Roles of 1-Cys peroxiredoxin in haem detoxification
in the human malaria parasite Plasmodium falciparum
Shin-ichiro Kawazu
1,2
, Nozomu Ikenoue
1
, Hitoshi Takemae
1,2
, Kanako Komaki-Yasuda
1,2
and Shigeyuki Kano
1
1 Research Institute, International Medical Center of Japan, Tokyo, Japan
2 Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan
Plasmodium falciparum is the parasite that causes falci-
parum malaria, one of the most debilitating and life-
threatening diseases in tropical regions of the world.
The trophozoite of the malaria parasite digests host
haemoglobin to obtain amino acids for metabolism
[1,2]. This process produces a large quantity of haem
(ferriprotoporphyrin IX; FP) in the parasite’s food
vacuole, but the parasite does not possess a haem-
oxygenase in the vacuole or cytosol. The parasite pro-
tects itself from noxious FP through two major
mechanisms. Most FP is polymerized into harmless
haemozoin (malaria pigment) in the food vacuole
[3–5], and the remainder is decomposed by glutathione
(GSH) in the cytosol [5–7]. The latter process produces
free iron, which can enter redox cycling and generate
O
2

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(Received 30 December 2004, revised 9
February 2005, accepted 14 February 2005)
doi:10.1111/j.1742-4658.2005.04611.x
In the present study, we investigated whether Plasmodium falciparum 1-Cys
peroxiredoxin (Prx) (Pf1-Cys-Prx), a cytosolic protein expressed at high lev-
els during the haem-digesting stage, can act as an antioxidant to cope with
the oxidative burden of haem (ferriprotoporphyrin IX; FP). Recombinant
Pf1-Cys-Prx protein (rPf1-Cys-Prx) competed with glutathione (GSH) for
FP and inhibited FP degradation by GSH. When rPf1-Cys-Prx was added
to GSH-mediated FP degradation, the amount of iron released was
reduced to 23% of the reaction without the protein (P<0.01). The rPf1-
Cys-Prx bound to FP–agarose at pH 7.4, which is the pH of the parasite
cytosol. The rPf1-Cys-Prx could completely protect glutamine synthetase
from inactivation by the dithiothreitol–Fe
3+
-dependent mixed-function oxi-
dation system, and it also protected enolase from inactivation by coincuba-
tion with FP ⁄ GSH. Incubation of white ghosts of human red blood cells
and FP with rPf1-Cys-Prx reduced formation of membrane associations
with FP to 75% of the incubation without the protein (P<0.01). The
findings of the present study suggest that Pf1-Cys-Prx protects the parasite
against oxidative stresses by binding to FP, slowing the rate of GSH-medi-
ated FP degradation and consequent iron generation, protecting proteins
from iron-derived reactive oxygen species, and interfering with formation
of membrane-associated FP.

Results and Discussion
Effect of Pf1-Cys-Prx on FP degradation by GSH
When FP was mixed with GSH, we observed a shift in
the maximal absorbance of FP from 390 to 370 nm
and a rapid decline in this peak absorbance, which
may be due to formation of GSH ⁄ FP complexes and
degradation of FP [6,16] (Fig. 1A). A similar shift in
the maximal absorbance was observed when FP was
degraded by GSH in the presence of recombinant
(r)Pf1-Cys-Prx (Fig. 1B); however, the rate of FP deg-
radation, as evaluated by decline in absorbance at
370 nm, was slower than that observed in the reaction
without recombinant protein (Fig. 1C). The rate of FP
degradation was [FP]-dependent with an apparent K
m
of 71 lm and a V
max
of 2.4 nmolÆmin
)1
(Fig. 1D). The
rate in the presence of rPf1-Cys-Prx was also [FP]-
dependent with an apparent K
m
of 100 lm (Fig. 1D).
The double-reciprocal plot showed the effect of com-
petitive inhibition of GSH by recombinant protein
(data not shown).
This modification of FP degradation was not due to
oxidation of GSH by recombinant Prx because the
recombinant protein did not oxidize GSH in either the

300, 600, 1200 s). (B) FP degradation by
GSH in (A) was observed in the presence of
rPf1-Cys-Prx. The absorption spectra were
taken as described in (A) except for an addi-
tional measurement at 2400 s (C) Decrea-
ses in the absorbance at 370 nm in (A, d)
and (B, s). (D) [FP]-dependent rates of FP
degradation by GSH (decrease in absorb-
ance at 370 nmÆmin
)1
) were measured at
RT without (d) or with addition of rPf1-
Cys-Prx (s). Data are representative (A–C)
or means (D) of three experiments.
S i. Kawazu et al. Roles of Prx in heme degradation of P. falciparum
FEBS Journal 272 (2005) 1784–1791 ª 2005 FEBS 1785
protein bound to the agarose. This binding was con-
firmed by the observation that only a trace of the pro-
tein was left in the unbound fraction. Pre-incubation of
the recombinant protein with FP abolished most of this
binding, suggesting that binding of rPf1-Cys-Prx to the
agarose was FP specific. Recombinant PfTPx-1 (2-Cys
Prx) protein (rPfTPx-1), which was included as a negat-
ive control, also bound to FP–agarose, but the binding
was not affected by preincubation of the protein with
FP. These results also indicate that Pf1-Cys-Prx can
bind to FP at pH 7.4, which is the pH of the parasite
cytosol. The fact that the majority of Pf1-Cys-Prx is pre-
sent in the cytosolic fraction of the parasite lysate sug-
gests that Prx is localized in the cytosol (Fig. 3B).

2
O
2
(300 s). Bovine erythrocyte GPx ( ) was used as
a positive control. The rates of substrate reductions by rPf1-Cys-Prx
were equivalent to those of nonenzymatic reaction (data not
shown). Data are representative of three experiments.
A
B
Fig. 3. Binding of Pf1-Cys-Prx to FP. (A) rPf1-Cys-Prx (r1-Cys Prx)
and rPfTPx-1 (r2-Cys Prx) were bound to FP-agarose in NaCl ⁄ P
i
pH 7.4 at RT. The protein was mixed with agarose directly or after
preincubation with FP. Bound and unbound proteins to agarose
were separated by SDS ⁄ PAGE (15% acrylamide), and the gel was
stained with Coomassie brilliant blue. Molecular mass markers
(kDa) are indicated on the left. Lane 1, 10% (1 lg each) of the pro-
teins mixed with agarose (input); lane 2, proteins bound to the
agarose without preincubation; lane 3, proteins bound to agarose
after preincubation; lane 4, unbound proteins recovered from
agarose when binding was performed without preincubation; lane
5, unbound proteins recovered from the agarose when binding was
performed after preincubation. (B) A homogenate of parasite cells
was centrifuged for separation of the parasite cytosol as a soluble
fraction. Homogenate, soluble fraction and pellet were separated
by SDS ⁄ PAGE (12.5% acrylamide), and the proteins were probed
with rabbit anti-[rPf1-Cys-Prx (1-Cys Prx)] IgG. Molecular mass
markers (kDa) are indicated on the left. Lane 1, homogenate
(25 lg); lane 2, pellet corresponding to 25 lg homogenate; lane 3,
soluble fraction corresponding to 25 lg homogenate.

in the inactivation of glutamine synthetase (GS) by the
mixed-function oxidation (MFO) system, which gener-
ates ROS by auto-oxidation of thiol in the presence of
iron, was evaluated (Fig. 4A). The dithiothreitol ⁄ Fe
3+
-
dependent MFO system reduced GS activity to
8.6 ± 2.5% of the initial value. When rPf1-Cys-Prx
was added to the system, complete protection against
inactivation was observed and the initial GS activity
was maintained (116.5 ± 7.3% of the initial activity).
The protective effect was abolished when the recom-
binant protein was heat-inactivated by autoclaving,
suggesting that the native structure is required for the
protective effect. The ability of recombinant Pf1-Cys-
Prx to reduce H
2
O
2
in vitro has been reported [13,17],
and therefore, this peroxidase activity may contribute
to protection of GS from the MFO-derived radicals.
In this system, dithiothreitol in the MFO could act as
a donor for rPf1-Cys-Prx.
Pf1-Cys-Prx could protect yeast enolase from inacti-
vation by coincubation with FP ⁄ GSH (Fig. 4B). The
FP ⁄ GSH coincubation reduced enolase activity to
4.6 ± 5.0% of the initial value. When rPf1-Cys-Prx
was added to the system, complete protection against
inactivation was observed (124.4 ± 29% of the initial

and HG + autoclaved rPf1-Cys-Prx, respectively. Data are mean +
SD of three experiments.
S i. Kawazu et al. Roles of Prx in heme degradation of P. falciparum
FEBS Journal 272 (2005) 1784–1791 ª 2005 FEBS 1787
peroxidase activity. The identity of the physiological
electron donor for 1-Cys Prx remains controversial in
general [18,19] and for P. falciparum 1-Cys-Prx in par-
ticular [13,17,20]. The thiol dependency of Pf1-Cys-Prx
requires further study [8,9,20], and such information
will provide further insights into the physiological
functions of this protein. On the other hand, Prx is
known to be multifunctional, and its molecular chaper-
one function has recently been demonstrated in yeast
2-Cys Prx [21]. This function of Pf1-Cys-Prx and its
contribution to the protector protein activity should be
investigated, and experiments are in progress in our
laboratory.
Pf1-Cys-Prx interferes with membrane-associated
FP formation
If Pf1-Cys-Prx slows FP degradation by GSH, free FP
in the parasite cytosol during haemoglobin digestion
may readily move into the cell membrane and alter
permeability. To examine the possibility that Pf1-Cys-
Prx affects membrane association of FP, white ghosts
of human red blood cell (RBC) and FP were coincu-
bated with or without recombinant Prx. When white
ghosts and 10 lm FP were incubated at 37 °C for
7 min at pH 7.0, more than half (6.3 ± 0.15 lm)of
the FP was contained in the ghost fraction. Incubation
of ghosts and FP with recombinant Prx protein

The FCR-3 strain of P. falciparum was cultured according
to the modified method of Trager and Jensen [22]. Parasites
in the trophozoite ⁄ schizont stages of development were
obtained from sorbitol-synchronized cultures by treating
cultures with 5% d-sorbitol [23].
Preparation and purification of recombinant
protein
The coding sequence for Pf1-Cys-Prx was amplified from
cDNA of the blood-stage P. falciparum with primers
5¢-GCGAATTC
ATGGCTTACCATTTAGGAGC-3¢ and
5¢-GCGAATTC
TTACATTTGAACAAATCTTA-3¢. The
primers, which contain EcoRI sites (italics) adjacent to the
initiation and the termination codons (underline), were
designed on the basis of the sequence reported previously
[13]. PCR products were digested with EcoRI to create
cohesive ends for ligation into the pGEX-6P-1 expression
vector (Amersham Biosciences, Piscataway, NJ, USA). The
recombinant plasmid, with the cDNA inserted in the cor-
rect orientation, was transformed into Escherichia coli
strain BL21. The fusion protein with N-terminal GST was
expressed by induction of the bacterial culture with 0.3 mm
isopropyl-b-d-thiogalactoside. The protein was purified by
Glutathione Sepharose
TM
4B column chromatography
(GST-Glutathione Affinity System, Amersham Biosciences).
The GST-tag of the fusion protein was removed with Pre-
Scission

600-s intervals with an Ultrospec 3000 spectrophotometer
(Amersham Biosciences). The [FP]-dependent degradation
rate was measured at 370 nm for 50 s.
GPx activity assays
GPx activity of rPf1-Cys-Prx was examined by monitoring
oxidation of NADPH in a GPx ⁄ GSH ⁄ GSH reductase (GR)
system at 340 nm at RT as described previously [24].
Briefly, assay solution containing 1 mm GSH, 4 lm rPf1-
Cys-Prx and 5 U yeast GR (Oriental Yeast, Tokyo, Japan)
was preincubated for 10 min at RT. After addition of
NADPH (0.3 mm), hydroperoxide-independent oxidation
was monitored for 3 min, and GPx activity was examined
with 75 lm t-butylhydroperoxide (Sigma-Aldrich) and
75 lm H
2
O
2
as substrates. The assay system was checked
with bovine erythrocyte GPx (0.5 U; Sigma-Aldrich) as a
positive control, and the nonenzymatic reaction rate was
observed by replacing the enzyme with buffer (0.1 m potas-
sium phosphate, 1 mm EDTA, pH 7.0).
Binding of Pf1-Cys-Prx to FP-agarose
FP-agarose binding was performed as described by Cam-
panale et al. [25] but with minor modifications. Briefly,
hemin–agarose (20 lL; Sigma-Aldrich) was washed three
times with NaCl ⁄ P
i
by centrifugation (5000 g, 5 min, 4 °C).
For competition binding assay, FP was prepared as a

grinder. The homogenate was cleared of cell debris (500 g,
5 min, 4 °C) and then centrifuged (100 000 g, 1 h, 4 °C) in
an Optima
TM
TLX Ultracentrifuge (Beckman, Palo Alto,
CA, USA). The supernatant was used as the soluble frac-
tion, and the pellet was resuspended in the original volume
of NaCl ⁄ P
i
. Homogenate, soluble fraction and pellet were
mixed with SDS ⁄ PAGE sample buffer [26]. After separation
by SDS ⁄ PAGE (12.5% acrylamide), the proteins were
transferred electrophoretically to polyvinylidene difluoride
sheets (Immobilon
TM
-P; Millipore, Billerica, MA, USA)
and reacted with the IgG fraction of rabbit antisera to rPf1-
Cys-Prx (25 lgÆmL
)1
) [14]. The blot was developed with
horseradish peroxidase-conjugated antirabbit IgG antibody
(1 : 1250; Cappel, Aurora, OH, USA) and ECL
TM
detec-
tion reagents (Amersham Biosciences).
Assay for iron release from FP
FP decomposition mixture containing 30 lm FP, 3 mm
GSH and 0.2 m Hepes pH 7.0 was incubated both with and
without rPf1-Cys-Prx (4 lm)at37°C for 5 min. Free iron
was then measured by the Ferrozine method [6,27]. Briefly,

Kim et al. [28] with slight modifications. Inactivation mix-
tures (50 lL) containing 50 mm Hepes pH 7.0, 10 mm
dithiothreitol, 3 lm FeCl
3
and 0.5 lg GS were preincubated
with or without rPf1-Cys-Prx (4 lm)at30°C for 30 min.
Remaining GS activity was measured by adding 1 mL of
assay solution containing 0.4 mm ADP, 150 mm glutamine,
10 mm K-ASO
4
,20mm NH
2
OH, 0.4 mm MnCl
2
and
100 mm Hepes pH 7.4. The reaction was incubated at
30 °C for 30 min and then terminated by addition of
0.45 mL stop solution (25 mL stop solution contained
1.375 g FeCl
3
Æ6H
2
O, 0.5 g trichloroacetic acid, 10 mL
S i. Kawazu et al. Roles of Prx in heme degradation of P. falciparum
FEBS Journal 272 (2005) 1784–1791 ª 2005 FEBS 1789
HCl). Absorbance of c-glutamylhydroxamate-Fe
3+
com-
plex was measured at 540 nm. The GSH ⁄ FP-mediated
inactivation was performed as follows using yeast enolase

gation (15 000 g, 20 min, 4 °C), washed once with 0.2 m
Hepes pH 7.0 and dissolved in 1 mL 0.2 m Hepes pH 7.0
containing 1% (w ⁄ v) SDS. The absorbance was measured
at 400 nm. FP concentration was calculated from a calibra-
tion curve generated with standard FP solutions that had
been prepared by dissolving FP at 1–10 lm in 0.2 m Hepes
pH 7.0 containing 1% (w ⁄ v) SDS.
Acknowledgements
This work was supported by a Grant-in-Aid for Scien-
tific Research on Priority Areas (2) (16017318 to
S.I.K) from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan and
by a Grant for Precursory Research for Embryonic
Science and Technology, Japan Science and Technol-
ogy Agency (to S.I.K).
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