falciparum has been shown recently to possess two major functional redox systems: a thioredoxin system [7,8] comprising NADPH, thioredoxin reductase TrxR, thioredoxin Trx [8,9] and thior
Trang 1P R I O R I T Y P A P E R
Plasmoredoxin, a novel redox-active protein unique
for malarial parasites
Katja Becker1, Stefan M Kanzok2, Rimma Iozef1, Marina Fischer1, R Heiner Schirmer2
and Stefan Rahlfs1
1
Interdisciplinary Research Center, Justus-Liebig-University, D-35392 Gießen, Germany;2Biochemistry Center,
Ruprecht–Karls-University, D-69120 Heidelberg, Germany
Thioredoxins are a group of small redox-active proteins
involved in cellular redox regulatory processes as well as
antioxidant defense Thioredoxin, glutaredoxin, and
try-paredoxin are members of the thioredoxin superfamily and
share structural and functional characteristics In the
mal-arial parasite, Plasmodium falciparum, a functional
thio-redoxin and glutathione system have been demonstrated
and are considered to be attractive targets for antimalarial
drug development
Here we describe the identification and characterization of
a novel 22 kDa redox-active protein in P falciparum As
demonstrated by in silico sequence analyses, the protein,
named plasmoredoxin (Plrx), is highly conserved but found
exclusively in malarial parasites It is a member of the
thio-redoxin superfamily but clusters separately from other
members in a phylogenetic tree We amplified the gene from
a gametocyte cDNA library and overexpressed it in E coli
The purified gene product can be reduced by glutathione but
much faster by dithiols like thioredoxin, glutaredoxin, try-panothione and tryparedoxin Reduced Plrx is active in an insulin-reduction assay and reduces glutathione disulfide with a rate constant of 640M )1Æs)1at pH 6.9 and 25C; glutathione-dependent reduction of H2O2and hydroxyethyl disulfide by Plrx is negligible Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis As demonstrated
by Western blotting, the protein is present in blood-stage forms of malarial parasites
Based on these results, plasmoredoxin offers the oppor-tunity to improve diagnostic tools based on PCR or immunological reactions It may also represent a specific target for antimalarial drug development and is of phylo-genetic interest
Keywords: antioxidant; malaria; Plasmodium falciparum; redox-metabolism; thioredoxin superfamily
The malarial parasite, Plasmodium falciparum is
respon-sible for more than 2 million deaths per year and novel
antiparasitic drugs are urgently and continuously required
[1,2] Malarial parasites are exposed to high fluxes of
reactive oxygen species (ROS) and for this reason, proteins
involved in antioxidant defense are promising targets for
antimalarial drug development [3–6] P falciparum has been
shown recently to possess two major functional redox
systems: a thioredoxin system [7,8] comprising NADPH,
thioredoxin reductase (TrxR), thioredoxin (Trx) [8,9] and
thioredoxin dependent peroxidases (TPx) [10–14] and a
glutathione system comprising NADPH, glutathione reductase (GR) [15], glutathione, glutathione S-transferase [16] and glutaredoxin (Grx) [17]
The thioredoxin superfamily includes the redox-active proteins thioredoxin, glutaredoxin, tryparedoxin, protein disulfide isomerase and DsbA (disulfide bond forming proteins of bacteria) [18,19] All members of this family share the thioredoxin-fold consisting of a central five-stranded b-sheet surrounded by four a-helices [20], and an active site with two conserved cysteine residues that specify the biological activity of the protein [18,19] Thioredoxins are a group of small ( 12 kDa) proteins with the classical active site sequence, CGPC They contribute to a range of essential cellular functions including protection from ROS, reduction of enzymes such as ribonucleotide reductase and thioredoxin peroxidase, and regulation of transcription factors [18–21] Mammalian Trx have been shown to function as cellular growth factors, to modulate apoptosis and to be highly expressed and secreted by certain tumor cells [22]
Glutaredoxins with a similar size are part of the glutathione system and characterized by the active site sequence, CPYC They also protect against oxidative damage, serve as hydrogen-donors for ribonucleotide reductase and are associated with transcriptional control [17,19,21,23] As shown for yeast cells, at least one out of
Correspondence to K Becker, Interdisciplinary Research Center,
Heinrich-Buff-Ring 26–32, Justus-Liebig-University,
D-35392 Gießen, Germany,
Fax: + 49 641 9939129; Tel.: + 49 641 9939120.
E-mail: becker.katja@gmx.de
Abbreviations: BSA, bovine serum albumin; GR, glutathione
reductase; Grx, glutaredoxin; GSH, glutathione, reduced;
GSSG, glutathione, oxidized; GST, glutathione S-transferase;
Plrx, plasmoredoxin; Trx, thioredoxin; TrxR, thioredoxin reductase;
TPx, thioredoxin dependent peroxidase.
Note: K.B and S.M.K contributed equally to this work.
(Received 2 December 2002, revised 28 January 2003,
accepted 3 February 2003)
Trang 2four Trx and Grx genes has to be present for viability [24].
The presence of both thioredoxins and glutaredoxins in
different organisms, together with the conservation of their
active sites through evolution, point to the importance of
these antioxidative and regulatory proteins for central
cellular functions As a third family of redox-active proteins
with functions comparable to Trx and Grx, tryparedoxins
have been described in trypanosomes and crithidiae,
unicellular parasites lacking a glutathione system [25,26]
Here we describe the identification and characterization
of a novel functional redox-active protein in the malarial
parasite, P falciparum Together with thioredoxins,
gluta-redoxins and trypagluta-redoxins, this protein represents a
member of the thioredoxin superfamily The presence of
the protein is restricted to malarial parasites where it is likely
to be involved in ribonucleotide reduction and glutathione
homeostasis
Materials and methods
PCR
Perfect match primers (forward: 5¢-ATGGCGTGCC
AAGTTGATAA-3¢; reverse: 5¢-TGCTGTCTGTAACCA
CACA-3¢) were designed and PCR was carried out with a
P falciparumgametocyte cDNA as a template; the forward
primer introduced a BamHI restriction site, the reverse
primer a PstI restriction site The PCR conditions were
chosen as follows: (a) 94C, 30 sec; (b) 80 C, hold; (c)
94C, 30 sec; (d) 60 C, 30 sec; (e) 72 C, 2 min; (f)
30· steps c–e; (g) 72 C, 3 min; (h) 15 C, hold The
amplified 570 bp PCR product was digested with the
corresponding restriction enzymes, purified and cloned into
the expression vector, pQE30, that had been cleaved
previously with BamHI/PstI The resulting
plasmid-con-struct was sequenced and showed 100% identity to the
genome sequence
Overexpression and purification of PfPlrx
The Qiagen expression-system (pQE30 vector, that adds an
N-terminal hexahistidyl-tag to the protein for
affinity-purification, and M15 E coli expression cells) was used for
overexpression and purification of Plrx The relative
mole-cular mass of the pure protein (as judged by silver stained
SDS/PAGE and gel filtration using a calibrated
Sepha-dex G-75 column) was 21.4 kDa (calculated 21 684 Da)
The calculated absorption coefficient, e280nm, of PfPlrx w as
determined to be 31.4 mM )1Æcm)1
Immunoblotting
Intraerythrocytic stages of P falciparum were cultured
in vitro as described previously [17] Rabbit antiserum
raised against recombinant PfPlrx was obtained from
BioScience, Go¨ttingen, Germany The reaction of the
antibodies with authentic PfPlrx in P falciparum
tropho-zoite extracts, as well as with recombinant protein, was
studied by Western blotting Samples were subjected to 12%
SDS/PAGE and then blotted on a polyvinylidene difluoride
membrane using a semi-dry blot procedure (50 mA for
55 min) As a secondary antibody, peroxidase-conjugated
porcine anti-(rabbit Ig) Igs (Dako Diagnostika, Hamburg, Germany) were used
Enzyme assays Ribonucleotide reductase activity was determined from the rate of conversion of [3H]GDP into [3H]dGDP essentially as described for CDP reduction [27] The assay mixture (200 lL) contained 50 mM Hepes, 100 mM KCl, 6.4 mM
MgCl2, 500 lM GDP (including 1.25 lCi [3H]GDP),
100 lM dTTP, variable concentrations of PfPlrx, E coli thioredoxin, and Trypanosoma brucei thioredoxin, respect-ively T brucei R1 subunit (1 mU, 1.48 lM) w ith a 67-fold molar excess of the R2 subunit (99 lM) w as used (1 U corresponds to 1 lmol dGDP formation per min)1) The mixture was incubated at 37C for 20 min and the reaction was stopped by boiling for 10 min Precipitated protein was removed by centrifugation at 13 000 g, and products and educts were dephosphorylated by 45 min incubation with
10 U alkaline phosphatase Nucleoside, deoxynucleoside and free bases were then separated isocratically by HPLC
on an Aminex A9 anion exchange column (250· 4 mm) in
100 mMsodium borate, pH 8.3 [28,29]
Glutathione reductase [15], thioredoxin reductase [9] and trypanothione reductase activities [29,30] were determined spectrophotometrically at 340 nm monitoring the consump-tion of NADPH as described previously In these assays up
to 100 lMPfPlrx was tested as the substrate The detection limit in these assays is DA¼ 0.002Æmin)1, that corresponds
to an NADPH oxidation rate of 0.3 lMÆmin)1 and an activity of 0.3 mUÆmL)1of an NADPH dependent disulfide reductase The insulin-reduction assay is described in the legend to Fig 3; P falciparum thioredoxin used for this assay was expressed and purified as described previously [9] Reaction of PfPlrx with different reducing agents Reduction of PlrxS2by trypanothione and tryparedoxin
In trypanosomes and other Kinetoplastida, a major relay system of electron transferring reactions exists that comprises of NADPH, trypanothione reductase, trypano-thione, tryparedoxin and a terminal acceptor such as ribonucleotide reductase [30] The interactions of Plrx with this system were tested as described for the GHOST assay [7] Briefly, 1 mL assay mixture at 25C w ere used The compounds were added in the following order: buffer (40 mMHepes, 1 mMEDTA at pH 7.5), NADPH (200 lM
final concentration), Trypanosoma cruzi trypanothione reductase (80 nM¼ 0.25 enzyme units), PlrxS2, trypanothi-one disulfide (20–50 lM) and T brucei tryparedoxin disul-fide (4.5 lM) In a series of assays, the order of additions was changed so that PlrxS2was added at differing steps in the sequence of additions
In the course of the reaction sequence, essentially, each disulfide is reduced completely to the corresponding dithiol, NADPH oxidation being the driving force After each addition to the assay mixture, the absorbance decrease at
340 nm due to NADPH oxidation was registered and the rate of the respective reaction was calculated according
to the equation: v¼ Dc · min)1¼ DA/(1 min · e · 1 cm) [lM· min)1], where the e)value for NADPH is 6.22 m )1Æcm)1 From a given value of v, the rate constant
Trang 3kwas determined using the equation for a second order
reaction: k¼ v/{[R(SH)2]· [PlrxS2]} Assay conditions for
the reduction of Plrx by other reducing agents are given in
the legend to Table 1
Reduction of GSSG by PfPlrx
PfPlrx was prereduced with 1 mM dithiothreitol The
protein was then separated rapidly from excess
dithiothre-itol by affinity chromatography using Ni-nitrilotriacetic acid
agarose Reduced PfPlrx (12.5 or 25 lM) was then
incuba-ted for 30 s and 15 min at 4C and 25 C, respectively, with
GSSG (25 or 50 lM) in 50 mMpotassium phosphate, 1 mM
EDTA, 200 mM KCl at pH 6.9 This incubation was
followed by addition of 100 lMNADPH and 50 mUÆmL)1
human glutathione reductase in order to determine the
concentration of residual GSSG PfPlrx was found to reduce
GSSG in a nonenzymatic reaction The rate constant, k,
of this reaction was calculated as v/[Plrx(SH)2]·
[GSSG]· min on the basis of the following experiment
Reduction of 25 lM GSSG (25C for 30 s) with PfPlrx
(12.5 lM) led to 19 lMresidual GSSG; this corresponds to
the reduction of 12 lM GSSG per min Thus, k was
calculated to be 0.0384 lM )1Æmin)1 In parallel experiments,
we removed Plrx after the reaction with GSSG using
Ni-nitrilotriacetic acid agarose Subsequently, the thiol
content, representing the formed GSH, was measured in
the solution
Results and discussion
In the genome of the malarial parasite, P falciparum [31] a
gene showing sequence similarities with thioredoxin genes
was identified The sequence consisted of an exon
contain-ing 537 bp located on chromosome 3 The gene was
amplified by PCR using a gametocyte cDNA as a template,
sequenced, cloned into an expression vector, and
over-expressed in E coli The deduced amino acid sequence
(PfPlrx; accession number AAF87222) comprised 179
residues (22 kDa) and contained the unique active site
motif, WCKYC, when compared with other members of the thioredoxin superfamily The novel protein was named plasmoredoxin (Plrx) Putative plasmoredoxins of compar-able size were also identified by in silico analyses in the genomes of the Plasmodium species, P vivax [32], P berg-hei, P yoelii, and P knowlesi (this paper) The correspond-ing amino acid sequence alignments showed identities of 67.4, 66.9, 72.6 and 67.2% with P falciparum plasmo-redoxin (Fig 1) The identity of PfPlrx with other members
of the thioredoxin superfamily, for example PfTrx (31.4%)
or PfGrx (27.5%) were significantly lower Apart from members of this superfamily, the highest degrees of identity (31.3 and 32.6%) were with ResA (P35160), a respiration regulating protein of Bacillus subtilis, and HelX (M96013),
a putative periplasmic disulfide oxidoreductase of the photosynthetic bacterium, Rhodobacter capsulatus, respect-ively Homology modelling based on the SWISS PROT
program resulted in a partial three-dimensional structure
of Plrx Residues 43–94, representing 28% of the complete amino acid sequence were modelled and indicated a characteristic thioredoxin fold including the active site sequence, WCKYC In a reconstructed phylogenetic tree, plasmoredoxins cluster as one group separate from thio-redoxins, glutaredoxins and tryparedoxins (Fig 2) Within the plasmoredoxins, the rodent parasites P yoelii and
P berghei Plrx share the highest degree of amino acid identity (91%), followed by the P vivax/P knowlesi pair with 87.6% P falciparum are in between these two groups This result suggests a close relationship between P knowlesi that infects monkeys and P vivax that causes tertian malaria in man
Interestingly, two similar sequence annotations (Gen-Bank accesson numbers, NP_473166 and CAB38989) were available that proposed a large protein of 2417 and 2396 amino acids, respectively, with a putative structural function
in the cytoskeleton of P falciparum These annotations suggested that plasmoredoxin might be part of this large protein as a possible second exon To check this possibility,
a PCR with exon-overlapping primers [one primer in putative exon 1 (the big structural protein), the other primer
Table 1 Reduction of PlrxS 2 by dithiols and glutathione at 25 °C.
Reductant k · 10 3 [l M )1 Æmin)1] k [ M )1 Æs)1] Conditions
Dihydrolipoamide 2.0 33 pH 7.4, 50 m M phosphate, 1 m M EDTA
P falciparum glutaredoxin b 14 230 pH 7.4, 100 m M Tris
P falciparum thioredoxinc 2.2 37 pH 7.4, 100 m M phosphate
T brucei brucei tryparedoxin d 30 503 pH 7.5, 40 m M Hepes
a
Assays were performed in 100 m M Tris, 1 m M EDTA, at different pH values adjusted at 25 C, in the presence of 200 l M NADPH,
1 UÆmL)1PfGR, 0.5–10 m M GSH, and 50 l M PlrxS 2 For the reaction of PlrxS 2 with glutathione, the limited data set did not allow us to distinguish between pseudosecond and third order kinetics b Assays were performed as above but in the presence of 1 m M GSH and 10–60 l M PfGrx At Grx concentrations ‡ 20 l M no clear increase in DAÆmin)1 value was detected The rate constant was therefore calculated on the basis of the value determined for 10 l M Grx c The reaction of Plrx (25–50 l M ) with thioredoxin (100 l M ) was determined
in 100 m M potassium phosphate, 2 m M EDTA, pH 7.4 in the presence of 1 UÆmL)1PfTrxR and 200 l M NADPH d Assay conditions for the reaction of Plrx with the trypanothione system are given in the Materials and methods section.
Trang 4in putative exon 2 (the plasmoredoxin)] was performed
using PfcDNA as a template Under various PCR
condi-tions, however, no product was obtained indicating that
PfPlrx is unlikely to represent a part of the protein encoded
by exon 1 and indeed, very recently both former sequence
predictions were updated and split into two parts resulting
in a putative protein of 2226 amino acids and a second predicted protein of 179 amino acids representing plasmo-redoxin
As summarized in Table 1, PfPlrx can be reduced by different dithiols as well as by GSH Most effective were
P falciparum glutaredoxin and T brucei tryparedoxin
Fig 1 Alignment of the amino acid sequence of plasmoredoxin from Plasmodium falciparum with putative homologues of different Plasmodium species.
Pf, P falciparum (GenBank AAF87222); Pv, P vivax (GenBank AAF99466); Py, P yoelii (GenBank EAA16465; gnl|py|TIGR_c5m141); Pk,
P knowlesi (gnl|pk|Sanger_PKN.0.004551); Pb, P berghei (gnl|pbgss|UFL_249PbC01, gnl|pbgss|UFL_204PbH08, gnl|pbgss|UFL_225PbD05), this sequence was generated from three different genomic clones and is likely to lack a small fragment of the sequence Identical amino acids are highlighted, the putative active site is boxed.
Fig 2 Phylogenetic relations of plasmoredoxins, thioredoxins, glutaredoxins, and tryparedoxins Plasmoredoxins represent a novel family of redox-active proteins belonging to the thioredoxin superfamily The sequence comparisons were carried out using the CLUSTAL W program of the EMBL European Bioinformatics Institute (www2.ebi.ac.uk./clustalW/) Pk, P knowlesi; Pv, P vivax; Pf, P falciparum; Py, P yoelii; Pb, P berghei; Hs, Homo sapiens; Ec, E coli; Tb, T brucei; Cf, Crithidia fasciculata; Tc, T cruzi; Plrx, plasmoredoxin; Trx, thioredoxin; Trp, tryparedoxin; Grx, glutaredoxin.
Trang 5Whether the reduction of Plrx by GSH is physiologically
significant might be questioned as the pseudosecond order
rate constant was only 1.6M )1Æs)1 at pH 7.4 and 25C
Concentration-dependent redox activity of Plrx was
dem-onstrated by its ability to cleave disulfide bonds of insulin
when using dithiothreitol as a source of reducing equivalents
(Fig 3) In this assay, P falciparum thioredoxin served as a
positive control and dithiothreitol as well as bovine serum
albumin as negative controls Using the glutathione system
as a primary source of reducing equivalents, the
insulin-reduction by 5 lMPlrx was too slow to be detected at the
physiological pH of 7.4 but a clear reaction was apparent at
pH 8.0
Interestingly, PfPlrx was found to be no substrate for
thioredoxin reductase from P falciparum, E coli, and man;
of glutathione reductase from P falciparum and man and
trypanothione reductase from T cruzi In each case, the
specific activity was below the detection limit of
25 mUÆmg)1enzyme protein
To test whether Plrx modulates glutathione reductase and
thioredoxin reductase activity, respectively, Plrx was
pre-reduced by incubation with 2 mMdithiothreitol Residual
dithiothreitol was removed by affinity chromatography on a
Ni-nitrilotriacetic acid column Directly after elution, 20 lM
Plrx(SH)2was added to a standard GR assay, pH 6.9 [15],
and a TrxR assay, pH 7.4, containing 20 lM PfTrx [9],
respectively The addition of reduced Plrx did not influence the reaction catalysed by the disulfide reductases at 25C The ability of PfPlrx to reduce hydroxyethyl disulfide GSH-dependently was tested in an assay system typically used for characterizing glutaredoxins [17] The assay (in
100 mM Tris, 1 mM EDTA, pH 8.0) contained 100 lM
NADPH, 0.25 UÆmL)1 PfGR, 1 mM GSH as well as different concentrations of PfGrx and PfPlrx, and was started with 735 lMhydroxyethyl disulfide In a reference cuvette containing no Grx/Plrx, the spontaneous reaction between GSH and hydroxyethyl disulfide was accounted for Grx (20 nM) produced an DAÆmin)1 value of 0.051, corresponding to a kcatof 410 min)1(see also [17]) Plrx (25 and 75 lM) resulted in DAÆmin)1values of 0.025 and 0.070, respectively, corresponding to a kcatof 0.15 min)1 Thus, the GSH-dependent hydroxyethyl disulfide reducing activity of PfPlrx is by a factor of almost 3000 lower than the activity of PfGrx1 [17]
Peroxidase activity of PfPlrx was tested in 100 mMTris,
1 mM EDTA, pH 7.4 (or 8.0) in the presence of 200 lM
NADPH, 1 UÆmL)1PfGR, 2 mMGSH and 50 lMPlrxS2 After 15 min preincubation, which guaranteed the reduc-tion of PlrxS2, 200 lM H2O2 was added The resulting DAÆmin)1value was higher by£ 0.01 than the one of the controls carried out in the absence of Plrx at both pH values This indicated an extremely slowreaction between Plrx(SH)2and H2O2– the second order rate constant being
£ 1.6 · 10)4 lM)1Æmin)1 – when comparing Plrx with known peroxidases of P falciparum [10–14]
Plasmoredoxin, in its dithiothreitol-reduced form, was tested successfully as a hydrogen donor for T brucei ribonucleotide reductase This result points to an in vivo contribution of PfPlrx to DNA synthesis The reduction of ribonucleotide reductase is, in most organisms, produced by Trx and Grx; in Trypanosomes, tryparedoxin was shown to have a comparable function [18,19,25,29]
Reduced PfPlrx was furthermore shown to reduce quantitatively glutathione disulfide A 15-min incubation
of 25 lMPfPlrx with 50 lMGSSG resulted in the formation
of 50 lM GSH, as indicated by a decrease of the GSSG concentration from 50)25 lM The concomitant determi-nation of 44.2 lMGSH makes it unlikely that glutathion-ylated Plrx is a major reaction product The following reaction scheme is therefore proposed:
PfPlrxðSHÞ2 þ GSSG ! PfPlrx ðS-SÞ þ 2 GSH According to the data obtained with different substrate concentrations and incubation times, the lower limit of the k-value for this chemical reaction can be estimated as 0.01 lM )1Æmin)1at 4C and of 0.04 lM )1Æmin)1at 25C For many thioredoxins (with the notable exception of PfTrx) the corresponding rate constant is£ 0.01 lM )1Æmin)1
at 25C [9]
The reduction of GSSG is, in most organisms, conducted
by the NADPH-dependent flavoenzyme, glutathione reductase (GR) [3] However, we have shown recently that insects including Drosophila melanogaster and Anophe-les gambiaelack a genuine GR although they contain high concentrations of glutathione [33] In this context, a nonenzymatic reduction of GSSG by reduced thioredoxin was described for different organisms and proposed to have
Fig 3 Insulin-reduction activity of PfPlrx in comparison with PfTrx In
this assay, the precipitation of reduced insulin B-chains is followed at
600 nm One ml of reaction mixture contained 0.17 m M porcine insulin
in 50 m M Tris/HCl, 2 m M EDTA at pH 7.4 The reaction was started
at 25 C by adding 1 m M dithiothreitol in the presence of 2 l M PfTrx
(closed square), 2 l M PfPlrx (closed triangle) or 5 l M PfPlrx (cross).
1 m M dithiothreitol without protein (closed diamond) served as a
negative control Addition of 5 l M bovine serum albumin to the
dithiothreitol control gave identical results In additional assays,
dithiothreitol was replaced by a reducing system consisting of either
200 l M NADPH, 1 UÆmL)1PfGR, 2 or 10 m M GSH, 5 l M Plrx or of
200 l M NADPH, 1 UÆmL)1PfTrxR, 5 l M Plrx at pH 6.9, 7.4 and 8.0.
Only at pH 8.0 and 10 m M GSH was a clear insulin reducing activity
observed within 30 min (open square) At pH 8.0 the reduction of
5 l M Plrx by 1 m M dithiothreitol (closed circle) was also more efficient
than at pH 7.4.
Trang 6in vivorelevance [9,33] Obviously, PfPlrx, as a member of
the thioredoxin superfamily, is also able to fulfil this
function The stoichiometric reaction observed for PfPlrx
and GSSG may contribute to antioxidant defense and
specific redox regulatory processes in malarial parasites that
growand multiply in an environment of high oxygen
tension [34]
P falciparum plasmoredoxin is a member of a novel
family of redox active proteins belonging to the thioredoxin
superfamily PfPlrx is larger than classical thioredoxins,
glutaredoxins and tryparedoxins, it shares, however, typical
structural and functional characteristics with the other three
groups The reactions of P falciparum plasmoredoxin with
rabbit IgG raised against the recombinant protein were
demonstrated by Western blotting As shown in Fig 4,
single bands of expected sizes (24 kDa, due to the His-tag
and 22 kDa) appeared when probing the recombinant
protein and a trophozoite extract of P falciparum This
result, and the fact that PfPlrx was amplified from a cDNA
library, indicate that the gene is transcribed and the protein
is present in blood-stage forms of the parasite that cause
malaria in the human host Considering the above may
allowa unique avenue for developing diagnostic tools based
on PCR or immunological methods Many organisms,
including E coli, D melanogaster, yeast and man possess
more than one Trx or Grx As indicated by our studies,
P falciparumpossesses at least one thioredoxin [9], at least
two glutaredoxin-like proteins [8,17] and the newly
discov-ered plasmoredoxin In this context it is furthermore
interesting to note that until nowthe gene of only one
glutathione S-transferase has been detected in the genome
of P falciparum [16] and that no glutathione-dependent
peroxidase has been discovered so far The gene believed to
represent a GPx, was found to code for a thioredoxin
dependent peroxidase [12] Taken together, these data might
indicate that P falciparum does not use glutathione
dependent reactions to the extent described for other
organisms In other words, malarial parasites might have
developed a unique additional defense line against oxidative stress, and an additional source of reducing equivalents for deoxyribonucleotide synthesis as well as for signalling processes Indeed, the multiplication rate of P falciparum
is among the fastest in eukaryotic organisms Potential roles
of plasmoredoxin in redox metabolism of P falciparum are delineated in Fig 5
Fig 4 Western blot of P falciparum plasmoredoxin Lane 1: Recom-binantly produced P falciparum plasmoredoxin (200 ng); lane 2: extract of the P falciparum strain 3D7 (18 lg total protein) The molecular masses of the standard proteins on lane 3 are given on the right hand side.
Fig 5 The putative roles of P falciparum plasmoredoxin (Plrx) in redox metabolism of the parasite Only proteins/pathways that have been verified to exist in P falciparum are shown NADPH represents the major source
of reducing equivalents in the infected erythrocyte Both, thioredoxin reductase (TrxR) and glutathione reductase (GR) reduce their respective substrates, thioredoxin (Trx) and glutathione disulfide (GSSG) by using NADPH Trx reduces Trx-dependent peroxi-dases as well as ribonucleotide reductase (RiboR) Reduced glutathione (GSH) serves
as a substrate of glutathione S-transferase (GST) or reduces glutaredoxin, which in turn
is able to provide RiboR with electrons Plasmoredoxin is, like thioredoxin, able to reduce RiboR as well as GSSG and can be reduced by GSH, Trx, and glutaredoxin.
Trang 7According to our data, Plrx seems to be present in
Plasmodiumspecies only, rendering the protein and/or the
gene a specific diagnostic tool for clinical and
epidemiolo-gical studies Furthermore, Plrx as an antioxidant protein
that is also involved in DNA synthesis may represent a
potential drug target
Acknowledgements
The technical assistance of Elisabeth Fischer, Petra Harwaldt and Beate
Hecker is acknowledged The authors wish to thank R L
Krauth-Siegel, Heidelberg, for performing the ribonucleotide reductase assays
and for kindly providing the components of the tryparedoxin-reducing
system We also thank Julia K Ulschmid and Scott Mulrooney for
helpful discussion Sequence data for P vivax/berghei/falciparum was
obtained from the University of Florida Gene Sequence Tag Project
Website at: http://parasite.vetmed.ufl.edu Funding was provided by
the National Institute of Allergy and Infectious Diseases (for P berghei
and P vivax data) and University of Florida Division of Sponsored
Research and the Burroughs Wellcome Fund (for P falciparum data).
Sequence data for P falciparum chromosome 3 was obtained from The
Sanger Centre website at
http://www.sanger.ac.uk/Projects/P_falcipa-rum/ Sequencing of P falciparum chromosome 3 was accomplished as
part of the Malaria Genome Project with support by The Wellcome
Trust Our work on redox metabolism of malarial parasites is
supported by the Deutsche Forschungsgemeinschaft (grants, SFB 535
to K B and Schi 102/8-1 to R H S.).
References
1 Olliaro, P (2001) Mode of action and mechanisms of resistance
for antimalarial drugs Pharmacol Ther 89, 207–219.
2 Greenwood, B & Mutabingwa, T (2002) Malaria in 2002 Nature
415, 670–672.
3 Schirmer, R.H., Mu¨ller, J.G & Krauth-Siegel, R.L (1995)
Disulfide-reductase inhibitors as chemotherapeutic agents: the
design of drugs for trypanosomiasis and malaria Angew Chem.
Int Ed Engl 34, 141–154.
4 Krauth-Siegel, R.L & Coombs, G.H (1999) Enzymes of parasite
thiol metabolism as drug targets Parasitol Today 15, 404–409.
5 Becker, K., Gromer, S., Schirmer, R.H & Mu¨ller, S (2000)
Thioredoxin reductase as a pathophysiological factor and drug
target Eur J Biochem 267, 6118–6125.
6 Davioud-Charvet, E., Delarue, S., Biot, C., Schwo¨bel, B.,
Boehme, C.C., Mu¨ssigbrodt, A., Maes, L., Sergheraert, C.,
Grellier, P., Schirmer, R.H & Becker, K (2001) A prodrug form
of a Plasmodium falciparum glutathione reductase inhibitor
con-jugated with a 4-anilinoquinoline J Med Chem 44, 4268–4276.
7 Kanzok, S.M., Rahlfs, S., Becker, K & Schirmer, R.H (2002)
Thioredoxin, thioredoxin reductase, and thioredoxin peroxidase
of the malaria parasite Plasmodium falciparum Methods Enzymol.
347, 370–381.
8 Rahlfs, S., Schirmer, R.H & Becker, K (2002) The thioredoxin
system of Plasmodium falciparum and other parasites Cell Mol.
Life Sci 59, 1024–1041.
9 Kanzok, S.M., Schirmer, R.H., Tu¨rbachova, I., Iozef, R &
Becker, K (2000) The thioredoxin system of the malaria parasite
Plasmodium falciparum Glutathione reduction revisited J Biol.
Chem 275, 40180–40186.
10 Kawazu, S., Tsuji, N., Hatabu, T., Kawai, S., Matsumoto, Y &
Kano, S (2000) Molecular cloning and characterization of a
peroxiredoxin from the human malaria parasite Plasmodium
fal-ciparum Mol Biochem Parasitol 109, 165–169.
11 Rahlfs, S & Becker, K (2001) Thioredoxin peroxidases of the malarial parasite Plasmodium falciparum Eur J Biochem 268, 1404–1409.
12 Sztajer, H., Gamain, B., Aumann, K.D., Slomianny, C., Becker, K., Brigelius-Flohe, R & Flohe´, L (2001) The putative glu-tathione peroxidase gene of Plasmodium falciparum codes for a thioredoxin peroxidase J Biol Chem 276, 7397–7403.
13 Krnajski, Z., Walter, R.D & Mu¨ller, S (2001) Isolation and functional analysis of two thioredoxin peroxidases (peroxiredox-ins) from Plasmodium falciparum Mol Biochem Parasitol 113, 303–308.
14 Kaw azu, S., Komaki, K., Tsuji, N., Kaw ai, S., Ikenoue, N., Hatabu, T., Ishikawa, H., Matsumoto, Y., Himeno, K & Kano,
S (2001) Molecular characterization of a 2-Cys peroxiredoxin from the human malaria parasite Plasmodium falciparum Mol Biochem Parasitol 116, 73–79.
15 Fa¨rber, P.M., Arscott, L.D., Williams, C.H Jr, Becker, K & Schirmer, R.H (1998) Recombinant Plasmodium falciparum glu-tathione reductase is inhibited by the antimalarial dye methylene blue FEBS Lett 422, 311–314.
16 Harwaldt, P., Rahlfs, S & Becker, K (2002) Glutathione S-transferase of the malarial parasite Plasmodium falciparum Characterization of a potential drug target Biol Chem 383, 821– 830.
17 Rahlfs, S., Fischer, M & Becker, K (2001) Plasmodium falci-parum possesses a classical glutaredoxin and a second, gluta-redoxin-like protein with a PICOT homology domain J Biol Chem 276, 37133–37140.
18 Arne´r, E.S & Holmgren, A (2000) Physiological functions of thioredoxin and thioredoxin reductase Eur J Biochem 267, 6102–6109.
19 Holmgren, A (2000) Antioxidant function of thioredoxin and glutaredoxin Antioxid Redox Signal 2, 811–820.
20 Martin, J.L (1995) Thioredoxin – a fold for all reasons Structure
3, 245–250.
21 Hirota, K., Matsui, M., Murata, M., Takashima, Y., Cheng, F.S., Itoh, T., Fukuda, K & Yodoi, J (2000) Nucleoredoxin, gluta-redoxin, and thioredoxin differentially regulate NF-kappaB, AP-1, and CREB activation in HEK293 cells Biochem Biophys Res Commun 274, 177–182.
22 Powis, G., Mustacich, D & Coon, A (2000) The role of the redox protein thioredoxin in cell growth and cancer Free Radic Biol Med 29, 312–322.
23 Luikenhuis, S., Perrone, G., Daw es, I.W & Grant, C (1998) The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species Mol Biol Cell 9, 1081–1091.
24 Draculic, T., Dawes, I.W & Grant, C.M (2000) A single gluta-redoxin or thiogluta-redoxin gene is essential for viability in the yeast Saccharomyces cerevisiae Mol Microbiol 36, 1167–1174.
25 Gommel, D.U., Nogoceke, E., Morr, M., Kiess, M., Kalisz, H.M.
& Flohe´, L (1997) Catalytic characteristics of tryparedoxin Eur.
J Biochem 248, 913–918.
26 Nogoceke, E., Gommel, D.U., Kiess, M., Kalisz, H.M & Flohe´,
L (1997) A unique cascade of oxidoreductases catalyses trypa-nothione-mediated peroxide metabolism in Crithidia fasciculata Biol Chem 378, 827–836.
27 Engstrom, Y., Eriksson, S., Thelander, L & Akerman, M (1979) Ribonucleotide reductase from calf thymus Purification and properties Biochemistry 18, 2941–2948.
28 Willing, A., Follmann, H & Auling, G (1988) Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme Eur J Biochem 170, 603–611.
Trang 829 Reckenfelderba¨umer, N., Lu¨demann, H., Schmidt, H., Steverding,
D & Krauth-Siegel, R.L (2000) Identification and functional
characterization of thioredoxin from Trypanosoma brucei brucei.
J Biol Chem 275, 7547–7552.
30 Lu¨demann, H., Dormeyer, M., Sticherling, C., Stallmann, D.,
Follmann, H & Krauth-Siegel, R.L (1998) Trypanosoma brucei
tryparedoxin, a thioredoxin-like protein in African trypanosomes.
FEBS Lett 431, 381–385.
31 Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M.,
Hyman, R.W., Carlton, J.M., Pain, A., Nelson, K.E., Bowman,
S., Paulsen, I.T., James, K., Eisen, J.A., Rutherford, K., Salzberg,
S.L., Craig, A., Kyes, S., Chan, M.S., Nene, V., Shallom, S.J., Suh,
B., Peterson, J., Angiuoli, S., Pertea, M., Allen, J., Selengut, J.,
Haft, D., Mather, M.W., Vaidya, A.B., Martin, D.M., Fairlamb,
A.H., Fraunholz, M.J., Roos, D.S., Ralph, S.A., McFadden, G.I.,
Cummings, L.M., Subramanian, G.M., Mungall, C., Venter, J.C.,
Carucci, D.J., Hoffman, S.L., Newbold, C., Davis, R.W., Fraser,
C.M & Barrell, B (2002) Genome sequence of the human malaria
parasite Plasmodium falciparum Nature 419, 498–511.
32 Carlton, J.M., Angiuoli, S.V., Suh, B.B., Kooij, T.W., Pertea, M.,
Silva, J.C., Ermolaeva, M.D., Allen, J.E., Selengut, J.D., Koo,
H.L., Peterson, J.D., Pop, M., Kosack, D.S., Shumw ay, M.F., Bidwell, S.L., Shallom, S.J., van Aken, S.E., Riedmuller, S.B., Feldblyum, T.V., Cho, J.K., Quackenbush, J., Sedegah, M., Shoaibi, A., Cummings, L.M., Florens, L., Yates, J.R., Raine, J.D., Sinden, R.E., Harris, M.A., Cunningham, D.A., Preiser, P.R., Bergman, L.W., Vaidya, A.B., van Lin, L.H., Janse, C.J., Waters, A.P., Smith, H.O., White, O.R., Salzberg, S.L., Venter, J.C., Fraser, C.M., Hoffman, S.L., Gardner, M.J & Carucci, D.J (2002) Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii Nature 419, 512– 519.
33 Kanzok, S.M., Fechner, A., Bauer, H., Ulschmid, J.K., Mu¨ller, H.M., Botella-Munoz, J., Schneuw ly, S., Schirmer, R & Becker, K (2001) Substitution of the thioredoxin system for glutathione reductase in Drosophila melanogaster Science 291, 643–646.
34 Schirmer, R.H., Krauth-Siegel, R.L & Schulz, G.E (1989) Glu-tathione reductase In Coenzymes and Cofactors: GluGlu-tathione, (Dolphin, D., Avramovic, O & Poulson, R., eds), Vol 3 Part A,
pp 553–596 Wiley & Sons, NewYork, USA.