Fine mapping of the Duffy antigen binding site for the Plasmodium vivax Duffy-binding protein

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<ul><li><p>Molecular &amp; Biochemical Parasitology 144 (2005) 100103</p><p>Short communication</p><p>Fine mapping of the Duffy antigen binding p</p><p>C adauS line L</p><p>a INSER enis Dteur, PaPasteur,</p><p>April 2005</p><p>Keywords: D site</p><p>Plasmodparasite amcontrast toassociatedpublic heal[1]. With thdrugs [2] aoverused invaccines arof pre-erytthe host blo[36]. Blocstanding of the molecular interactions between the parasiteligand and the red blood cell (RBC) receptor. P. vivax, andthe related simian malaria parasite, Plasmodiumknowlesi, arestrictly depantigen fortial attachmapical end tbetween thred blood cgroup antigidentified aendothelialand was thfor chemokin the regu</p><p> CorresponE-mail ad</p><p>ite ligins (Duffy--EBPciparuar extg two</p><p>escribon IIy-bindin me</p><p>ndentdomain located between the fourth and the eighth cysteines(C4C8) of PvDBP-RII was shown to be necessary for bind-ing to erythrocyte receptor [23]. Accordingly, the mutagen-</p><p>0166-6851/$doi:10.1016/jendent on interaction with the Duffy blood groupinvasion of human erythrocytes [7,8]. After an ini-ent to the erythrocyte, the merozoite reorients its</p><p>owards the erythrocyte surface and a tight junctione Duffy antigen and the parasite ligand enables theell invasion by the parasite [9]. The Duffy blooden, is carried by a 4045 kDa glycoprotein, alsos a chemokine receptor expressed on RBC andcells of post-capillary venules of various tissues</p><p>erefore named DARC, for Duffy antigen/receptorines [10]. Several studies argue for a role of DARClation of the inflammation process [1115]. The</p><p>ding author. Tel.: +33 1 44 49 30 93; fax: +33 1 43 06 50 19.dress: (Y. Colin).</p><p>esis analysis in this domain have identified residues directlyinvolved in the binding and others mediating the specificityof the interaction [24,25].</p><p>The binding site on DARC protein used by both P. vivaxand P. knowlesi ligands maps to 35 amino acids (Ala8-Asp42)located in the N-terminal extracellular domain of the recep-tor [26]. Interestingly, the P. vivax specific binding site isrestricted to one DARC extracellular domain, whereas a closeassociation between the four extracellular domains of thereceptor is needed for chemokines binding [27,28].</p><p>In the present study, alanine-scan mutagenesis of recom-binant DARC protein was performed to identify N-terminalamino acids directly involved in the interaction with PvDBP-RII on RBC. Region II of the P. vivax DBP was expressedas a soluble protein in an insect cell/baculovirus expres-</p><p> see front matter 2005 Elsevier B.V. All rights reserved..molbiopara.2005.04.016vivax Duffy-bindinhristophe Tournamille a, Anne Filipe a, Cyril Bhirley Longacre c, Jean-Pierre Cartron a, Caro</p><p>M U665, Institut National de la Transfusion Sanguine, Universite Paris 7 Db Unite dImmunologie Structurale, Institut Pas</p><p>c Laboratoire de Vaccinologie Parasitaire, Institut</p><p>Received 3 February 2005; received in revised form 19Available online 1 July 2</p><p>uffy antigen; DARC; Plasmodium vivax; Duffy-binding protein; Interaction</p><p>ium vivax is the most widely distributed malariaongst the four species that infect humans. InPlasmodium falciparum, P. vivax is not directlywith a high level of mortality but it remains ath problem in South America, Asia and Oceaniae emergence of resistance to current antimalarialnd the increase of Anopheles lines insensitive tosecticides, the development of new strategies fore being studied. Some of them emphasize the rolehrocytic malaria vaccines, while others focus onod stage of the parasite during its human life cycleking erythrocyte invasion requires a better under-</p><p>parasprotethe D(DBLP. falsimiltaininVI, dRegiDuffdomadepeg site for the Plasmodiumroteint b, Marie-Madeleine Riottot b,e Van Kim a, Yves Colin a,</p><p>iderot, 6 rue Alexandre Cabanel, Paris F-75015, Franceris F-75015, FranceParis F-75015, France</p><p>005; accepted 30 April 2005</p><p>ands, P. vivax or P. knowlesi -Duffy-bindingBPs) [16,17], which bind to DARC, belong to</p><p>binding like erythrocyte-binding protein family) [18] that includes other P. knowlesi EBPs and them EBPs [19,20]. All these DBL-EBPs present a</p><p>racellular domain divided into six regions con-conserved cysteine-rich domains, region II and</p><p>ed as the Duffy-binding like domains (DBL) [21].(RII, 320 amino acids) of the 140 kDa P. vivaxing protein (PvDBP) was identified as the bindingdiating erythrocyte invasion by the Duffy antigen-pathway [22]. More precisely, the 170 amino-acid</p></li><li><p>C. Tournamille et al. / Molecular &amp; Biochemical Parasitology 144 (2005) 100103 101</p><p>Fig. 1. Mappend. The threefragments of DbeadsTM 4B (II of Pv-DBPsoluble recomThe cell-cultuwas eluted bychromatograptechnique afteN-terminal ofplasmid. For5g of PvDBwere eluted inGST-DARC/Ncapacity of wto PvDBP-RIwith 5g of PDARC/Nter pwere eluted wwith the 2.9.7the capacity othree indepenand DARC/N800g of Q1proteins (not</p><p>sion systemsoluble 40was perforRBCs. Onnot shownteins werecorrespondof DARC;the Fy6 lin2C3, i3A, Bing of N-terminal residues of DARC involved in the binding to PvDBP-RII. (A) ReDARC/Nter regions that were fused with the glutathione S-transferase protein (GSTARC were cloned in the pGEX-5X-3 plasmid. The GST-DARC/Nter proteins expre</p><p>Amersham-Biosciences). (B) Direct-interaction of GST-DARC/Nter proteins with(amino acids 201536 of the PvDBP, accession number M37514), was cloned in tbinant protein containing a C-terminal hexahistidine-tag was expressed in the bacre supernatant was dialysed 3 times for 14 h against 20 mM Tris pH 7.5; 0.5 M NaC10 mM Imidazol, 20 mM Tris pH 8.0 and 0.5 M NaCl, concentrated on a YM-1</p><p>hy (Pharmacia, S200, 16/60) equilibrated with 20 mM Tris pH 7.5; 0.5 M NaCl. Thr immunising mice with the soluble PvDBP-RII recombinant protein (LongacreDARC were obtained by in vitro mutagenesis (Quick change site-directed mutag</p><p>direct protein/protein interaction, 1.2 mg of the different GST-DARC/Nter proteinP-RII in 1 PBS containing protease inhibitors (Roche) for 4 h at 4 C. After twglutathione 20 mM, Tris 50 mM pH 8, separated on 10% SDS-PAGE gels and trter proteins was detected by probing the membrane with the 2.9.7 mAb. Critical r</p><p>eight and mutated GST-DARC/Nter proteins to PvDBP-RII. Mutations E9AN17AI (not shown). (C) Displacement of erythrocyte-bound PvDBP-RII by GST-DARCvDBP-RII for 1 h at 4 C in PBS-BSA 1%. After two washes in PBS-BSA 1%, b</p><p>roteins for 1 h at 4 C in PBS-BSA 1%. Erythrocytes were collected by centrifugaith 300 mM NaCl, separated on 10% SDS-PAGE gels and transferred on nitrocellmAb, for 50g (left lane) and 800g (right lane) of each GST-DARC/Nter proteif weight and mutated GST-DARC/Nter proteins to displace PvDBP-RII from erythdent experiments was performed. The signals obtained in presence of 800g of GSter1-N27A proteins were 510% of the signals obtained in presence of 50g of th9A to W26A alanine mutants, GST-DARC/Nter3 and empty GST proteins were 80shown). Alanine-replacement mutants E9AN17A and S28AY30A displace PvDB</p><p>. To assess the specific binding activity of thekDa PvDBP-RII, an erythrocyte-binding assaymed using Duffy-positive and Duffy-negative</p><p>ly Duffy-positive RBC bound PvDBP-RII (data). Three different GST-DARC/Nter fusion pro-</p><p>constructed: GST-DARC/Nter1 (Met1-Ser60)s to the whole N-terminal extracellular domainGST-DARC/Nter2 (Met1-Tyr30) encompassesear epitope 19-QLDFEDVW-26 recognized byG6, MIMA-107 and MINA-108 MAbs [2831]</p><p>and GST-Dear epitopeMIMA-19teins werebacterial exFig. 1A). Dthat GST-DDARC/Nteplace PvDB(Fig. 1B anpresentation of the amino-acid sequence of DARC N-terminal) and the S18-S28 peptide are outlined. PCR amplified cDNAssed in E. coli BL21 were purified with glutathione-Sepharose</p><p>soluble PvDBP-RII. PCR amplified fragment encoding regionhe baculovirus expression vector pVL 1393 (Invitrogen). Theculovirus/insect cell system (Longacre et al., in preparation).l and applied to a metal-affinity column (Talon). PvDBP-RII0 Centricon column (Amicon) and purified by gel filtratione 2.9.7 anti-PvDBP-RII mAb was obtained by the hybridomaet al., in preparation). Alanine replacement mutants of theenesis kit, Stratagen) of the GST-DARC/Nter1 recombinant</p><p>s bound to glutathione-sepharose beads were incubated witho washes in 1 PBS containing protease inhibitors, proteinsansferred on nitrocellulose-membrane. PvDBP-RII bound toesidues of DARC were determined by comparing the bindingand of S28AY30A had no deleterious effect on the binding</p><p>/Nter proteins. 1 109 Duffy-positive RBCs were incubatedinding reactions were incubated with 50 and 800g of GST-tion through bis(2-ethylhexyl) phthalate, and bound proteinsulose-membrane. PvDBP-RII eluted from RBC was revealedns. Critical residues of DARC were determined by comparingrocytes. Densitometry scanning of the autoradiography from</p><p>T-DARC/Nter1, GST-DARC/Nter2, GST-DARC/Nter1-S18Ae same proteins, whereas the signals obtained in presence of110% of the signal obtained in presence of 50g of the sameP-RII as the wt GST-DARC/Nter1 protein (not shown).</p><p>ARC/Nter3 (Ser29-Ser60) contains the Fya lin-41-DGDYGANLE-46 recognized by 655 and</p><p>MAbs [28,31] (Fig. 1A). The DARC/Nter pro-expressed as soluble GST fusion proteins in apression system (as described in the legends ofirect interaction and displacement assays show</p><p>ARC/Nter1 and GST-DARC/Nter2, but not GST-r3, were able to bind PvDBP-RII and to dis-P-RII binding on the erythrocytes Duffy-positive</p><p>d C). These results are in accordance with previous</p></li><li><p>102 C. Tournamille et al. / Molecular &amp; Biochemical Parasitology 144 (2005) 100103</p><p>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 thever our re</p><p>and Tyr30.acids direcwe performbetween GFig. 1B, muD24) and fin the losstrol. In adddisplace PvConverselyinteractionthat the Q1not able tothese residFinally, weing to Duffying the Fy6obtained (dstrate that ttope is notimplicatedcellular dombinding. Coof the Q19Aaccountedchanges. Wnot impairmonoclonadent of the</p><p>Interestiresidues Qtope (Fig.previouslyprimate erywe demonsthis Fy6 epof homologvation thator non-polthe introduifies the strrespectivelPvDBP-RIcompensatin the neigh(night mon</p><p>RecentlRII proteinrecognitionpolar residthese resul</p><p>Table 1Effect of amino acids E9AY30 mutations on the interactions between theN-terminal region of DARC and PvDBP-RIIa</p><p>ARC/Nprotein</p><p>E9AL10AS11AP12AS13AT14AE15AN16AS17AS18A-Q19A-L20A-D21A-F22A-E23A-D24A-V25A-W26AN27AS28AS29AY30A</p><p>e capacdisplacld-type GST-DARC/Nter1 and the mock control GST. The results arehree binding experiments (NT: not tested).</p><p>, on both receptor and ligand, are probably involved inionic and hydrophobic interactions and therefore their</p><p>erties may influence functional conformations and con-te to the specificity of the interaction. It is anticipatedata on the tertiary structure of the DARCPvDBP-RIIlex will eventually elucidate the nature of the contacts</p><p>een these two proteins.ecause the blood stage is critical for the parasite lifeand because P. vivax and the related simian parasite, P.lesi, totally depend on interaction with Duffy antigen forn erythrocyte invasion, knowledge of these molecular</p><p>actions will provide essential information for the devel-nt of new strategies against P. vivax malaria. Recom-t vaccines based on PvDBP-RII are currently being</p><p>loped [35], but artificial peptides targeting DARC pro-which is the invariant side of the interaction, mightsent a complementary approach to block P. vivax bloodinfection, provided that they are not rapidly cleared</p><p>the circulation. Once this paper was ready to be submit-r publication, Choe et al. [36] reported that sulphation</p><p>rosine 41 is essential for high affinity association ofbinant PvDBP-RII with recombinant cells expressinghole DARC protein. The apparent discrepancy with</p><p>resent study might be explained if we consider that thepitope Q19-W26 represents, stricto senso, the PvDBP-e smaller peptide D21-D42 does not [26]. How-sults restricted PvRII binding site between Ala8Thus, in order to define precisely DARC amino</p><p>tly implicated in the interaction with PvDBP-RII,ed site-directed mutagenesis of all amino acids</p><p>lu9 and Tyr30 on GST-DARC/Nter1. As shown intation of six residues, two polar charged (D21 and</p><p>our non-polar (L20, F22, V25 and W26), resultedof PvDBP-RII binding, as for the mock GST con-ition, these GST-DARC/Nter1 mutants could notDBP-RII on the erythrocytes as shown in Fig. 1C., S18A or N27A mutations did not influence director displacement of PvDBP-RII. It is noteworthy9A and E23A mutants bound normally but weredisplace PvDBP-RII, suggesting that mutation ofues could modulate the affinity of the interaction.performed inhibition assays of PvDBP-RII bind--positive RBC by the Ser18-Ser28 peptide includ-epitope sequence (Fig. 1A). No inhibition was</p><p>ata not shown). Altogether these findings demon-he region located at the N-terminal side of Fy6 epi-directly involved in the binding, but is potentiallyin conformation and presentation of DARC extra-</p><p>ain, and thus is essential for an efficient P. vivaxnversely, it is unlikely that the lack of interactionto W26A alanine replacement mutants could be</p><p>by a non-specific effect based on protein foldinge have previously shown that these mutations dothe binding of DARC-transfected cells to anti-Fyl antibodies and/or chemokines which are depen-overall structure integrity of the protein [27,28].ngly, we delineate the PvRII binding site between19 and W26, which strictly matches the Fy6 epi-1 and Table 1), recognized by the BG6 mAbshown to have the capacity to block non-humanthrocyte invasion by P. vivax, in vitro [32]. Heretrate that PvDBP-RII binding site is restricted toitope in human DARC. Moreover, comparisonous DARC sequences [33] reinforces the obser-</p><p>, in non-human primates, the absence of chargedar residues (squirrel monkey, rhesus monkey) orction of a proline residue which potentially mod-ucture (brown capuchin), at or near Fy6 epitope,y, are related to the absence of interaction betweenI and non-human primate erythrocytes, whereasion of the requisite charged and non-polar residuesbouring region of Fy6 permits P. vivax interactionkey) [17,34].y, mutagenesis analysis performed on the PvDBP-</p><p>have shown that residues essential for DARCare invariant, comprising charged and uncharged</p><p>ues, and non-polar residues [24,25]. Altogether,ts indicate that side chains at these residue posi-</p><p>GST-Dfusion</p><p>Nter1Nter2Nter3Nter1-Nter1-Nter1-Nter1-Nter1-Nter1-Nter1-Nter1-Nter1-Nter1-Nter1Nter1Nter1Nter1Nter1Nter1Nter1Nter1Nter1-Nter1-Nter1-Nter1-</p><p>a Thand tothe wifrom t</p><p>tionsbothproptributhat dcompbetw</p><p>Bcycleknowhumainteropmebinandeveteinreprestagefromted foof tyrecom</p><p>the wour pFy6 eters</p><p>Direct interactionwith PvDBP-RII</p><p>Displacement of PvDBP-RII bound on Fy-pos RBC</p><p>+ ++ + + ++ ++ ++ ++ +NT NT+ ++ ++ ++ ++ + +/ + ++ ++ ++ +</p><p>ity of GST-DARC/Nter1 mutants to bind directly PvDBP-RIIe PvDBP-RII on Fy-pos RBC was relative to that obtained for</p></li><li><p>C. Tournamille et al. / Molecular &amp; Biochemical Parasitology 144 (2005) 100103 103</p><p>RII binding site of DARC, and that sulpha...</p></li></ul>