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Plasmodium falciparum Histidine-richProtein-2 (PfHRP2) Modulates the RedoxActivity of Ferri-protoporphyrin IX(FePPIX) : PEROXIDASE-LIKEACTIVITY OF THE PfHRP2-FePPIXCOMPLEX

Ryuichi Mashima, Leann Tilley, Mary-Anne

Siomos, Vicki Papalexis, Mark J. Raftery andRoland StockerJ. Biol. Chem. 2002, 277:14514-14520.

doi:10.1074/jbc.M109386200 originally published online February 21, 2002

Access the most updated version of this article at doi: 10.1074/jbc.M109386200

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 17, Issue of April 26, pp. 1451414520, 2002 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Plasmodium falciparum Histidine-rich Protein-2 (PfHRP2)Modulates the Redox Activity of Ferri-protoporphyrin IX (FePPIX)PEROXIDASE-LIKE ACTIVITY OF THEPfHRP2-FePPIX COMPLEX*

Received for publication, September 28, 2001, and in revised form, February 18, 2002Published, JBC Papers in Press, February 21, 2002, DOI 10.1074/jbc.M109386200

Ryuichi Mashima, Leann Tilley, Mary-Anne Siomos, Vicki Papalexis, Mark J. Rafteryll,and Roland Stocker**

From the Biochemistry Group, The Heart Research Institute, 145 Missenden Road, Camperdown, New South Wales 2050, theDepartment of Biochemistry, La Trobe University, Bundoora, Victoria 3086, and the IlCytokine Research Unit, School of Pathology,University of New South Wales, Kensington, New South Wales 2052, Australia

Histidine-rich protein-2 from Plasmodium falciparum(PfHRP2) binds up to 50 molecules of ferri-protoporphyrin IX(FePPIX) (Choi, C. Y., Cerda, J. F., Chu, H. A., Babcock, G. T.,

and Marletta, M. A. (1999) Biochemistry 38, 1691616924). Wereasoned that the PfHRP2-FePPIX complex has antioxidantproperties that could be beneficial to the parasite. Therefore, weexamined whether binding to PfHRP2 modulated the redox

properties of FePPIX. We observed that PfHRP2 completelyinhibited the auto-oxidation of ascorbate mediated by free FeP-PIX. We also investigated the peroxidase activity ofPfHRP2-FePPIX using 13-hydroperoxy-9,11-octadienoate (18:2-OOH) assubstrate. Reaction ofPfHRP2-FePPIX with 18:2-OOH in thepresence of added reducing agents gave 13-hydroxy-9,11-octadienoate (18:2-OH) as a major product and 13-keto-9,11-

octadienoate (18:2=O) and 9,12,13-trihydroxy-10-octadecaenoate as minor products. Binding of FePPIX to

PfHRP2 lowered the rate of decomposition of 18:2-OOH andincreased the 18:2-OH to 18:2=O ratio. Similar to otherauthentic peroxidases, phenols, amines, and biological

reductants like ascorbate promoted 18:2-OH production, andNaCN inhibited 18:2-OH production. Thioanisole also acted as a

reductant and was converted to thioanisole sulfoxide, suggesting

formation of compound I during the reaction. These data showthat PfHRP2 modulates the redox activity of FePPIX and thatthe PfHRP2-FePPIX complex may have previouslyunrecognized antioxidant properties.

During the blood stage of malaria infection, the parasite feedson red cell proteins, principally hemoglobin. Degradation ofhemoglobin takes place in the food vacuole, and this process isinitiated by the conversion of oxyhemoglobin to methemoglobinwith the concomitant production of superoxide anion thatdismutates to H2O2 (1). About 80% of the hemoglobin is degradedduring the intra-erythrocytic development of the para-

* This work was supported by the Australian National Health andMedical Research Council. The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked advertisement in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

Present address: Centre for Thrombosis and Vascular Research, Schoolof Medical Sciences, University of New South Wales, UNSW Sydney, NewSouth Wales 2052, Australia.

** To whom correspondence should be addressed: Centre for Thrombosisand Vascular Research, School of Medical Sciences, University of NewSouth Wales, UNSW Sydney, New South Wales 2052, Australia. E-mail:[email protected].

site (2), corresponding to -15 mmol/liter H2O2 being produced.H2O2 is a potentially harmful oxidant that can cause damage to

protein and lipid in the presence of transition metals andreducing substances. Indeed, parasitized red cells are exposed tooxidative stress (reviewed in Ref. 3).

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A second toxic insult comes from ferri-protoporphyrin IX(FePPIX)1 released as the byproduct of methemoglobin degra-dation (1). Free FePPIX is toxic because of its detergent-like(4) and redox properties (5). The parasite detoxifies FePPIXvia nonenzymatic processes, including reaction with H2O2 andglutathione, that lead to the accumulation of iron (6). Inaddition, FePPIX is crystallized to a granular pigment, knownas hemozoin (f3-hematin) (7). This process is initiated andaccelerated by histidine-rich proteins, of which Plasmodiumfalciparum histidine-rich protein-2 (PfHRP2) is characterizedbest. Histidine comprises 34% of the amino acid residues ofPfHRP2 (8, 9), and for effective conversion of FePPIX to f3-

hematin, the protein requires an acidic pH that exists in thefood vacuole (10, 11).

PfHRP2 is also present outside the food vacuole, i.e. under-neath the red cell membrane and in the erythrocyte cytoplasm(12). This indicates a biological role forPfHRP2 at neutral pHwhere it acts as an efficient FePPIX-binding protein rather than

promoting f3-hematin formation (11). Up to -50 mol of FePPIXbinds tightly per mol ofPfHRP2 through hexa-coordination (13)to the repetitive amino acid sequence AHHAHHAAD (14).Although the spectroscopic properties of the PfHRP2-FePPIXcomplex are well characterized (13), its biochemical propertiesare poorly understood. We reasoned that the coordinated bindingto PfHRP2 modulates the redox activity of FePPIX and that thePfHRP2-FePPIX complex may possess peroxidase activity.

FePPIX-containing peroxidases reduce hydroperoxides(ROOH) via heterolysis to the corresponding alcohol (ROH) andcompound I (Reaction 1), an intermediate with an oxidationstate two electrons higher than that of the resting enzyme. Incontrast, free FePPIX and the heme proteins cytochrome P-450and hemoglobin decompose ROOH through homolysis to

1 The abbreviations used are: FePPIX, ferri-protoporphyrin IX; 18:2=O, 13-ketooctadeca-9,11-dienoic acid; 18:2-OH, 13-hydroxyoctadeca-9,11-dienoic acid; 18:2-OOH, 13-hydroperoxyoctadeca-9,11-dienoicacid; 20:4-OOH, 15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid; HPLC,high-performance liquid chromatography; OPD, o-phenylenediamine;PBS, phosphate-buffered saline; PfHRP2, P. falciparum histidine-richprotein-2; PLPC, 1-palmitoyl-2-l inoleoyl phosphatidylcholine; PLPC-OOH, 1-palmitoyl-2-linoleoyl phosphatidylcholine hydroperoxide; PPIX,protoporphyrin IX; SnPPIX, tin PPIX; ZnPPIX, zinc (II) PPIX; ROOH,hydroperoxide; ROH, corresponding alcohol; RO~, alkoxyl radical; AH2,
mailto:[email protected]:[email protected]:[email protected]://www.jbc.org/mailto:[email protected]:[email protected]://www.jbc.org/


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hydrogen donor.


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Plasmodium falciparum Histidine-rich Protein-2 14515

FIG. 1. Auto-oxidation of ascorbate. Ascorbate (100AM) was incubatedat 37 C in PBS containing 5 tM FePPIX (0), 1 AMPfHRP2 (), PfHRP2-FePPIX (1 WMPfHRP2 and 5 AM FePPIX, ), and PBS (). The data areexpressed as the means S.D. (n = 3).

alkoxyl radical (RO') and an oxo-ferryl species (Reaction 2)(15). Catalytic reduction of ROOH requires consecutive one-electron reduction of the FePPIX peroxidase intermediatescompound I (Reaction 3) and compound II (the one-electronoxidized form of the enzyme) (Reaction 4), and this is achieved

by hydrogen donors (AH2) such as amines and phenols.

Alternatively, compound I can be reduced by sulfur- andnitrogen-containing compounds such as thioanisole throughoxygen transfer (Reaction 5) (16, 17).

ROOH+Fe(III)peroxidase>ROH+ compound I

ROOH+Fe(III)>RO' +Fe(IV)= O

Compound I+AH2>Compound II+AH'

Compound II+AH2>Fe(III)peroxidase+AH'

Compound I +X>Fe(III)peroxidase +X=O

REACTIONS 15

In the present study, we investigated the redox properties of

the PfHRP2-FePPIX complex. We show that binding to Pf-HRP2 decreases the pro-oxidant properties and increases the

peroxidase activity of FePPIX, raising the possibili ty that Pf-HRP2 acts as a previously unrecognized antioxidant by atten-uating alkoxyl and hydroxyl radical formation.

EXPERIMENTAL PROCEDURES

MaterialsLinoleic acid, arachidonic acid, o-phenylenediamine(OPD), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), GSH, N,

N'-diphenyl-1,4-phenylenediamine, phenol, gallic acid, salicylic acid,1,2,4-aminotriazole, ferri-protoporphyrin IX (hematin, FePPIX), andthioanisole were obtained from Sigma. NaCN and NaN3 were fromMerck, 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) was fromAvanti Polar-Lipids Inc. (Alabaster, AL), and guaiacol was from Fluka(Buchs, Switzerland). Bilirubin conjugate, protoporphyrin IX (PPIX), andtin protoporphyrin IX (SnPPIX) were from Porphyrin Products (Logan,

UT), and Trolox and zinc (II) protoporphyrin IX (ZnPPIX) fromAldrich. [1-14C]Linoleic acid (55 mCi/mmol) and PD-10 columns werefrom Amersham Biosciences. Unless specified otherwise, aqueous bufferswere stored over Chelex-100 (Bio-Rad) prior to use. 13-Hydroxy9,11-octadienoate (18:2-OH) was prepared from 18:2-OOH as described (18).13-Keto-9,11-octadienoate (18:2=O) was prepared by reduction of 18:2-OOH with acetylchloride in pyridine or purchased from Cayman (AnnArbor, MI).

Expression and Purification of Recombinant PfHRP2BL21/DE3Escherichia coli (Stratagene, La Jolla, CA) transfected with the pET-8cexpression vector containing the recombinant PfHRP2 sequence (kindlydonated by Drs. D. Sullivan and D. Goldberg, Howard Hughes MedicalInstitute) were grown overnight, and protein expression was induced byisopropyl-3-D-thiogalactopyranoside (0.84 mM). After incubation at 37C for 4 h, the harvested cells were sonicated (XL2000, Daintree), and

PfHRP2 was purified from the supernatant using a Ni 2+-agarose column(Novagen) eluted with 20 mM Tris-HCl (pH 7.9) containing 1 M

imidazole. The recombinant PfHRP2 used in this study contains 93histidine residues and binds 46 molecules of FePPIX/protein (13). Un-


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FIG. 2. Degradation of 18:2-OOH by FePPIX and PfHRP2-FeP-PIX. 18:2-OOH (5 AM) was reacted at 37 C with FePPIX alone () or

PfHRP2-FePPIX (1 WM PfHRP2 and 1 tM FePPIX, ) in 50 mM sodiumphosphate buffer (pH 7.2) in the presence of 100 toM OPD. At the timesindicated, the 18:2-OOH concentration was determined as described underExperimental Procedures. The data are expressed as the means S.D. (n =3). Where error bars cannot be seen, they are smaller than the size of thesymbols.Downloaded from http://www.jbc.org/by guest on June 23, 2013

less otherwise specified, reconstitution was performed for 5 min at 37C in 50 mM sodium phosphate buffer (pH 7.2) containing equimolaramounts ofPfHRP2 and FePPIX (13). For some experiments PfHRP2(1.1AM) was reconstituted with different metallo-porphyrins (100 AM)in 200 mM sodium phosphate buffer (pH 7.2) for 5 min at 37 C.Excess metallo-porphyrin was then removed by gel filtration (PD-10column) using PBS as eluent.

Preparation of Lipid Hydroperoxides15-Hydroperoxyeicosa5,8,11,13-tetraenoic acid (20:4-OOH), 18:2-OOH, and 1-palmitoyl-2-linoleoylphosphatidylcholine hydroperoxide (PLPC-OOH) were prepared bylipoxygenase-catalyzed oxidation and purified by solid phase extraction(C18 cartridge, Waters, Milford, MA) followed by preparative reversed-phase HPLC using an LC-18 column (20 X 250 mm, 5 Am, Supelco,Bellofonte, PA) (19). 18:2-OOH and 20:4-OOH were eluted withmethanol/water/acetic acid (950:50:1), whereas PLPC-OOH was elutedusing 0.02% triethylamine in methanol at 8.0 ml/min. [1-14C]18: 2-OOH (55mCi/mmol) was prepared similarly, except that an analytical LC-18 column

(4.6 X 250 mm, 5Am, Supelco) was used, and the flow rate of the mobilephase was 1.0 ml/min. The fractions containing the respective hydroperoxidewere collected, dried, and redissolved in meth-

anol for determination of the concentration at 234 nm using =28,000 cm' (20).

Auto-oxidation and Co-oxidation ExperimentsThe time-dependentdecay of ascorbate was used to examine the ability ofPfHRP2 to rendercatalytic metals inactive (21). Briefly, ascorbate (100 AM) was incubated at37 C in nonchelexed PBS containing the additives specified, and the decayof ascorbate followed at 265 nm. For co-oxidation experiments, PLPC (100oM) was incubated in PBS containing 1 AMPfHRP2, 5AM FePPIX, 100AMOPD at 37 C, and the time-dependent formation of PLPC-OOH wasdetermined as described below.

Peroxidase Activity ofPfHRP2The time-dependent accumulation of18:2-OH and 18:2=O from 5 18:2-OOH was monitored at 37 C inPBS containing 1 mM OPD and PfHRP2 reconstituted with FePPIX or the

metallo-porphyrin indicated. Control experiments contained either FePPIX-freePfHRP2, FePPIX alone, or water as vehicle. At the times indicated, theconcentrations of 18:2-OH, 18:2-OOH, and 18:2=O were determined asdescribed below. Where indicated, NaCN, NaN3, or 1,2,4- aminotriazole (010 mM) was added, in which case the peroxidase activity of 1 AMPfHRP2and 1 FePPIX was examined using 100AM 18:2-OOH and 500AM OPD in50 mM sodium phosphate (pH 7.2) and an incubation period of 15 min.

Steady-state KineticsThe substrate specificity of FePPIX-bound Pf-HRP2 was examined for 5 min at 37 C in 50 m Mphosphate buffer (pH 7.2)containing 1AMPfHRP2, 1AM FePPIX, 100AM 18:2-OOH, and 0200AMof the reducing substrate indicated. Km and Vmax were determined from therespective Michaelis-Menten plots obtained under the same conditions using100AM OPD and 0200AM 18:2-OOH.

Involvement of Compound I in Peroxidase Activity of PfHRP2Theinvolvement of compound I in the peroxidase activity ofPfHRP2 wasassessed by the conversion of thioanisole to thioanisole sulfoxide (15).Briefly, FePPIX-containingPfHRP2 (3AM each) was incubated at 37 C inPBS containing 200 AM 18:2-OOH and 1.6 mM thioanisole. Theconcentration of thioanisole sulfoxide was determined by HPLC using anLC-18 column (4.6 X 150 mm, 3Am, Supelco) eluted with methanol/ water(40:60) at 0.8 ml/min and monitored at 254 nm. Under these


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14516 Plasmodium falciparum Histidine-rich Protein-2

TABLE IReaction of 18:2-OOH with PfHRP2-FePPIX

The peroxidase-like activities ofPfHRP2-FePPIX (0.1AMPfHRP2 and 5AM FePPIX), FePPIX (5 tM), or GSH peroxidase (0.16AM in selenium) wasdetermined following incubation for 5 min at 37 C in 50 mM sodium phosphate buffer (pH 7.2) using 200AM 18:2-OOH and 1 mM reducing substrate (OPDfor both PfHRP2-FePPIX and FePPIX and GSH for GSH peroxidase). Concentrations of 18:2-OOH and its metabolites were determined by HPLC on anLC18 column (4.6 X 150 mm, 3Am) with acetonitrile/water/acetic acid (60:40:0.1) at 1.0 ml/min. Quantitation was performed by UV detection at 234 nmfor 18:2-OH and 18:2-OOH and 280 nm for 18:2=O. The data are expressed as the means S.D. of three experiments.

Cata lys tProducts Turnover

18:2-OH/18:2=O

18:2-OH 18:2=O 18:2-OHb

18:2=Ob

18:2-OH 18:2=OttM gM (%) (%)

PfHRP2-FePPIX 73 6 22 1 41 3 12 1 15 1 4 0 3.4 0.4FePPIX 42 2c 27 6 22 1c 14 3 8 0 5 1 1.6 0.2c

GSH peroxidase 94 11 NDd 86 11 NAe 2344 277 NA NAa Turnover number was calculated by the amount of product per amount of catalyst.b Yield of products (in percentages) was calculated by products formed per 18:2-OOH degraded.p

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