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Synthetic peptides corresponding to a repetitive sequence of malarial histidine rich protein bind haem and inhibit haemozoin formation in vitro
Molecular and Biochemical Parasitology 90 (1997) 281 – 287
Synthetic peptides corresponding to a repetitive sequence of malarial histidine rich protein bind haem and inhibit haemozoin formation in vitro Amit V. Pandey 1,a, Ratanmani Joshi b, Babu L. Tekwani a,*, Ram L. Singh c, Virender S. Chauhan b b
a Di6ision of Biochemistry, Central Drug Research Institute, Lucknow 226001, India Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Road, New Delhi 110 067, India c Department of Biochemistry, R.M.L. A6adh Uni6ersity, Faizabad 224 001, India
Received 17 February 1997; received in revised form 24 June 1997; accepted 10 September 1997
Abstract Synthetic peptides containing a repetitive hexapeptide sequence (Ala-His-His-Ala-Ala-Asp) of malarial histidinerich protein II were evaluated for binding with haem in vitro. The pattern of haem binding suggested that each repeat unit of this sequence provides one binding site for haem. Chloroquine inhibited the haem – peptide complex formation with preferential formation of a haem–chloroquine complex. In vitro studies on haem polymerisation showed that none of the peptides could initiate haemozoin formation. However, they could inhibit haemozoin formation promoted by a malarial parasite extract, possibly by competitively binding free haem. These results indicate this hexapeptide sequence represents the haem binding site of the malarial histidine-rich protein and possibly the site of nucleation for haem polymerisation. © 1997 Elsevier Science B.V. Keywords: Malaria; Haemoglobin; Haem; Haemozoin; Histidine-rich protein; Chloroquine
1. Introduction
* Corresponding author. Fax: +91 522 223405; e-mail: [email protected] 1 Present address: Malaria Group, ICGEB, Aruna Asaf Ali Road, New Delhi 110 067, India.
Malaria kills more than 2 million people every year and around half the world population is at the risk of infection. Emergence of drug resistance to chloroquine which has been the drug of choice for treatment of malaria has further complicated
0166-6851/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 6 8 5 1 ( 9 7 ) 0 0 1 6 1 - 8
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A.V. Pandey et al. / Molecular and Biochemical Parasitology 90 (1997) 281–287
the problem. Despite being in use for more than half a century the mechanism of action of chloroquine is still not properly understood [1]. Lysosomal damage, interaction with parasite DNA, inhibition of polyamine biosynthesis, formation of complexes with haem, inhibition of phospholipase and vacuolar proteases and change of the vacuolar pH are among the prominent effects of chloroquine ascribed to its antimalarial action [2,3]. However, not all of these could explain the selective action of chloroquine on those stages of parasite life cycle which actively degrade haemoglobin [4]. In the past few years formation of haemozoin pigment by the malaria parasite has emerged as a potential biochemical target for the antimalarial drugs [5]. During the intraerythrocytic stage malaria parasite digests haemoglobin inside its food vacuole by a variety of proteases [6 – 10]. Toxic haem released as a by-product of this reaction is converted into haemozoin pigment [11 – 13]. An enzyme ‘haem polymerase’ which could be inhibited by chloroquine, was initially proposed as a catalyst of haem polymerisation and the biochemical target of chloroquine in malaria parasite [14–16]. Some studies described this reaction as a spontaneous chemical process which occurs in the absence of protein or parasite derived material [17–21]. We have recently shown that under physiological conditions haem polymerisation is not spontaneous [1]. In another report published recently Fitch and Chou [22] have also found that there is no spontaneous haemozoin formation and characterised a heat labile as well as a heat stimulable haem polymerase activity in the extracts of Plasmodium berghei. In a recent study a malarial histidine rich protein (HRP II) localised in the digestive vacuole of the malaria parasite has been found to provide a nucleus for the haem polymerisation [23]. This protein binds 17 units of haem per molecule. A major part of malarial HRP II consists of multiple repeats of a hexapeptide unit (Ala-His-His-Ala-Ala-Asp) [24]. Since the involvement of histidine pairs is known to have a role in haem binding we thought it would be useful to examine the properties of this peptide unit which could be the nucleating site where
haemozoin formation occurs. We designed peptides based on this sequence to study its haem binding and polymerisation property. Peptides containing one, two and three units of the sequence (namely HRP IIA (Gly-Ala-His-His-AlaAla-Asp-Gly-Cys), IIB (Cys-Gly-Ala-His-HisAla-Ala-Asp-Ala-His-His-Ala-Ala-Asp-Gly-Cys) and IIC (Cys-Gly-Ala-His-His-Ala-Ala-Asp-AlaHis-His-Ala-Ala-Asp-Ala-His-His-Ala-Ala-AspGly-Cys), respectively) were used for this study.
2. Materials and methods
2.1. Materials Haemin and SDS were purchased from Sigma (St. Louis, MO). All other chemicals were of analytical grade procured from local suppliers. Peptides were synthesized manually by solid phase method on Wang-resin. After cleavage from the resin, peptides were lyophilized twice and purified by high performance liquid chromatography on semi-preparatory reverse phase, C18-bondapak column (gradient used 10% CH3CN–90% CH3CN in 30 min). Finally, the peptides were characterised by amino acid analysis and mass spectroscopy.
2.2. Parasite and experimental host Male Swiss albino mice weighing 15–20 g were obtained from the Laboratory Animals division of the Institute. The animals were maintained on a commercial pellet diet and housed under standard conditions. Plasmodium yoelii nigeriensis routinely maintained by serial blood passages was used for infection. Mice were infected by intraperitoneal passage of 1× 107 infected erythrocytes diluted in 0.5 ml of sterile acid citrate dextrose (citric acid 7.3 g, sodium citrate 22 g, dextrose 24.5 g, dissolved in 1000 ml distilled water). Parasitaemia was monitored by microscopic examination of Giemsa stained thin blood smears. Blood was collected at high level of parasitaemia (\50%) in sterile acid citrate dextrose.
A.V. Pandey et al. / Molecular and Biochemical Parasitology 90 (1997) 281–287
2.3. Haem binding assay Binding experiments were done as described earlier [23] using 20 nmol of peptide in a total volume of 1.0 ml with the haem concentration varied from 0 to 80 mM. Equal amounts of haem were added to the reference cuvette. Spectra were recorded on a Hitachi 557 double beam double wavelength spectrophotometer. In some experiments sodium dithionite was also included in the reaction mixture to maintain reducing conditions. Interaction of chloroquine with the haem – peptide complex formation was studied by thin layer chromatography (TLC) on precoated silica gel TLC plates (0.2 mm thickness, F254, silica gel precoated plastic sheets, obtained from Merck, Germany) using N,N-dimethylformamide:chloroform (4:1) as solvent.
2.4. Haem polymerisation assay Haem polymerisation activity was assayed in P. yoelii lysates based on the method described previously [15]. Plasma and buffy coat were removed from the blood of infected mice by centrifugation at 500× g for 10 min. The erythrocyte pellet was washed once with phosphate buffer saline (10 mM, pH 7.4) and suspended in four volumes of the same buffer containing glucose (0.9% w/v). The lysate was prepared by freezing the suspension in liquid nitrogen drop by drop. The frozen droplets of the lysate were stored in aliquots at − 70°C till further use. When required an aliquot of the lysate was thawed and centrifuged at 16 000× g for 20 min at 4°C. The pellet was resuspended in sodium acetate buffer (100 mM, pH 5.0) and used for the haem polymerisation assay. The assay mixture contained 50 ml of the parasite extract, 100 mM haemin as the substrate and acetate buffer (100 mM, pH 5.0) in a total volume of 1.0 ml. Two controls were always run simultaneously, one without substrate and another without parasite extract, containing only 100 mM haemin suspended in acetate buffer. Peptides were dissolved in distilled water just before use and 50 ml containing the appropriate concentration was added to the reaction mixture. Each assay was set up at least in triplicate and incu-
283
bated at 37°C for 4 h in a constantly shaking water bath. The reaction was stopped by centrifugation at 16 000× g for 5 min and pellets, if any, were resuspended in Tris–HCl (100 mM, pH 7.4) containing 2.5% SDS. The pellets were washed twice with Tris–HCl and once with bicarbonate buffer (100 mM, pH 9.0). The final pellet thus obtained was of polymerised haem (haemozoin). For quantitation of haemozoin, the pellets were solubilised in 2 N NaOH and spectra were recorded using 2.5% SDS as solvent on a Hitachi 557 spectrophotometer between 360 and 700 nm. A millimolar extinction coefficient of 91 mM − 1 cm − 1 at 400 nm was used to quantitate haemozoin in the form of haem as described previously [25].
3. Results and discussion Peptides were found to bind with haem as observed by spectral analysis and TLC. Binding of haem with peptides is evident from the shift in absorption maxima of haem in difference spectra. Haem alone has an absorption maximum at 400 nm. The spectrum of the peptide–haem complex is typical of the high spin state of haem, with a major absorption peak at 415 nm (Fig. 1). In the subsequent experiments absorption at 415 nm was used to study haem binding. We observed that the
Fig. 1. Spectra of haem – peptide complex. Concentration of peptide HRP IIC was 20 mM and haem 10 mM. An aliquot of 10 mM haem was added in the reference.
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itself. When these peptides were incubated with free haem alone under acidic conditions (acetate buffer, 100 mM, pH 5.0) no haemozoin formation occurred (Fig. 3) indicating that the peptides could not simulate the haem polymerisation property of the malarial HRP II [23]. Haem alone or with purified malaria pigment (haemozoin) could not initiate haem polymerisation. HRP IIC was also unable to polymerise haem either alone or in combination with purified haemozoin. Addition of purified haemozoin in the reaction with parasite extract was found to have no effect on haem polymerisation. However, the peptides caused significant inhibition of haem polymerisation activity of P. yoelii extracts in a concentration-dependent manner (Fig. 4), suggesting a competition for haem binding with the malarial protein. These observations indicate that this highly repetitive sequence may be the nucleus for the binding of haem. However, for initiating the polymerisation of haem some additional sites of HRP II may be necessary as the repeat peptides alone could not induce haem polymerisation.
Fig. 2. Interaction of haem with synthetic HRP peptides. (A) Binding of haem to HRP IIC. Each point represents the mean9 SD of triplicate observations. HRP IIC (20 nmol) was titrated against haem in increments of 5 nmol. Total increase in the volume during experiment was less than 5%. (B) Titration of different peptides with haem. The number of hexamer units in the peptides is plotted against the number of haem bound per mole of peptide.
number of repeat units in peptides directly correlate with the number of haem units bound per molecule of peptide (Fig. 2A,B). Binding of haem with peptides was observed under non-reducing as well as reducing conditions suggesting that polymerisation or cyclization was not responsible for the observed effects. All the subsequent experiments were conducted under non-reducing conditions only. We then examined the effect of these peptides on the process of haem polymerisation
Fig. 3. Effect of peptides and haemozoin on haem polymerisation. Different components were added as displayed in the table below the bar diagram. Concentrations of components was as follows: haem, 100 mM; peptide, 100 mM; purified haemozoin, 5 nmol (equivalent of haem); parasite extract containing 2.5 nmol of haemozoin. Other conditions were as described in Section 2. Results are displayed as mean9SD of triplicate experiments.
A.V. Pandey et al. / Molecular and Biochemical Parasitology 90 (1997) 281–287
Fig. 4. Inhibition of Plasmodium yoelii haem polymerisation activity by HRP II peptides. Each bar represents mean 9 SD of triplicate experiments. Haemozoin formed in the control during reaction was 4.5 nmol.
Haem–peptide complex formation was also studied by TLC (Fig. 5). Haem alone shows significant migration (lane 4), however, the haem – peptide complex did not move under this solvent system (lane 1). The haem – chloroquine complex (lane 3) could be resolved from free haem and the haem peptide–complex. When chloroquine was included in the reaction mixtures containing haem and peptides, formation of a haem – chloroquine complex was favored and the formation of a haem–peptide complex was impaired as observed by TLC (lane 2). This inhibition of the peptide – haem complex formation may suggest a mechanism of inhibition of haem polymerisation by chloroquine. The presence of chloroquine leads to haem–chloroquine complex formation which stops the process of further chain elongation of haem polymer. In a recent report HRP II-mediated haem polymerisation was found to be inhibited by incorporation of the haem – chloroquine complex in the growing haemozoin molecule [26]. However, it is not clear how many haem units are required to make the pigment non-toxic to the parasite. Moreover, the polymer with the haem – chloroquine complex attached at the end may still retain toxicity towards the parasite. The present study suggests that inhibition of haem binding to these sites may be the mechanism of action of the
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chloroquine, leading to the generation of a larger pool of toxic haem, which kills the malaria parasite [3]. Recently a chloroquine importer has been characterised from P. falciparum [27]. Uptake of chloroquine by P. falciparum-infected erythrocytes was found to be energy dependent and chloroquine resistance was linked with the difference in the kinetics of drug accumulation. These observations are in agreement with the previous reports that similar concentrations of chloroquine are required to inhibit the haem polymerisation activity of both chloroquine resistant and susceptible strains of P. falciparum [2]. These studies taken together suggest that resistance to chloroquine is likely to be due to insufficient accumulation of the drug inside the malarial food vacuole, the site of action. Some other studies have reported that changes in the side chain of chloroquine molecule may revert the resistance. Molecules containing shortened or elongated side
Fig. 5. Interaction of chloroquine with haem – peptide complex formation. Lanes: 1, haem – peptide complex; 2, haem – peptide interaction in presence of chloroquine; 3, haem – chloroquine complex; 4, haem. Concentrations of components were as follows: peptide HRP IIC, 40 mM; haem, 20 mM; chloroquine, 40 mM. O, origin; SF, solvent front.
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chains were found to be active against chloroquine resistant strains of P. falciparum in vitro [28,29]. Further studies are required to understand the structure of the haem – chloroquine and haem–HRP II complexes which might lead to generation of antimalarial compounds with better haem binding capacity [30]. Together these studies suggest that accumulation of quinoline drugs not only depends on the weak base properties but also on the presence of some drug receptor inside the parasite food vacuole [31,32]. A chloroquine binding protein has been identified [31]. Haem has been proposed as a possible receptor for quinoline antimalarials [3]. Moreover, all the blood schizontocidal drugs (except pyrimethamine which has a different well defined mechanism) bind to haem and inhibit haem polymerisation [2]. Studies on structural and mechanistic aspects of haem binding to these peptides might provide the key for understanding the process of haem polymerisation and the design of novel antimalarials, for which the need is pressing due to fast spreading resistance to most antimalarial drugs available at present.
Acknowledgements The authors thank the Director, Central Drug Research Institute, Lucknow, India and the Director, International Centre for Genetic Engineering and Biotechnology, New Delhi, India for providing necessary facilities. We acknowledge Dr S.K. Puri for critically reading the manuscript and Professor O.P. Shukla and Dr V.C. Pandey for helpful discussions. AVP received financial support in the form of a Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), India. This is CDRI communication no. 5643.
References [1] Pandey AV, Tekwani BL. Formation of haemozoin/bhaematin under physiological conditions is not spontaneous. FEBS Lett 1996;393:189–92.
[2] Slater AFG. Chloroquine: mechanism of drug action and resistance in Plasmodium falciparum. Pharmacol Ther 1993;57:203 – 35. [3] Orjih AU, Banyal HS, Chelvi R, et al. Haemin lyses malaria parasite. Science 1981;214:667 – 9. [4] Pandey AV, Tekwani BL. Depolymerisation of malarial haemozoin: a novel reaction initiated by blood schizontocidal antimalarials. FEBS Lett 1997;402:236 – 40. [5] Asawamahasakda W, Ittarat I, Chang CC, et al. Effect of antimalarials and protease inhibitors on plasmodial haemozoin production. Mol Biochem Parasitol 1994;67:183 – 91. [6] Silva AM, Le AY, Gulnik SV, et al. Structure and inhibition of plasmepsin II, a haemoglobin-degrading enzyme from Plasmodium falciparum. Proc Natl Acad Sci USA 1996;93:10034– 9. [7] Moon RP, Tyas L, Certa U, et al. Expression and characterisation of plasmepsin I from Plasmodium falciparum. Eur J Biochem 1997;244:552 – 60. [8] Francis SE, Banerjee R, Goldberg DE. Biosynthesis and maturation of the malaria aspartic hemoglobinases plasmepsins I and II. J Biol Chem 1997;272:14961– 8. [9] Rosenthal PJ, Meshnick SR. Haemoglobin catabolism and iron utilisation by the malaria parasites. Mol Biochem Parasitol 1996;83:131 – 9. [10] Francis SE, Gluzman IY, Oksman A, et al. Characterisation of native falcipain, an enzyme involved in Plasmodium falciparum haemoglobin degradation. Mol Biochem Parasitol 1996;83:189 – 200. [11] Fitch CD, Kanjananggulpan P. The state of ferriprotoporphyrin IX in malaria pigment. J Biol Chem 1987;262:15552– 5. [12] Slater AFG, Swiggard WJ, Orton BR, et al. An iron carboxylate bond links the haem units of malaria pigment. Proc Natl Acad Sci USA 1991;88:325 – 9. [13] Bohle DS, Dinnebier RE, Madsen SK, et al. Characterisation of the products of the haem detoxification pathway in malarial late trophozoites by X-ray diffraction. J Biol Chem 1997;272:713 – 6. [14] Slater AFG, Cerami A. Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites. Nature 1992;355:167 – 9. [15] Chou AC, Fitch CD. Control of haem polymerase by chloroquine and other quinoline derivatives. Biochem Biophys Res Commun 1993;195:422 – 7. [16] Wellems TE. How chloroquine works. Nature 1992;355:108 – 9. [17] Egan TJ, Ross DC, Adams PA. Quinoline antimalarial drugs inhibit spontaneous formation of b-haematin (malaria pigment). FEBS Lett 1994;352:54 – 7. [18] Adams PA, Egan TJ, Ross DC, et al. The chemical mechanism of b-haematin formation studied by Mo¨ssbauer spectroscopy. Biochem J 1996;318:25 – 7. [19] Dorn A, Stoffel R, Matile H, et al. Malarial haemozoin/ b-haematin supports haem polymerisation in the absence of protein. Nature 1995;374:269 – 71.
A.V. Pandey et al. / Molecular and Biochemical Parasitology 90 (1997) 281–287 [20] Bendrat K, Berger BJ, Cerami A. Haem polymerisation in malaria. Nature 1995;378:138. [21] Pandey AV, Tekwani BL, Pandey VC. Characterisation of haemozoin from liver and spleen of mice infected with Plasmodium yoelii a rodent malaria parasite. Biomed Res 1995;16:115 – 20. [22] Fitch CD, Chou AC. Heat labile and heat stimulable haem polymerase activities in Plasmodium berghei. Mol Biochem Parasitol 1996;82:261–4. [23] Sullivan DJ, Gluzman IY, Goldberg DE. Plasmodium haemozoin formation mediated by histidine-rich proteins. Science 1996;271:219–21. [24] Wellems TE, Howard RJ. Homologous genes encode two distinct histidine rich proteins in a cloned isolate of Plasmodium falciparum. Proc Natl Acad Sci USA 1986;83:6065 – 9. [25] Asakura T, Minakata K, Adachi K, et al. Denatured haemoglobin in sickle erythrocytes. J Clin Invest 1977;59:633 – 40. [26] Sullivan DJ, Gluzman IY, Goldberg DE. On the molecular mode of chloroquine’s action. Proc Natl Acad Sci USA 1996;93:11865–70.
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[27] Sanchez CP, Wunsch S, Lanzer M. Identification of a chloroquine importer in Plasmodium falciparum difference in import kinetics are genetically linked with the chloroquine-resistant phenotype. J Biol Chem 1997;272:2652– 8. [28] De D, Krogstad FM, Cogswell FB, et al. Aminoquinolines that circumvent resistance in Plasmodium falciparum in vitro. Am J Trop Med Hyg 1996;55:579 – 83. [29] Ridley RG, Hofheinz W, Matile H, et al. 4-Aminoquinoline analogs of chloroquine with shortened side chains retain activity against chloroquine-resistant Plasmodium falciparum. Antimicrob Agents Chemother 1996;40:1846 – 54. [30] Hawley SR, Bray PG, Park BK, et al. Amodiaquine accumulation in Plasmodium falciparum as a possible explanation for its superior antimalarial activity over chloroquine. Mol Biochem Parasitol 1996;80:15 – 25. [31] Foley M, Deady W, Ng K, et al. Photoaffinity labelling of chloroquine binding proteins in Plasmodium falciparum. J Biol Chem 1996;269:6955– 61. [32] Adams PA, Berman PAM, Egan TJ, et al. The iron environment in haem and haem-antimalarial complexes of pharmacological interest. J Inorg Biochem 1996;63:69 – 77.
Synthetic peptides corresponding to a repetitive sequence of malarial histidine rich protein bind haem and inhibit haemozoin formation in vitro Amit V. Pandey 1,a, Ratanmani Joshi b, Babu L. Tekwani a,*, Ram L. Singh c, Virender S. Chauhan b b
a Di6ision of Biochemistry, Central Drug Research Institute, Lucknow 226001, India Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Road, New Delhi 110 067, India c Department of Biochemistry, R.M.L. A6adh Uni6ersity, Faizabad 224 001, India
Received 17 February 1997; received in revised form 24 June 1997; accepted 10 September 1997
Abstract Synthetic peptides containing a repetitive hexapeptide sequence (Ala-His-His-Ala-Ala-Asp) of malarial histidinerich protein II were evaluated for binding with haem in vitro. The pattern of haem binding suggested that each repeat unit of this sequence provides one binding site for haem. Chloroquine inhibited the haem – peptide complex formation with preferential formation of a haem–chloroquine complex. In vitro studies on haem polymerisation showed that none of the peptides could initiate haemozoin formation. However, they could inhibit haemozoin formation promoted by a malarial parasite extract, possibly by competitively binding free haem. These results indicate this hexapeptide sequence represents the haem binding site of the malarial histidine-rich protein and possibly the site of nucleation for haem polymerisation. © 1997 Elsevier Science B.V. Keywords: Malaria; Haemoglobin; Haem; Haemozoin; Histidine-rich protein; Chloroquine
1. Introduction
* Corresponding author. Fax: +91 522 223405; e-mail: [email protected] 1 Present address: Malaria Group, ICGEB, Aruna Asaf Ali Road, New Delhi 110 067, India.
Malaria kills more than 2 million people every year and around half the world population is at the risk of infection. Emergence of drug resistance to chloroquine which has been the drug of choice for treatment of malaria has further complicated
0166-6851/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 6 8 5 1 ( 9 7 ) 0 0 1 6 1 - 8
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A.V. Pandey et al. / Molecular and Biochemical Parasitology 90 (1997) 281–287
the problem. Despite being in use for more than half a century the mechanism of action of chloroquine is still not properly understood [1]. Lysosomal damage, interaction with parasite DNA, inhibition of polyamine biosynthesis, formation of complexes with haem, inhibition of phospholipase and vacuolar proteases and change of the vacuolar pH are among the prominent effects of chloroquine ascribed to its antimalarial action [2,3]. However, not all of these could explain the selective action of chloroquine on those stages of parasite life cycle which actively degrade haemoglobin [4]. In the past few years formation of haemozoin pigment by the malaria parasite has emerged as a potential biochemical target for the antimalarial drugs [5]. During the intraerythrocytic stage malaria parasite digests haemoglobin inside its food vacuole by a variety of proteases [6 – 10]. Toxic haem released as a by-product of this reaction is converted into haemozoin pigment [11 – 13]. An enzyme ‘haem polymerase’ which could be inhibited by chloroquine, was initially proposed as a catalyst of haem polymerisation and the biochemical target of chloroquine in malaria parasite [14–16]. Some studies described this reaction as a spontaneous chemical process which occurs in the absence of protein or parasite derived material [17–21]. We have recently shown that under physiological conditions haem polymerisation is not spontaneous [1]. In another report published recently Fitch and Chou [22] have also found that there is no spontaneous haemozoin formation and characterised a heat labile as well as a heat stimulable haem polymerase activity in the extracts of Plasmodium berghei. In a recent study a malarial histidine rich protein (HRP II) localised in the digestive vacuole of the malaria parasite has been found to provide a nucleus for the haem polymerisation [23]. This protein binds 17 units of haem per molecule. A major part of malarial HRP II consists of multiple repeats of a hexapeptide unit (Ala-His-His-Ala-Ala-Asp) [24]. Since the involvement of histidine pairs is known to have a role in haem binding we thought it would be useful to examine the properties of this peptide unit which could be the nucleating site where
haemozoin formation occurs. We designed peptides based on this sequence to study its haem binding and polymerisation property. Peptides containing one, two and three units of the sequence (namely HRP IIA (Gly-Ala-His-His-AlaAla-Asp-Gly-Cys), IIB (Cys-Gly-Ala-His-HisAla-Ala-Asp-Ala-His-His-Ala-Ala-Asp-Gly-Cys) and IIC (Cys-Gly-Ala-His-His-Ala-Ala-Asp-AlaHis-His-Ala-Ala-Asp-Ala-His-His-Ala-Ala-AspGly-Cys), respectively) were used for this study.
2. Materials and methods
2.1. Materials Haemin and SDS were purchased from Sigma (St. Louis, MO). All other chemicals were of analytical grade procured from local suppliers. Peptides were synthesized manually by solid phase method on Wang-resin. After cleavage from the resin, peptides were lyophilized twice and purified by high performance liquid chromatography on semi-preparatory reverse phase, C18-bondapak column (gradient used 10% CH3CN–90% CH3CN in 30 min). Finally, the peptides were characterised by amino acid analysis and mass spectroscopy.
2.2. Parasite and experimental host Male Swiss albino mice weighing 15–20 g were obtained from the Laboratory Animals division of the Institute. The animals were maintained on a commercial pellet diet and housed under standard conditions. Plasmodium yoelii nigeriensis routinely maintained by serial blood passages was used for infection. Mice were infected by intraperitoneal passage of 1× 107 infected erythrocytes diluted in 0.5 ml of sterile acid citrate dextrose (citric acid 7.3 g, sodium citrate 22 g, dextrose 24.5 g, dissolved in 1000 ml distilled water). Parasitaemia was monitored by microscopic examination of Giemsa stained thin blood smears. Blood was collected at high level of parasitaemia (\50%) in sterile acid citrate dextrose.
A.V. Pandey et al. / Molecular and Biochemical Parasitology 90 (1997) 281–287
2.3. Haem binding assay Binding experiments were done as described earlier [23] using 20 nmol of peptide in a total volume of 1.0 ml with the haem concentration varied from 0 to 80 mM. Equal amounts of haem were added to the reference cuvette. Spectra were recorded on a Hitachi 557 double beam double wavelength spectrophotometer. In some experiments sodium dithionite was also included in the reaction mixture to maintain reducing conditions. Interaction of chloroquine with the haem – peptide complex formation was studied by thin layer chromatography (TLC) on precoated silica gel TLC plates (0.2 mm thickness, F254, silica gel precoated plastic sheets, obtained from Merck, Germany) using N,N-dimethylformamide:chloroform (4:1) as solvent.
2.4. Haem polymerisation assay Haem polymerisation activity was assayed in P. yoelii lysates based on the method described previously [15]. Plasma and buffy coat were removed from the blood of infected mice by centrifugation at 500× g for 10 min. The erythrocyte pellet was washed once with phosphate buffer saline (10 mM, pH 7.4) and suspended in four volumes of the same buffer containing glucose (0.9% w/v). The lysate was prepared by freezing the suspension in liquid nitrogen drop by drop. The frozen droplets of the lysate were stored in aliquots at − 70°C till further use. When required an aliquot of the lysate was thawed and centrifuged at 16 000× g for 20 min at 4°C. The pellet was resuspended in sodium acetate buffer (100 mM, pH 5.0) and used for the haem polymerisation assay. The assay mixture contained 50 ml of the parasite extract, 100 mM haemin as the substrate and acetate buffer (100 mM, pH 5.0) in a total volume of 1.0 ml. Two controls were always run simultaneously, one without substrate and another without parasite extract, containing only 100 mM haemin suspended in acetate buffer. Peptides were dissolved in distilled water just before use and 50 ml containing the appropriate concentration was added to the reaction mixture. Each assay was set up at least in triplicate and incu-
283
bated at 37°C for 4 h in a constantly shaking water bath. The reaction was stopped by centrifugation at 16 000× g for 5 min and pellets, if any, were resuspended in Tris–HCl (100 mM, pH 7.4) containing 2.5% SDS. The pellets were washed twice with Tris–HCl and once with bicarbonate buffer (100 mM, pH 9.0). The final pellet thus obtained was of polymerised haem (haemozoin). For quantitation of haemozoin, the pellets were solubilised in 2 N NaOH and spectra were recorded using 2.5% SDS as solvent on a Hitachi 557 spectrophotometer between 360 and 700 nm. A millimolar extinction coefficient of 91 mM − 1 cm − 1 at 400 nm was used to quantitate haemozoin in the form of haem as described previously [25].
3. Results and discussion Peptides were found to bind with haem as observed by spectral analysis and TLC. Binding of haem with peptides is evident from the shift in absorption maxima of haem in difference spectra. Haem alone has an absorption maximum at 400 nm. The spectrum of the peptide–haem complex is typical of the high spin state of haem, with a major absorption peak at 415 nm (Fig. 1). In the subsequent experiments absorption at 415 nm was used to study haem binding. We observed that the
Fig. 1. Spectra of haem – peptide complex. Concentration of peptide HRP IIC was 20 mM and haem 10 mM. An aliquot of 10 mM haem was added in the reference.
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itself. When these peptides were incubated with free haem alone under acidic conditions (acetate buffer, 100 mM, pH 5.0) no haemozoin formation occurred (Fig. 3) indicating that the peptides could not simulate the haem polymerisation property of the malarial HRP II [23]. Haem alone or with purified malaria pigment (haemozoin) could not initiate haem polymerisation. HRP IIC was also unable to polymerise haem either alone or in combination with purified haemozoin. Addition of purified haemozoin in the reaction with parasite extract was found to have no effect on haem polymerisation. However, the peptides caused significant inhibition of haem polymerisation activity of P. yoelii extracts in a concentration-dependent manner (Fig. 4), suggesting a competition for haem binding with the malarial protein. These observations indicate that this highly repetitive sequence may be the nucleus for the binding of haem. However, for initiating the polymerisation of haem some additional sites of HRP II may be necessary as the repeat peptides alone could not induce haem polymerisation.
Fig. 2. Interaction of haem with synthetic HRP peptides. (A) Binding of haem to HRP IIC. Each point represents the mean9 SD of triplicate observations. HRP IIC (20 nmol) was titrated against haem in increments of 5 nmol. Total increase in the volume during experiment was less than 5%. (B) Titration of different peptides with haem. The number of hexamer units in the peptides is plotted against the number of haem bound per mole of peptide.
number of repeat units in peptides directly correlate with the number of haem units bound per molecule of peptide (Fig. 2A,B). Binding of haem with peptides was observed under non-reducing as well as reducing conditions suggesting that polymerisation or cyclization was not responsible for the observed effects. All the subsequent experiments were conducted under non-reducing conditions only. We then examined the effect of these peptides on the process of haem polymerisation
Fig. 3. Effect of peptides and haemozoin on haem polymerisation. Different components were added as displayed in the table below the bar diagram. Concentrations of components was as follows: haem, 100 mM; peptide, 100 mM; purified haemozoin, 5 nmol (equivalent of haem); parasite extract containing 2.5 nmol of haemozoin. Other conditions were as described in Section 2. Results are displayed as mean9SD of triplicate experiments.
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Fig. 4. Inhibition of Plasmodium yoelii haem polymerisation activity by HRP II peptides. Each bar represents mean 9 SD of triplicate experiments. Haemozoin formed in the control during reaction was 4.5 nmol.
Haem–peptide complex formation was also studied by TLC (Fig. 5). Haem alone shows significant migration (lane 4), however, the haem – peptide complex did not move under this solvent system (lane 1). The haem – chloroquine complex (lane 3) could be resolved from free haem and the haem peptide–complex. When chloroquine was included in the reaction mixtures containing haem and peptides, formation of a haem – chloroquine complex was favored and the formation of a haem–peptide complex was impaired as observed by TLC (lane 2). This inhibition of the peptide – haem complex formation may suggest a mechanism of inhibition of haem polymerisation by chloroquine. The presence of chloroquine leads to haem–chloroquine complex formation which stops the process of further chain elongation of haem polymer. In a recent report HRP II-mediated haem polymerisation was found to be inhibited by incorporation of the haem – chloroquine complex in the growing haemozoin molecule [26]. However, it is not clear how many haem units are required to make the pigment non-toxic to the parasite. Moreover, the polymer with the haem – chloroquine complex attached at the end may still retain toxicity towards the parasite. The present study suggests that inhibition of haem binding to these sites may be the mechanism of action of the
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chloroquine, leading to the generation of a larger pool of toxic haem, which kills the malaria parasite [3]. Recently a chloroquine importer has been characterised from P. falciparum [27]. Uptake of chloroquine by P. falciparum-infected erythrocytes was found to be energy dependent and chloroquine resistance was linked with the difference in the kinetics of drug accumulation. These observations are in agreement with the previous reports that similar concentrations of chloroquine are required to inhibit the haem polymerisation activity of both chloroquine resistant and susceptible strains of P. falciparum [2]. These studies taken together suggest that resistance to chloroquine is likely to be due to insufficient accumulation of the drug inside the malarial food vacuole, the site of action. Some other studies have reported that changes in the side chain of chloroquine molecule may revert the resistance. Molecules containing shortened or elongated side
Fig. 5. Interaction of chloroquine with haem – peptide complex formation. Lanes: 1, haem – peptide complex; 2, haem – peptide interaction in presence of chloroquine; 3, haem – chloroquine complex; 4, haem. Concentrations of components were as follows: peptide HRP IIC, 40 mM; haem, 20 mM; chloroquine, 40 mM. O, origin; SF, solvent front.
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chains were found to be active against chloroquine resistant strains of P. falciparum in vitro [28,29]. Further studies are required to understand the structure of the haem – chloroquine and haem–HRP II complexes which might lead to generation of antimalarial compounds with better haem binding capacity [30]. Together these studies suggest that accumulation of quinoline drugs not only depends on the weak base properties but also on the presence of some drug receptor inside the parasite food vacuole [31,32]. A chloroquine binding protein has been identified [31]. Haem has been proposed as a possible receptor for quinoline antimalarials [3]. Moreover, all the blood schizontocidal drugs (except pyrimethamine which has a different well defined mechanism) bind to haem and inhibit haem polymerisation [2]. Studies on structural and mechanistic aspects of haem binding to these peptides might provide the key for understanding the process of haem polymerisation and the design of novel antimalarials, for which the need is pressing due to fast spreading resistance to most antimalarial drugs available at present.
Acknowledgements The authors thank the Director, Central Drug Research Institute, Lucknow, India and the Director, International Centre for Genetic Engineering and Biotechnology, New Delhi, India for providing necessary facilities. We acknowledge Dr S.K. Puri for critically reading the manuscript and Professor O.P. Shukla and Dr V.C. Pandey for helpful discussions. AVP received financial support in the form of a Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), India. This is CDRI communication no. 5643.
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