Molecular & Biochemical Parasitology 144 (2005) 100–103

Short communication

Fine mapping of the Duffy antigen binding site for thePlasmodiumvivax Duffy-binding protein

Christophe Tournamillea, Anne Filipea, Cyril Badautb, Marie-Madeleine Riottotb,Shirley Longacrec, Jean-Pierre Cartrona, Caroline Le Van Kima, Yves Colina,∗

a INSERM U665, Institut National de la Transfusion Sanguine, Universite Paris 7 Denis Diderot, 6 rue Alexandre Cabanel, Paris F-75015, Franceb Unite d’Immunologie Structurale, Institut Pasteur, Paris F-75015, France

c Laboratoire de Vaccinologie Parasitaire, Institut Pasteur, Paris F-75015, France

Received 3 February 2005; received in revised form 19 April 2005; accepted 30 April 2005Available online 1 July 2005

Keywords: Duffy antigen; DARC;Plasmodium vivax; Duffy-binding protein; Interaction site

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Plasmodium vivax is the most widely distributed malariaarasite amongst the four species that infect humans. Inontrast toPlasmodium falciparum, P. vivax is not directlyssociated with a high level of mortality but it remains aublic health problem in South America, Asia and Oceania

1]. With the emergence of resistance to current antimalarialrugs[2] and the increase of Anopheles lines insensitive toverused insecticides, the development of new strategies foraccines are being studied. Some of them emphasize the rolef pre-erythrocytic malaria vaccines, while others focus on

he host blood stage of the parasite during its human life cycle3–6]. Blocking erythrocyte invasion requires a better under-tanding of the molecular interactions between the parasite

igand and the red blood cell (RBC) receptor.P. vivax, andhe related simian malaria parasite,Plasmodium knowlesi, aretrictly dependent on interaction with the Duffy blood groupntigen for invasion of human erythrocytes[7,8]. After an ini-

ial attachment to the erythrocyte, the merozoite reorients itspical end towards the erythrocyte surface and a tight junctionetween the Duffy antigen and the parasite ligand enables theed blood cell invasion by the parasite[9]. The Duffy bloodroup antigen, is carried by a 40–45 kDa glycoprotein, also

dentified as a chemokine receptor expressed on RBC and

parasite ligands,P. vivax or P. knowlesi �-Duffy-bindingproteins (DBPs)[16,17], which bind to DARC, belong tothe Duffy-binding like erythrocyte-binding protein famil(DBL-EBP)[18] that includes otherP. knowlesi EBPs and theP. falciparum EBPs[19,20]. All these DBL-EBPs presentsimilar extracellular domain divided into six regions cotaining two conserved cysteine-rich domains, region II aVI, described as the Duffy-binding like domains (DBL)[21].Region II (RII, 320 amino acids) of the 140 kDaP. vivaxDuffy-binding protein (PvDBP) was identified as the bindindomain mediating erythrocyte invasion by the Duffy antigedependent pathway[22]. More precisely, the 170 amino-acidomain located between the fourth and the eighth cyste(C4–C8) of PvDBP-RII was shown to be necessary for biing to erythrocyte receptor[23]. Accordingly, the mutagenesis analysis in this domain have identified residues direinvolved in the binding and others mediating the specificof the interaction[24,25].

The binding site on DARC protein used by bothP. vivaxandP. knowlesi ligands maps to 35 amino acids (Ala8-Asp4located in the N-terminal extracellular domain of the rector [26]. Interestingly, theP. vivax specific binding site isrestricted to one DARC extracellular domain, whereas a c

ndothelial cells of post-capillary venules of various tissuesnd was therefore named DARC, for Duffy antigen/receptor

or chemokines[10]. Several studies argue for a role of DARC

0 19.

association between the four extracellular domains of thereceptor is needed for chemokines binding[27,28].

In the present study, alanine-scan mutagenesis of recom-b nala P-R da res-

d.

n the regulation of the inflammation process[11–15]. The

∗ Corresponding author. Tel.: +33 1 44 49 30 93; fax: +33 1 43 06 5E-mail address: colin@idf.inserm.fr (Y. Colin).

166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserveoi:10.1016/j.molbiopara.2005.04.016

inant DARC protein was performed to identify N-termimino acids directly involved in the interaction with PvDBII on RBC. Region II of theP. vivax DBP was expresses a soluble protein in an insect cell/baculovirus exp

C. Tournamille et al. / Molecular & Biochemical Parasitology 144 (2005) 100–103 101

Fig. 1. Mapping of N-terminal residues of DARC involved in the binding to PvDBP-RII. (A) Representation of the amino-acid sequence of DARC N-terminalend. The three DARC/Nter regions that were fused with the glutathioneS-transferase protein (GST) and the S18-S28 peptide are outlined. PCR amplified cDNAfragments of DARC were cloned in the pGEX-5X-3 plasmid. The GST-DARC/Nter proteins expressed inE. coli BL21 were purified with glutathione-SepharosebeadsTM 4B (Amersham-Biosciences). (B) Direct-interaction of GST-DARC/Nter proteins with soluble PvDBP-RII. PCR amplified fragment encoding regionII of Pv-DBP (amino acids 201–536 of the PvDBP, accession number M37514), was cloned in the baculovirus expression vector pVL 1393 (Invitrogen). Thesoluble recombinant protein containing a C-terminal hexahistidine-tag was expressed in the bacculovirus/insect cell system (Longacre et al., in preparation).The cell-culture supernatant was dialysed 3 times for 14 h against 20 mM Tris pH 7.5; 0.5 M NaCl and applied to a metal-affinity column (Talon). PvDBP-RIIwas eluted by 10 mM Imidazol, 20 mM Tris pH 8.0 and 0.5 M NaCl, concentrated on a YM-10 Centricon column (Amicon) and purified by gel filtrationchromatography (Pharmacia, S200, 16/60) equilibrated with 20 mM Tris pH 7.5; 0.5 M NaCl. The 2.9.7 anti-PvDBP-RII mAb was obtained by the hybridomatechnique after immunising mice with the soluble PvDBP-RII recombinant protein (Longacre et al., in preparation). Alanine replacement mutants of theN-terminal of DARC were obtained by in vitro mutagenesis (Quick change site-directed mutagenesis kit, Stratagen) of the GST-DARC/Nter1 recombinantplasmid. For direct protein/protein interaction, 1.2 mg of the different GST-DARC/Nter proteins bound to glutathione-sepharose beads were incubated with5�g of PvDBP-RII in 1× PBS containing protease inhibitors (Roche) for 4 h at 4◦C. After two washes in 1× PBS containing protease inhibitors, proteinswere eluted in glutathione 20 mM, Tris 50 mM pH 8, separated on 10% SDS-PAGE gels and transferred on nitrocellulose-membrane. PvDBP-RII bound toGST-DARC/Nter proteins was detected by probing the membrane with the 2.9.7 mAb. Critical residues of DARC were determined by comparing the bindingcapacity of weight and mutated GST-DARC/Nter proteins to PvDBP-RII. Mutations E9A–N17A and of S28A–Y30A had no deleterious effect on the bindingto PvDBP-RII (not shown). (C) Displacement of erythrocyte-bound PvDBP-RII by GST-DARC/Nter proteins. 1× 109 Duffy-positive RBCs were incubatedwith 5�g of PvDBP-RII for 1 h at 4◦C in PBS-BSA 1%. After two washes in PBS-BSA 1%, binding reactions were incubated with 50 and 800�g of GST-DARC/Nter proteins for 1 h at 4◦C in PBS-BSA 1%. Erythrocytes were collected by centrifugation through bis(2-ethylhexyl) phthalate, and bound proteinswere eluted with 300 mM NaCl, separated on 10% SDS-PAGE gels and transferred on nitrocellulose-membrane. PvDBP-RII eluted from RBC was revealedwith the 2.9.7 mAb, for 50�g (left lane) and 800�g (right lane) of each GST-DARC/Nter proteins. Critical residues of DARC were determined by comparingthe capacity of weight and mutated GST-DARC/Nter proteins to displace PvDBP-RII from erythrocytes. Densitometry scanning of the autoradiography fromthree independent experiments was performed. The signals obtained in presence of 800�g of GST-DARC/Nter1, GST-DARC/Nter2, GST-DARC/Nter1-S18Aand DARC/Nter1-N27A proteins were 5–10% of the signals obtained in presence of 50�g of the same proteins, whereas the signals obtained in presence of800�g of Q19A to W26A alanine mutants, GST-DARC/Nter3 and empty GST proteins were 80–110% of the signal obtained in presence of 50�g of the sameproteins (not shown). Alanine-replacement mutants E9A–N17A and S28A–Y30A displace PvDBP-RII as the wt GST-DARC/Nter1 protein (not shown).

sion system. To assess the specific binding activity of thesoluble 40 kDa PvDBP-RII, an erythrocyte-binding assaywas performed using Duffy-positive and Duffy-negativeRBCs. Only Duffy-positive RBC bound PvDBP-RII (datanot shown). Three different GST-DARC/Nter fusion pro-teins were constructed: GST-DARC/Nter1 (Met1-Ser60)corresponds to the whole N-terminal extracellular domainof DARC; GST-DARC/Nter2 (Met1-Tyr30) encompassesthe Fy6 linear epitope 19-QLDFEDVW-26 recognized by2C3, i3A, BG6, MIMA-107 and MINA-108 MAbs[28–31]

and GST-DARC/Nter3 (Ser29-Ser60) contains the Fya lin-ear epitope 41-DGDYGANLE-46 recognized by 655 andMIMA-19 MAbs [28,31] (Fig. 1A). The DARC/Nter pro-teins were expressed as soluble GST fusion proteins in abacterial expression system (as described in the legends ofFig. 1A). Direct interaction and displacement assays showthat GST-DARC/Nter1 and GST-DARC/Nter2, but not GST-DARC/Nter3, were able to bind PvDBP-RII and to dis-place PvDBP-RII binding on the erythrocytes Duffy-positive(Fig. 1B and C). These results are in accordance with previous

102 C. Tournamille et al. / Molecular & Biochemical Parasitology 144 (2005) 100–103

studies showing the ability of the 35 amino-acid N-terminalpeptide (Ala8-Asp42) to inhibit Duffy-positive erythrocytebinding to transfected COS cells expressing PvDBP-RII,whereas the smaller peptide D21-D42 does not[26]. How-ever our results restricted PvRII binding site between Ala8and Tyr30. Thus, in order to define precisely DARC aminoacids directly implicated in the interaction with PvDBP-RII,we performed site-directed mutagenesis of all amino acidsbetween Glu9 and Tyr30 on GST-DARC/Nter1. As shown inFig. 1B, mutation of six residues, two polar charged (D21 andD24) and four non-polar (L20, F22, V25 and W26), resultedin the loss of PvDBP-RII binding, as for the mock GST con-trol. In addition, these GST-DARC/Nter1 mutants could notdisplace PvDBP-RII on the erythrocytes as shown inFig. 1C.Conversely, S18A or N27A mutations did not influence directinteraction or displacement of PvDBP-RII. It is noteworthythat the Q19A and E23A mutants bound normally but werenot able to displace PvDBP-RII, suggesting that mutation ofthese residues could modulate the affinity of the interaction.Finally, we performed inhibition assays of PvDBP-RII bind-ing to Duffy-positive RBC by the Ser18-Ser28 peptide includ-ing the Fy6 epitope sequence (Fig. 1A). No inhibition wasobtained (data not shown). Altogether these findings demon-strate that the region located at the N-terminal side of Fy6 epi-tope is not directly involved in the binding, but is potentiallyimplicated in conformation and presentation of DARC extra-cb iono d bea ldingc ns don -Fym pen-d

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BP-R RCr rgedp ,t posi-

Table 1Effect of amino acids E9A–Y30 mutations on the interactions between theN-terminal region of DARC and PvDBP-RIIa

GST-DARC/Nterfusion proteins

Direct interactionwith PvDBP-RII

Displacement of PvDBP-RII bound on Fy-pos RBC

Nter1 + +Nter2 + +Nter3 − −Nter1-E9A + +Nter1-L10A + +Nter1-S11A + +Nter1-P12A + +Nter1-S13A + +Nter1-T14A NT NTNter1-E15A + +Nter1-N16A + +Nter1-S17A + +Nter1-S18A + +Nter1-Q19A + −Nter1-L20A − −Nter1-D21A − −Nter1-F22A − −Nter1-E23A + −Nter1-D24A − +/−Nter1-V25A − −Nter1-W26A − −Nter1-N27A + +Nter1-S28A + +Nter1-S29A + +Nter1-Y30A + +

a The capacity of GST-DARC/Nter1 mutants to bind directly PvDBP-RIIand to displace PvDBP-RII on Fy-pos RBC was relative to that obtained forthe wild-type GST-DARC/Nter1 and the mock control GST. The results arefrom three binding experiments (NT: not tested).

tions, on both receptor and ligand, are probably involved inboth ionic and hydrophobic interactions and therefore theirproperties may influence functional conformations and con-tribute to the specificity of the interaction. It is anticipatedthat data on the tertiary structure of the DARC–PvDBP-RIIcomplex will eventually elucidate the nature of the contactsbetween these two proteins.

Because the blood stage is critical for the parasite lifecycle and becauseP. vivax and the related simian parasite,P.knowlesi, totally depend on interaction with Duffy antigen forhuman erythrocyte invasion, knowledge of these molecularinteractions will provide essential information for the devel-opment of new strategies againstP. vivax malaria. Recom-binant vaccines based on PvDBP-RII are currently beingdeveloped[35], but “artificial peptides” targeting DARC pro-tein which is the “invariant side” of the interaction, mightrepresent a complementary approach to blockP. vivax bloodstage infection, provided that they are not rapidly clearedfrom the circulation. Once this paper was ready to be submit-ted for publication, Choe et al.[36] reported that sulphationof tyrosine 41 is essential for high affinity association ofrecombinant PvDBP-RII with recombinant cells expressingthe whole DARC protein. The apparent discrepancy withour present study might be explained if we consider that theFy6 epitope Q19-W26 represents, stricto senso, the PvDBP-

ellular domain, and thus is essential for an efficientP. vivaxinding. Conversely, it is unlikely that the lack of interactf the Q19A to W26A alanine replacement mutants coulccounted by a non-specific effect based on protein fohanges. We have previously shown that these mutatioot impair the binding of DARC-transfected cells to antionoclonal antibodies and/or chemokines which are deent of the overall structure integrity of the protein[27,28].

Interestingly, we delineate the PvRII binding site betwesidues Q19 and W26, which strictly matches the Fy6ope (Fig. 1 and Table 1), recognized by the BG6 mAreviously shown to have the capacity to block non-hurimate erythrocyte invasion byP. vivax, in vitro [32]. Heree demonstrate that PvDBP-RII binding site is restricte

his Fy6 epitope in human DARC. Moreover, comparif homologous DARC sequences[33] reinforces the obseation that, in non-human primates, the absence of char non-polar residues (squirrel monkey, rhesus monke

he introduction of a proline residue which potentially mfies the structure (brown capuchin), at or near Fy6 epitespectively, are related to the absence of interaction betvDBP-RII and non-human primate erythrocytes, wheompensation of the requisite charged and non-polar resn the neighbouring region of Fy6 permitsP. vivax interactionnight monkey)[17,34].

Recently, mutagenesis analysis performed on the PvDII protein have shown that residues essential for DA

ecognition are invariant, comprising charged and unchaolar residues, and non-polar residues[24,25]. Altogether

hese results indicate that side chains at these residue

C. Tournamille et al. / Molecular & Biochemical Parasitology 144 (2005) 100–103 103

RII binding site of DARC, and that sulphated tyrosine 41 isessential either to give an “open” conformation to the wholeN-terminal domain of DARC and/or to increase the affinityof the DARC/PvDBP-RII association.

Acknowledgement

We thank Graham Bentley, Unite d’Immunologie Struc-turale, Institut Pasteur (Paris, France) for critical reading ofthis paper.

References

[1] Mendis K, Sina BJ, Marchesini P, Carter R. The neglected burden ofPlasmodium vivax malaria. Am J Trop Med Hyg 2001;64:97–106.

[2] Collins WE, Jeffery GM. Primaquine resistance inPlasmodiumvivax. Am J Trop Med Hyg 1996;55:243–9.

[3] Michon P, Fraser T, Adams JH. Naturally acquired and vaccine-elicited antibodies block erythrocyte cytoadherence of thePlasmod-ium vivax Duffy binding protein. Infect Immun 2000;68:3164–71.

[4] Singh AP, Puri SK, Chitnis CE. Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding proteininhibit erythrocyte invasion. Mol Biochem Parasitol 2002;121:21–31.

[5] Stoute JA, Kester KE, Krzych U, et al. Long-term efficacy andimmune responses following immunization with the RTS, S malaria

. A60

ry-

xp

enttac

[ ulecepoidest

[ theion-

[ seines

[ ene–53

[ BA.n

[ turn

[ y oen

[site

[18] Adams JH, Sim BK, Dolan SA, Fang X, Kaslow DC, Miller LH.A family of erythrocyte binding proteins of malaria parasites. ProcNatl Acad Sci USA 1992;89:7085–9.

[19] Camus D, Hadley TJ. APlasmodium falciparum antigen that bindsto host erythrocytes and merozoites. Science 1985;230:553–6.

[20] Adams JH, Blair PL, Kaneko O, Peterson DS. An expandingebl family of Plasmodium falciparum. Trends Parasitol 2001;17:297–9.

[21] Adams JH, Fang X, Kaslow DC, Miller LH. Identification of a cryp-tic intron in thePlasmodium vivax Duffy binding protein gene. MolBiochem Parasitol 1992;56:181–3.

[22] Chitnis CE, Miller LH. Identification of the erythrocyte bindingdomains ofPlasmodium vivax and Plasmodium knowlesi proteinsinvolved in erythrocyte invasion. J Exp Med 1994;180:497–506.

[23] Ranjan A, Chitnis CE. Mapping regions containing binding residueswithin functional domains ofPlasmodium vivax and Plasmodiumknowlesi erythrocyte-binding proteins. Proc Natl Acad Sci USA1999;96:14067–72.

[24] Hans D, Pattnaik P, Bhattacharyya A, et al. Mapping binding residuesin the Plasmodium vivax domain that binds Duffy antigen during redcell invasion. Mol Microbiol 2005;55:1423–34.

[25] VanBuskirk KM, Sevova E, Adams JH. Conserved residues in thePlasmodium vivax Duffy-binding protein ligand domain are criti-cal for erythrocyte receptor recognition. Proc Natl Acad Sci USA2004;101:15754–9.

[26] Chitnis CE, Chaudhuri A, Horuk R, Pogo AO, Miller LH. Thedomain on the Duffy blood group antigen for bindingPlasmodiumvivax and P. knowlesi malarial parasites to erythrocytes. J Exp Med1996;184:1531–6.

[27] Tournamille C, Le Van Kim C, Gane P, et al. Close association of theorJ

norng

fher

ln-

.mar

--

aynd

l-ant

te

l

vaccine. J Infect Dis 1998;178:1139–44.[6] Pouniotis DS, Proudfoot O, Minigo G, Hanley JC, Plebanski M

new boost for malaria vaccines. Trends Parasitol 2004;20:157–[7] Miller LH, Mason SJ, Dvorak JA, McGinniss MH, Rothman IK. E

throcyte receptors for (Plasmodium knowlesi) malaria: Duffy bloodgroup determinants. Science 1975;189:561–3.

[8] Miller LH, Carter R. A review Innate resistance in malaria. EParasitol 1976;40:132–46.

[9] Miller LH, Aikawa M, Johnson JG, Shiroishi T. Interaction betwecytochalasin B-treated malarial parasites and erythrocytes. Ament and junction formation. J Exp Med 1979;149:172–84.

10] Hadley TJ, Lu ZH, Wasniowska K, et al. Postcapillary venendothelial cells in kidney express a multispecific chemokine retor that is structurally and functionally identical to the erythrisoform, which is the Duffy blood group antigen. J Clin Inv1994;94:985–91.

11] Luo H, Chaudhuri A, Zbrzezna V, He Y, Pogo AO. Deletion ofmurine Duffy gene (Dfy) reveals that the Duffy receptor is functally redundant. Mol Cell Biol 2000;20:3097–101.

12] Dawson TC, Lentsch AB, Wang Z, et al. Exaggerated responendotoxin in mice lacking the Duffy antigen/receptor for chemok(DARC). Blood 2000;96:1681–4.

13] Du J, Luan J, Liu H, et al. Potential role for Duffy antigchemokine-binding protein in angiogenesis and maintenanchomeostasis in response to stress. J Leukoc Biol 2002;71:141

14] Middleton J, Patterson AM, Gardner L, Schmutz C, AshtonLeukocyte extravasation: chemokine transport and presentatiothe endothelium. Blood 2002;100:3853–60.

15] Segerer S, Bohmig GA, Exner M, et al. When renal allograftsDARC. Transplantation 2003;75:1030–4.

16] Haynes JD, Dalton JP, Klotz FW, et al. Receptor-like specificita Plasmodium knowlesi malarial protein that binds to Duffy antigligands on erythrocytes. J Exp Med 1988;167:1873–81.

17] Wertheimer SP, Barnwell JW.Plasmodium vivax interaction with thehuman Duffy blood group glycoprotein: identification of a parareceptor-like protein. Exp Parasitol 1989;69:340–50.

.

h-

-

to

of.

by

f

first and fourth extracellular domains of the Duffy antigen/receptfor chemokines by a disulfide bond is required for ligand binding.Biol Chem 1997;272:16274–80.

[28] Tournamille C, Filipe A, Wasniowska K, et al. Structure–functioanalysis of the extracellular domains of the Duffy antigen/receptfor chemokines: characterization of antibody and chemokine bindisites. Br J Haematol 2003;122:1014–23.

[29] Wasniowska K, Blanchard D, Janvier D, et al. Identification othe Fy6 epitope recognized by two monoclonal antibodies in tN-terminal extracellular portion of the Duffy antigen receptor fochemokines. Mol Immunol 1996;33:917–23.

[30] Wasniowska K, Petit-LeRoux Y, Tournamille C, et al. Structuracharacterization of the epitope recognized by the new anti-Fy6 mooclonal antibody NaM 185-2C3. Transfus Med 2002;12:205–11.

[31] Wasniowska K, Lisowska E, Halverson GR, Chaudhuri A, Reid METhe Fya, Fy6 and Fy3 epitopes of the Duffy blood group systerecognized by new monoclonal antibodies: identification of a lineFy3 epitope. Br J Haematol 2004;124:118–22.

[32] Barnwell JW, Nichols ME, Rubinstein P. In vitro evaluation of therole of the Duffy blood group in erythrocyte invasion byPlasmodiumvivax. J Exp Med 1989;169:1795–802.

[33] Tournamille C, Blancher A, Le Van Kim C, et al. Sequence, evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines. Immunogenetics 2004;55:682–94.

[34] Nichols ME, Rubinstein P, Barnwell J, Rodriguez de CordobS, Rosenfield RE. A new human Duffy blood group specificitdefined by a murine monoclonal antibody. Immunogenetics aassociation with susceptibility toPlasmodium vivax. J Exp Med1987;166:776–85.

[35] Yazdani SS, Shakri AR, Mukherjee P, Baniwal SK, Chitnis CE. Evauation of immune responses elicited in mice against a recombinmalaria vaccine based onPlasmodium vivax Duffy binding protein.Vaccine 2004;22:3727–37.

[36] Choe H, Moore MJ, Owens CM, et al. Sulphated tyrosines mediaassociation of chemokines andPlasmodium vivax Duffy binding pro-tein with the Duffy antigen/receptor for chemokines (DARC). MoMicrobiol 2005;55:1413–22.