exon II (Fig.
4D), consistent with the model depicted in Fig.
4C.
Translation of the two EgTGR cDNAs revealed a different first AUG
codon for each cDNA in a favorable Kozak consensus sequence for
initiation of translation (Fig. 4A). The open reading frame encoded by the shorter cDNA is 597 amino acids long, whereas the longer cDNA contains an open reading frame of 624 amino acids coding for an extra 27 amino acid extension at the N terminus of the
polypeptide chain, which is absent in the translated sequence of the
shorter cDNA. An examination of the sequences with SignalP and
PSORT programs indicated the presence of a predicted mitochondrial translocation signal in the N-terminal region of the longer translated product (Fig. 4A). Thus, the shorter mRNA would encode a
leaderless TGR that by default would localize to the cytosol, and the
longer cDNA would encode a mitochondrial TGR variant. The
presequence present in the predicted mitTGR possesses all
the elements to be decoded by the trans-locases of the outer and inner
mitochondrial membranes and assures its correct sorting to the
mitochondrial matrix (35). It contains several arginines, hydroxylated
and hydrophobic residues, and no acidic residues (Fig. 4A).
In addition, the leader presequence forms an amphipathic
Because the gene product variants would localize to different cellular
compartments, we examined the presence of TGR in the different
subcellular fractions obtained by differential centrifugation of a
protoscolex extract (see "Experimental Procedures"). To this end,
the central fragment of EgTGR was cloned for expression as a His-tagged
fusion and the purified recombinant protein was used to raise a rabbit
antiserum. The Western blot showed the presence of a single band of 66 kDa in the total protoscolex extract in the cytosolic fraction, and in
the mitochondrial fraction, no band was observed in either the
microsomal or nuclear fractions (Fig. 5,
panel A). Likewise, thioredoxin reductase activity was significant in the cytosolic and mitochondrial fractions (15 and 13 milliunits min
The apparent molecular mass of the antibody recognized bands (66 kDa) is consistent with the predicted molecular masses of the
cytosolic TGR (65.54 kDa) and the mature
mitTGR (65.47 kDa) as well as with the band size of
the native purified enzyme (Fig. 3). It is worth mentioning that the
antiserum raised against the central fragment of the TGR did not
recognized bands of lower molecular masses indicative of TR. These
results together with the purification of the native enzyme would
suggest that in protoscolex the predominant Se-pyridine
nucleotide-disulfide oxidoreductase is the cloned TGR.
The characterized TGR cDNA and enzyme indicate that the EgTGR
is a selenoenzyme. Indeed, the deduced amino acid sequence contains the
C-terminal molecular signature of Se-TR, the cDNA contains the
SECIS element needed for decoding the UGASec codon, and the activity of the purified enzyme is inhibited by gold compound auronofin. E. granulosus is a cestode platyhelminth. The
presence of selenium in the C-terminal redox center of thioredoxin
reductases and TGR has also been described in the trematode
platyhelminth S. mansoni (20) and in C. elegans,
a nematode helminth (9). Thus, this selenium redox center seems to be
characteristic not only of mammals but also of helminth phyla,
suggesting that it might have appeared before the
protostomate/deuterostomate branching. In lower eukaryotic organisms
such as Plasmodium sp., the C-terminal redox center is
present but contains a pair of redox active Cys residues instead of
Cys-Sec (38). This finding suggests that Se-piridine-disulfide
oxidoreductases might have evolved after the appearance of metazoan
organisms. Intriguingly, the C-terminal redox centers of
Drosophila melanogaster and Anopheles gambiae TRs
do not possess the Cys-Sec redox pair but rather twin cysteines instead
(39). Thus, insect TRs appear to have reverted to the Cys-Cys redox
center. A less likely evolutionary scenario would be the independent
appearance of Se-TR in deuterostomate and protostomate organisms.
Similar to the presence of Sec in the C-terminal redox center of
thioredoxin reductases, it is interesting to note that the Grx-TR
domain fusion is present in helminth and vertebrates and appears to be
absent in Plasmodium sp. and insects.
Two trans-spliced TGR mRNA isoforms were isolated from E. granulosus protoscoleces. The fact that the sequences of both
cDNAs were identical with the exception of the absence of exon I in the shorter cDNA was considered strong evidence for both
cDNAs being derived from a single gene. The partial
characterization of EgTGR gene gave further confirmatory
evidence and suggested possible mechanisms for the generation of the
two mRNA variants. These alternative mRNAs may arise from
trans-splicing on a single primary transcript or on alternative
transcripts (see Fig. 4, B and C). The existence
of a TATA consensus sequence in the intron between exons I and II that
may serve as an alternative site for transcription initiation would
give support to the proposed mechanism that involves two alternative
EgTGR transcripts, each of them trans-spliced at the 5'-end.
Alternative transcription from a single gene followed by trans-splicing
has also been proposed to occur in E. granulosus elp gene (21).
Accumulated recent evidence suggests that alternative splicing and
alternative primary transcripts from single genes represent generalized
processes that increase the diversity of gene expression. Recent
studies on expression of mammalian TR and Grx genes illustrate this
phenomenon. Mammals possess three different TR genes, namely TR1, TR3, and TGR. Whereas TGR codes
for a thioredoxin glutathione reductase, TR1 and
TR3 were originally described as cytosolic and mitochondrial
enzymes, respectively (40). However, it has been recently demonstrated
that both TR1 and TR3 exhibit extensive heterogeneity because of differential transcript splicing (15, 16).
Alternative first exon splicing in human TR3 gene results in
the generation of mitochondrial and cytosolic proteins. A comparison among mouse, rat, and human revealed that the multiple isoforms are
conserved in mammals (16). Similar to what has been described in
mammals, a recent report (41) has revealed that one of the two
Drosophila TR genes codes for two isoforms of the
enzyme, a cytosolic and a mitochondrial one. Likewise, alternative
splicing forms of human, mouse, and rat Grx mRNA do occur. Two
recent reports (17, 42) indicate extensive variations in the
5'-sequences of Grx mRNAs because of alternative first exon
splicing. So far, the generation of gene product variants that localize
to different compartments from a single TGR gene has not
been previously reported. Indeed, mouse TGR originally isolated
from the testis has been reported to be present exclusively in the
microsomal fraction (19). The recently characterized TGR cDNA from
S. mansoni encodes a cytosolic TGR (20). In addition, our
results suggest that alternative transcription and trans-splicing
constitute mechanisms capable of contributing to increase in gene
product diversity for the thioredoxin and glutathione systems and
possibly more general mechanisms that enriches and
regulates the expression of the genomes from organisms
employing RNA trans-splicing.
The two cDNA isoforms encode a mitochondrial and a cytosolic TGR
variant. The immunoblot of subcellular fractions of E. granulosus protoscoleces revealed the presence of TGR in both
compartments. As already mentioned, the mitTGR variant
contains all of the elements to be trans-located to this compartment.
However, there remains an intriguing or at least curious feature of the
N-terminal presequence of mitTGR, the presence of four Cys
residues. In this regard, it is interesting to note that localization
of proteins has been reported to be dependent on the redox state of
thiol groups (43, 44). It is possible to speculate that the
mitochondrial presequence may act as a localization switch, becoming
available to the protein import machinery of the mitochondria only when
the Cys residues of the presequence are oxidized or reduced.
The cytosolic and the mitochondrial processing peptidases processed
mitochondrial TGR variants are expected to be almost identical regarding their biochemical properties, because they have the same
amino acid sequence with the exception of an extra N-terminal Ala
residue in the mitochondrial variant. Parasite material is the major
drawback of studying this organism and precluded purification and
further characterization of each TGR variant. We are currently attempting to clone both variants for expression in a eukaryotic vector.
As expected, the purified native enzyme possesses TR activity. In
addition, the purified TGR possesses Grx activity in a reaction mixture
containing yeast GR and glutathione. This activity was only partially
inhibited by 10 nM auronofin, a concentration that completely inhibited TR activity, and did not affect yeast GR. This
finding suggests that the glutaredoxin domain of TGR can accept
electrons from both the Cys-Sec redox center and the glutathione system. In other words, the Grx domain of TGR can also function independently of TR domains. The purified enzyme also possesses Grx
activity in the absence of yeast GR, this activity being completely inhibited by auronofin, indicating that the Cys-Sec redox center participates in the electron transfer toward oxidized glutathione. Because this assay is driven by the reduction of oxidized glutathione, this result implies that the enzyme also possesses GR activity. Indeed,
in GR assays, the purified enzyme was capable of reducing oxidized
glutathione, an activity inhibited by auronofin, a known selective
inhibitor of selenocysteine containing thioredoxin reductases. Because
conventional thioredoxin reductases (i.e. without a Grx domain) cannot reduce oxidized glutathione, it is reasonable to speculate that the transfer of electrons from the Cys-Sec redox center
toward oxidized glutathione occurs through the Grx domain. Thus, in
this situation, the electron flow in TGR would include a step in the
opposite direction to the flow in the GSH system. Because the standard
redox potentials of GSH ( Thus, taken together, the biochemical characterization of TGR indicates
that the Grx and TR domains of the enzyme can function either coupled
or independently of each other, because the Grx domain can accept
electrons from either TR domains or the glutathione system and the TR
domains can transfer electrons to either the fused Grx domain or to
E. granulosus thioredoxin.
In addition to the biochemical data, the results presented in this
article suggest that EgTGR seems to be the major Se-pyridine-disulfide oxidoreductase present in E. granulosus protoscolex. Indeed,
conventional TR seems to be absent in the larval worm. As already
mentioned, the only protein recognized by an antiserum raised against
the central fragment of EgTGR was the 66-kDa band that corresponds to
TGR. Consistently, purification of TR activity from protoscolex extracts resulted in the exclusive isolation of a 66-kDa band. It is
interesting to note that for the helminth parasite S. mansoni, it has been proposed that both GR and conventional TR may
be absent in the adult worm and that these functions would be carried
out exclusively by TGR (20).
Together, the thioredoxin and glutathione systems constitute mechanisms
of electron transfer to numerous substrates and substrate reductases,
participate in many cellular and metabolic processes, and play an
important role as a primary line of defense against oxidative damage.
The role of the thioredoxin and GSH systems in the mitochondria is not
fully understood. In yeast, the absence of mitochondrial Grx (Grx5)
leads to constitutive oxidative damage, causing iron accumulation in
the cell and inactivation of enzymes requiring iron/sulfur clusters for
their activity (46). E. granulosus is a parasite; thus, it
is subjected not only to endogenous oxidants but also to the oxidative
stress imposed by the cells of the immune system of its hosts.
Activated macrophages and neutrophils can release on the parasite
surface enormous amounts of superoxide anion and nitric oxide that give
rise to hydrogen peroxide and potent oxidants such as hydroxyl radical
and peroxynitrite. Thus, oxidative stress control is central to
parasite survival. We have previously reported the existence of
E. granulosus genes encoding leaderless thioredoxin and
thioredoxin peroxidase and the capacity of the larval worm to be
metabolically competent toward high hydrogen peroxide concentrations
(23) (47). An examination of a recent E. granulosus EST data
base (www.nematodes.org/Lopho/LophDB.php) (22) seems to reinforce the
idea that antioxidant defenses are also critical in the mitochondria.
Both a thioredoxin and a thioredoxin peroxidase with putative
mitochondrial trans-location signal were identified. Therefore,
E. granulosus would possess in the cytosolic and
mitochondrial compartments not only TGR but also Trx and thioredoxin peroxidase.
Thus, the results presented in this article indicate that
E. granulosus thioredoxin glutathione reductase is
a multifunctional parasite enzyme that possesses a functional Grx and
TR domain fusion capable of shuttling electrons to targets of both
thioredoxin and glutathione systems and that mitochondrial and
cytosolic variants of the enzyme are generated from a single
TGR gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 kanamycin.
Recombinant protein expression was induced at
A600 of 0.6 with 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h at
37 °C. Cells were harvested by centrifugation (8000 × g, 30 min), and the pellet was resuspended in 40 ml of lysis
buffer (50 mM phosphate buffer, pH 7.5, 10 mM
imidazole, 300 mM NaCl, 1 mg ml
1 lysozyme).
The bacterial suspensions were sonicated on ice and centrifuged
(14,000 × g, 30 min). The recombinant protein was found entirely in the pelleted fraction. The His-tagged protein was
recovered from the inclusion bodies by resuspending the pellet in 40 ml
of 10 mM Tris 1 mM EDTA, pH 7.8 (TE) TE
and centrifuged for 1 h at 14,000 × g. The
pellet was resuspended in 20 ml of TE buffer, 8 M urea and
incubated for 1 h at 4 °C, and the solution was centrifuged for
1 h at 14,000 × g. The solubilized protein recovered from the supernatant was refolded by sequential dialysis against TE buffer containing 2, 1, and 0.4 M urea and a
final dialysis against 10 mM phosphate buffer, pH 7.4, containing 150 mM NaCl (PBS). The recombinant protein was
analyzed in SDS-PAGE 12% gels under reducing conditions according to
standard techniques. Protein concentration was determined using the BCA
(Pierce), and the protein was subsequently used to immunize a rabbit.
Purification of recombinant EgTrx from a pMal-C2 (New England Biolabs)
plasmid carrying the full-length EgTrx gene was carried out
as described previously (23).
1 leupeptin.
Purification was carried out by a combination of salt precipitation,
ion-exchange chromatography, and affinity chromatography, and each step
was monitored for thioredoxin reductase activity using the
5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) reduction assay (see
below). Ammonium sulfate precipitation of the extract was carried out
in three steps of 30, 55, and 85% saturation. The 55% fraction
contained most of the thioredoxin reductase activity. The active
fraction was dialyzed against 10 mM TE, and applied to a
HiTrap Q-Sepharose HP column (Amersham Biosciences). Bound proteins
were eluted with increasing salt concentration (50, 100, 150, 200, and 300 mM NaCl steps). All of the thioredoxin
reductase activity eluted at 150 mM NaCl. This fraction was
dialyzed against TE, applied onto a 2',5'-ADP-agarose matrix (Sigma),
and washed with TE, 20 mM NaCl, and thioredoxin reductase
eluted with 200 mM NaCl. The purified protein was analyzed
in SDS-PAGE 10% gels under reducing conditions according to standard
techniques. Protein concentration was determined using BCA.
1 leupeptin, and
disrupted on ice in a Potter homogenizer. The suspension was
settled by gravity for 20 min to remove unbroken cells and small
protoscolex fragments. The procedure was repeated twice. Afterward, the
supernatant suspension was separated into four fractions by sequential
centrifugation essentially as described previously (19). The extract
was centrifuged at 900 × g for 30 min, the resulting
supernatant was centrifuged at 10,000 × g for 30 min,
and the remaining supernatant was centrifuged at 120,000 × g for 1 h. The first, second, and third pellet were considered to be the nuclear, mitochondrial, and microsomal fractions, respectively, whereas the final remaining solution was considered as
the cytosolic fraction. The nuclear, mitochondrial, and microsomal fractions were resuspended in the homogenization buffer and
recentrifuged twice before use. The cytosolic fraction was
recentrifuged once. Pelleted fractions were resuspended in 10 mM Tris, pH 7.6, 1 mM phenylmethylsulfonyl
fluoride, 10 mM EDTA, 5 µg/ml leupeptin, sonicated, and
stored at
70 °C until use. Cytosolic and mitochondrial markers
were used as controls of the subcellular fraction identity (see
the next two paragraphs).
1 bovine serum albumin, 5 mM DTNB, and
200 µM NADPH. The reaction was carried out at 25 °C
and monitored by the increase in absorbance at 412 nm. One enzyme unit
is defined as the NADPH-dependent production of 2 µM 2-nitro-5-thiobenzoic acid. For the insulin reduction assay, the purified enzyme was added to a reaction mixture containing 100 mM potassium phosphate, pH 7.0, 1.0 mg
ml
1 insulin, 20 µM recombinant EgTrx, and
200 µM NADPH. NADPH oxidation was followed at 340 nm. One
unit of enzyme was defined as the oxidation of 1 µM/min
NADPH/min at 25 °C.
1 bovine serum albumin, 0.7 mM
-hydroxyethyl disulfide (Acros Organics), and 0.4 units of yeast glutathione reductase (Sigma) was preincubated for 2 min. Afterward, the sample was added and then the reduction of
the mixed glutathione-hydroxyethyl disulfide followed by the oxidation
of NADPH at 340 nm. One unit of Grx activity was defined as the
oxidation of 1 µmol of NADPH/min at 25 °C. Enzyme assays were also
carried out as described above but omitting GR or GR and GSH from the
reaction mixture as indicated in each case.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (107K):
[in a new window]
Fig. 1.
Amino acid sequences of E. granulosus TGRs and homologues proteins. Alignment of
the deduced amino acid sequences of mitochondrial and cytosolic TGR
from E. granulosus (Eg-TGRmit and
Eg-TGRcyt, respectively) with sequences of mouse TGR
(GenBankTM accession number AAK31172) and S. mansoni (GenBankTM accession number AF395822) TGR
(Sman-TGR). Identical residues are shaded in
black, and conserved substitutions are shaded in
gray. The arrow below the sequences indicates the
glutaredoxin domain. The predicted cleavage site of the mitochondrial
signal peptide of Eg-TGRmit is indicated by a closed
triangle. The redox active residues of (i) the glutaredoxin
domain, (ii) the disulfide center in the pyridine nucleotide-disulfide
oxidoreductase domain, and (iii) the C-terminal center containing the
amino acid selenocysteine (denoted by U) are shown by
asterisks above sequences. ( ) denote gaps introduced to
optimize alignment. The cDNA sequences encoding EgTGRmit and
EgTGRcyt have been deposited in GenBankTM under accession
numbers AY147415 and AY147416, respectively.
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Fig. 2.
E. granulosus SECIS element.
Predicted secondary structure of the E. granulosus EgTGR
SECIS element present in the 3'-end untranslated region of EgTGR
cDNAs. ( ) indicates Watson Crick base-pairing; colon
indicates non-Watson Crick interactions at the SECIS core. Consensus
SECIS structural features in the stem loop according to Korotkov
et al. (33) are indicated. Conserved sequences in eukaryotic
SECIS elements present in the apical bulge and SECIS core are shown in
boldface. Nucleotide numbers referring to the cDNA
sequences of EgTGR deposited in GenBankTM are
indicated.
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Fig. 3.
Purified E. granulosus
TGR. SDS-PAGE analysis of the purified E. granulosus TGR under reducing conditions on a 10% resolving gel.
Lane 1, molecular mass markers (Low molecular mass
calibration kit for SDS electrophoresis, Amersham Biosciences, 97, 66, 45, 30, 20.1, and 14.4 kDa); lane 2, purified EgTGR
(sequential purification by ammonium sulfate precipitation, ion
exchange, and ADP-agarose affinity). The gel was silver-stained.
Specific enzymatic activities of EgTGR
1 mg
1, similar
to the activity reported for mouse TGR. This activity was partially
inhibited by auronofin. The purified EgTGR was also capable of reducing
glutathione-hydroxyethyl-mixed disulfide in the absence of yeast GR
with a Grx-specific activity of 1.0 unit min
1
mg
1, and this activity was completely inhibited by
auronofin. The
-hydroxyethyl reductase activity in the absence of
GSH and yeast GR was reduced to 50% of the activity in the presence of
GSH and was negligible when pretreated with auronofin (Table I).
View larger version (25K):
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Fig. 4.
Alternative mRNAs arising from
trans-splicing encode mitochondrial and cytosolic TGR variants.
A, PCR amplification of the 5'-end of TGR cDNA
from E. granulosus protoscoleces using as forward primer an
oligonucleotide derived from the SL exon sequence and a TGR-specific
sequence as reverse primer as described under "Experimental
Procedures." Two PCR products were amplified (right track
of the agarose gel and left track corresponds to molecular
mass markers, X174 Hae). The nucleotide sequences of the PCR
products revealed that both cDNAs contained the entire splice
leader exon (schematically depicted as a dark gray
box). The longer cDNA contained an 85-nucleotide exon
immediately downstream of the splice leader (exon I,
light gray box) followed by the downstream 3'-end sequence
(exon II and downstream exons, white box). The
shorter cDNA did not contain exon I. The SL exon was followed by exon
II and downstream exons. Translation of both cDNAs indicates that the
longer cDNA encodes a mitochondrial TGR variant, whereas the shorter
cDNA codes for a cytosolic TGR variant. The mitochondrial signal
peptide is underlined, and the predicted cleavage site by
the mitochondrial processing peptidase is indicated by a closed
triangle. B, proposed model of alternative
trans-splicing on a single primary transcript. Route a
involves trans-splicing of the SL exon on exon I and then cis-splicing
of exon I and exon II. Route b involves trans-splicing of
the SL exon on exon II. C, proposed model of trans-splicing
on alternative transcripts. From EgTGR gene, two primary
transcripts are synthesized from alternative transcription initiation
sites. Both transcripts are trans-spliced at the 5'-end, and
cis-splicing between exons I and II occurs in the longer transcript
only. This model implies the existence of a promoter sequence within
the intron between exons I and II. D, exon/intron boundaries
and TATA consensus sequence. The intron sequence between exons I and II
is shown in italics. Donor and acceptor splicing sites are
underlined. A TATA consensus sequence 80 nucleotides
upstream of exon II that may serve as alternative transcription
initiation site is also underlined.
-helix with
a positively charged surface on one side and a hydrophobic surface on
the other. The presequence of mitTGR also contains the
critical recognition elements for proteolytic cleavage by the
mitochondrial processing peptidases as follows: the distal and proximal
arginine residues including one located at the invariant
2 position,
proline residues between these regions, and an alanine residue at P1'
site (Fig. 4A) (36, 37). Thus, this presequence once in the
mitochondrial matrix would be removed by the mitochondrial processing
peptidases, giving rise to a mature polypeptide chain of 598 amino
acids (Fig. 4A) that would be identical to the cytosolic
variant with the exception of an extra N-terminal alanine residue
present in the mitTGR variant. The data also indicate that
the mitochondrial trans-location signal coded by the longer cDNA is
formed by the joining of the exons I and II (Fig. 4A).
1 mg
1, respectively) and
negligible in the microsomal and nuclear fractions. A control Western
blot performed using a monoclonal antibody raised against p29, a known
cytosolic protein present in E. granulosus protoscolex (24),
revealed a 29-kDa band only in the cytosolic fraction, suggesting that
there was no cross-contamination of the other subcellular fractions
(Fig. 5, panel B). In addition, citrate synthase activity
was measured in all of the fractions and found to be significant in the
mitochondrial extract exclusively (105 milliunits min
1
mg
1), indicating the identity of the mitochondrial
fraction and absence of cross-contamination of the other subcellular
fractions. These results strongly indicate that the two trans-spliced
mRNAs code for a cytosolic and a mitochondrial TGR variant.
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Fig. 5.
Subcellular localization of TGR by
immunoblot. Western blot of a total E. granulosus
larval worm extract (lane 5) and subcellular fractions of
the larval worm (lanes 1-4, microsomal, cytosolic,
mitochondrial, and nuclear fractions, respectively) probed with an
antiserum raised against a recombinant fragment of EgTGR (panel
A) and with a monoclonal antibody raised against P29, a known
cytosolic protein of E. granulosus (panel B). The
total larval worm extract and subcellular fractions obtained as
described under "Experimental Procedures," were resolved on 10%
gels under reducing conditions and then subjected to immunoblot. Single
bands of 66 kDa were recognized in the total extract and mitochondrial
and cytosolic fractions by anti-TGR antibodies. Single bands of 29 kDa
were recognized in the total extract and the cytosolic fraction by
anti-P29 antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
240 mV) and Grx (
233 to
198 mV) have
near values (45), the reduction of GSH by the Grx domain would be
thermodynamically favorable when the concentrations of reduced Grx and
oxidized glutathione increase. The interaction of the C-terminal redox
center with the Grx domain has been predicted based on molecular
modeling of TGR structure. This together with biochemical data has led
us to suggest that the direction of electron transfer in TGR is from
the Cys-Sec redox center toward the Grx domain and downstream electron
acceptor (19). Although not very efficient, EgTGR was also capable of reducing
-hydroxyethyldisulfide independently of GSH
(i.e. in the absence of exogenous GSH).
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ACKNOWLEDGEMENTS |
---|
We thank Professor Ricardo Ehrlich (Department of Biochemistry, Universidad de la República, Montevideo, Uruguay); Professor Murray Selkirk (Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom); Dr. Cecilia Fernández, Dr. Álvaro Díaz, Florencia Irigoín (Cátedra de Inmunología, Universidad de la República, Montevideo, Uruguay); and Dr. Klaus Brehm (Institut fur Hygiene und Mikrobiologie, Universität Würzburg, Würzburg, Germany) for helpful discussions and suggestions.
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FOOTNOTES |
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* This work was supported in part by University of Uruguay Comision Sectorial de Investigación Científica grant (to C. C. and G. S.), The Wellcome Trust through a Research and Development Award in Tropical Medicine, an International Foundation for Science grant (to G. S.), and a Programa de Desarrollo de las Ciencias Básicas fellowship (to A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY147415 and AY147416 for mitochondrial and cytosolic cDNAs respectively.
§ Present address: Instituto de Biología Molecular y Celular de Plantas, CSIC-Universidad Politécnica de Valencia, Avenida de los Naranjos s/n CP46022, Valencia, Spain.
To whom correspondence should be addressed. Tel.:
5982-4801196; Fax: 5982-4874320; E-mail: gsalin@fq.edu.uy.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M209266200
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ABBREVIATIONS |
---|
The abbreviations used are: Trx, thioredoxin; TR, thioredoxin reductase; GR, glutathione reductase; Grx, glutaredoxin; Sec, selenocysteine; TGR, thioredoxin glutathione reductase; PBS, phosphate-buffered saline; SL, splice leader; EgTGR, E. granulosus TGR; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); SECIS, selenocysteine incorporation signal; mit, mitochondrial; SE, selenium; TE, 10 mM Tris, 1 mM EDTA, pH 7.8.
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