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Biochemical Analysis of the Plasmodium falciparum Erythrocyte-binding Antigen-175 (EBA175)-Glycophorin-A Interaction

IMPLICATIONS FOR VACCINE DESIGN*
  • Madushi Wanaguru
    Affiliations
    From the Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH,

    the Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA
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  • Cécile Crosnier
    Affiliations
    From the Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH,

    the Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA
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  • Steven Johnson
    Affiliations
    the Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
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  • Julian C. Rayner
    Affiliations
    the Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA
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  • Gavin J. Wright
    Correspondence
    To whom correspondence should be addressed: Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 2DP, United Kingdom. Tel.: 44-1223-496852; Fax: 44-1223-496802
    Affiliations
    From the Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH,

    the Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the Wellcome Trust Grant 098051 and MRC Grant G0900888.
    ♦ This article was selected as a Paper of the Week.
Open AccessPublished:September 16, 2013DOI:https://doi.org/10.1074/jbc.M113.484840
      PfEBA175 has an important role in the invasion of human erythrocytes by Plasmodium falciparum and is therefore considered a high priority blood-stage malaria vaccine candidate. PfEBA175 mediates adhesion to erythrocytes through binding of the Duffy-binding-like (DBL) domains in its extracellular domain to Neu5Acα2–3Gal displayed on the O-linked glycans of glycophorin-A (GYPA). Because of the difficulties in expressing active full-length (FL) P. falciparum proteins in a recombinant form, previous analyses of the PfEBA175-GYPA interaction have largely focused on the DBL domains alone, and therefore they have not been performed in the context of the native protein sequence. Here, we express the entire ectodomain of PfEBA175 (PfEBA175 FL) in soluble form, allowing us to compare the biochemical and immunological properties with a fragment containing only the tandem DBL domains (“region II,” PfEBA175 RII). Recombinant PfEBA175 FL bound human erythrocytes in a trypsin and neuraminidase-sensitive manner and recognized Neu5Acα2–3Gal-containing glycans, confirming its biochemical activity. A quantitative binding analysis showed that PfEBA175 FL interacted with native GYPA with a KD ∼0.26 μm and is capable of self-association. By comparison, the RII fragment alone bound GYPA with a lower affinity demonstrating that regions outside of the DBL domains are important for interactions with GYPA; antibodies directed to these other regions also contributed to the inhibition of parasite invasion. These data demonstrate the importance of PfEBA175 regions other than the DBL domains in the interaction with GYPA and merit their inclusion in an EBA175-based vaccine.
      Background: The GYPA-PfEBA175 interaction is important for erythrocyte invasion by the malaria parasite.
      Results: The entire ectodomain of EBA175 interacted with GYPA with different biochemical parameters to the previously determined GYPA-binding fragment containing two DBL domains.
      Conclusion: Regions outside of the tandem DBL domains contribute to GYPA binding by EBA175.
      Significance: These findings may assist the design of an EBA175-based malaria vaccine.

      Introduction

      The human malaria parasite, Plasmodium falciparum, causes an estimated 300–500 million clinical cases and up to 800,000 deaths each year (
      • Murray C.J.
      • Rosenfeld L.C.
      • Lim S.S.
      • Andrews K.G.
      • Foreman K.J.
      • Haring D.
      • Fullman N.
      • Naghavi M.
      • Lozano R.
      • Lopez A.D.
      Global malaria mortality between 1980 and 2010: a systematic analysis.
      ,
      • World Health Organization
      ). Widespread implementation of control measures within the last decade, including artemisinin combination therapy and the use of insecticide-treated bed nets, has resulted in a significant decrease in the incidence of P. falciparum malaria in endemic countries (
      • Geels M.J.
      • Imoukhuede E.B.
      • Imbault N.
      • van Schooten H.
      • McWade T.
      • Troye-Blomberg M.
      • Dobbelaer R.
      • Craig A.G.
      • Leroy O.
      European Vaccine Initiative: lessons from developing malaria vaccines.
      ,
      • Greenwood B.M.
      • Targett G.A.
      Malaria vaccines and the new malaria agenda.
      ,
      • Snow R.W.
      • Guerra C.A.
      • Mutheu J.J.
      • Hay S.I.
      International funding for malaria control in relation to populations at risk of stable Plasmodium falciparum transmission.
      ). In the face of emerging resistance to artemisinin in parasites and pyrethroid insecticides in mosquito vectors, however, the need for an effective vaccine for long term control and prevention of malaria remains an important global health objective (
      • Geels M.J.
      • Imoukhuede E.B.
      • Imbault N.
      • van Schooten H.
      • McWade T.
      • Troye-Blomberg M.
      • Dobbelaer R.
      • Craig A.G.
      • Leroy O.
      European Vaccine Initiative: lessons from developing malaria vaccines.
      ,
      • Greenwood B.M.
      • Targett G.A.
      Malaria vaccines and the new malaria agenda.
      ,
      • Crompton P.D.
      • Pierce S.K.
      • Miller L.H.
      Advances and challenges in malaria vaccine development.
      ). Vaccines that target the blood stage of the infection are conceptually attractive because the parasite is directly exposed to the host humoral immune system, and it is this stage of the life cycle that is responsible for all the clinical symptoms of malaria (
      • Miller L.H.
      • Baruch D.I.
      • Marsh K.
      • Doumbo O.K.
      The pathogenic basis of malaria.
      ). The blood stage is initiated when an extracellular form of the parasite called the merozoite recognizes and invades host erythrocytes. Invasion is a complex process involving multiple interactions between host erythrocyte receptors and parasite ligands displayed on the merozoite surface. PfEBA175 was the first P. falciparum invasion ligand identified and interacts with the highly abundant erythrocyte surface sialoglycoprotein, glycophorin-A (GYPA)
      The abbreviations used are: GYPA, glycophorin-A; AVEXIS, avidity-based extracellular interaction screen; MALS, multiangle light-scattering; SEC, size exclusion chromatography; SPR, surface plasmon resonance; DBL, Duffy-binding-like; FL, full length; RII, region II; COMP, cartilage oligomeric matrix protein; HBS, HEPES-buffered saline.
      (
      • Camus D.
      • Hadley T.J.
      A Plasmodium falciparum antigen that binds to host erythrocytes and merozoites.
      ,
      • Orlandi P.A.
      • Sim B.K.
      • Chulay J.D.
      • Haynes J.D.
      Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum.
      ,
      • Sim B.K.
      • Chitnis C.E.
      • Wasniowska K.
      • Hadley T.J.
      • Miller L.H.
      Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum.
      ). PfEBA175 is considered a leading vaccine candidate because antibodies directed against PfEBA175 are present in malaria-immune individuals, and antibodies raised against recombinantly expressed fragments of PfEBA175 inhibit erythrocyte invasion by P. falciparum in vitro (
      • El Sahly H.M.
      • Patel S.M.
      • Atmar R.L.
      • Lanford T.A.
      • Dube T.
      • Thompson D.
      • Sim B.K.
      • Long C.
      • Keitel W.A.
      Safety and immunogenicity of a recombinant nonglycosylated erythrocyte binding antigen 175 Region II malaria vaccine in healthy adults living in an area where malaria is not endemic.
      ,
      • Jiang L.
      • Gaur D.
      • Mu J.
      • Zhou H.
      • Long C.A.
      • Miller L.H.
      Evidence for erythrocyte-binding antigen 175 as a component of a ligand-blocking blood-stage malaria vaccine.
      ,
      • Lopaticki S.
      • Maier A.G.
      • Thompson J.
      • Wilson D.W.
      • Tham W.H.
      • Triglia T.
      • Gout A.
      • Speed T.P.
      • Beeson J.G.
      • Healer J.
      • Cowman A.F.
      Reticulocyte and erythrocyte binding-like proteins function cooperatively in invasion of human erythrocytes by malaria parasites.
      ,
      • Pandey K.C.
      • Singh S.
      • Pattnaik P.
      • Pillai C.R.
      • Pillai U.
      • Lynn A.
      • Jain S.K.
      • Chitnis C.E.
      Bacterially expressed and refolded receptor binding domain of Plasmodium falciparum EBA-175 elicits invasion inhibitory antibodies.
      ,
      • Richards J.S.
      • Stanisic D.I.
      • Fowkes F.J.
      • Tavul L.
      • Dabod E.
      • Thompson J.K.
      • Kumar S.
      • Chitnis C.E.
      • Narum D.L.
      • Michon P.
      • Siba P.M.
      • Cowman A.F.
      • Mueller I.
      • Beeson J.G.
      Association between naturally acquired antibodies to erythrocyte-binding antigens of Plasmodium falciparum and protection from malaria and high-density parasitemia.
      ,
      • Zhang D.
      • Pan W.
      Evaluation of three Pichia pastoris-expressed Plasmodium falciparum merozoite proteins as a combination vaccine against infection with blood-stage parasites.
      ). EBA175 is a member of the erythrocyte-binding-like family of Plasmodium proteins, which include the Duffy-binding proteins of Plasmodium vivax and Plasmodium knowlesi (PvDBP and PkDBP) and the P. falciparum paralogs EBA140, EBA181, and EBL1. The members of the erythrocyte-binding-like family share a similar gene structure, and this homology has been used to define six regions, RI–RVI, in their ectodomains (Fig. 1A) (
      • Adams J.H.
      • Sim B.K.
      • Dolan S.A.
      • Fang X.
      • Kaslow D.C.
      • Miller L.H.
      A family of erythrocyte binding proteins of malaria parasites.
      ). A functional analysis of these regions showed that the RII fragment is capable of rosetting erythrocytes (
      • Sim B.K.
      • Chitnis C.E.
      • Wasniowska K.
      • Hadley T.J.
      • Miller L.H.
      Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum.
      ). The structure of RII, which comprises two tandem DBL domains (F1 and F2), has been determined by x-ray crystallography (
      • Tolia N.H.
      • Enemark E.J.
      • Sim B.K.
      • Joshua-Tor L.
      Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum.
      ).
      Figure thumbnail gr1
      FIGURE 1Recombinant PfEBA175 FL is soluble and immunologically active. A, schematic diagram of PfEBA175 FL highlighting the six regions of homology, RI–RVI, including RII that contains the DBL domains F1 and F2. The sequences are given only for the short peptide tags. Cd4, Ig-like domains 3 and 4 of rat Cd4 (∼25 kDa); Bio, peptide substrate for the biotin ligase BirA. The biotinylatable lysine residue is indicated in green. His, hexa-His. COMP + β-lac, pentamerization domain of the rat COMP protein and the ampicillin resistance protein β-lactamase. B, Western blot of unpurified cell culture supernatant containing biotinylated PfEBA175 FL. C, binding by ELISA of three anti-EBA175 monoclonal antibodies to untreated and heat-treated PfEBA175 FL. Negative control is Cd4 tag alone; data are mean ± S.D., n = 3. D, SEC elution profile of affinity-purified PfEBA175 FL with Coomassie G-250-stained denaturing and native gels of peak fractions (inset); P1, minor peak; P2, major peak. Column void volume = 24 ml; molecular mass standards are indicated with red arrows. E, MALS of purified PfEBA175 FL. The peaks correspond to SEC elution (absorbance at 280 nm on the right y axis); horizontal lines indicate the molecular mass (left y axis). PfEBA175 FL is shown in red with a 30-kDa monomeric control protein shown for comparison (black).
      The PfEBA175 receptor, GYPA, is a dimeric type I transmembrane protein carrying 15 closely clustered O-linked tetrasaccharides capped with sialic acid/N-acetylneuraminic acid (Neu5Ac) (
      • MacKenzie K.R.
      • Prestegard J.H.
      • Engelman D.M.
      A transmembrane helix dimer: structure and implications.
      ,
      • Tomita M.
      • Marchesi V.T.
      Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin.
      ). The “Neu5Acα2–3Gal” sequence of these glycans was shown to be essential for the recognition of GYPA by PfEBA175 (
      • Orlandi P.A.
      • Klotz F.W.
      • Haynes J.D.
      A malaria invasion receptor, the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum recognizes the terminal Neu5Ac(α2–3)Gal-sequences of glycophorin A.
      ). Co-crystallization of the RII fragment with a structural analog of this disaccharide, α-2,3-sialyllactose, revealed six putative glycan-binding sites at the dimer interface of the parasite protein, with four of the sites located within two channels that span the dimer and another two in a deep groove accessible only through a cavity at the top of the dimer (
      • Tolia N.H.
      • Enemark E.J.
      • Sim B.K.
      • Joshua-Tor L.
      Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum.
      ). Based on the locations of the glycan-binding sites in the RII fragment, and the predominance of the monomeric form of PfEBA175 RII in solution, Tolia and co-workers (
      • Batchelor J.D.
      • Zahm J.A.
      • Tolia N.H.
      Dimerization of Plasmodium vivax DBP is induced upon receptor binding and drives recognition of DARC.
      ) have proposed a “receptor-induced dimerization model” for the erythrocyte binding of PfEBA175, which postulates monomeric PfEBA175 assembling into a dimer around the dimeric extracellular region of GYPA during invasion. This model is also consistent with recent structural studies investigating the mode of binding of the PvDBP RII to its sulfotyrosine-carrying receptor, DARC.
      Although studies of the 68-kDa recombinant PfEBA175 RII have proven highly informative, native PfEBA175 is a much larger protein, being synthesized as a 190-kDa membrane-tethered precursor that is proteolytically cleaved during erythrocyte invasion to release the 175-kDa extracellular region (
      • Orlandi P.A.
      • Sim B.K.
      • Chulay J.D.
      • Haynes J.D.
      Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum.
      ). Because of the technical difficulties associated with expressing full-length Plasmodium proteins in a functionally active soluble recombinant form (
      • Birkholtz L.M.
      • Blatch G.
      • Coetzer T.L.
      • Hoppe H.C.
      • Human E.
      • Morris E.J.
      • Ngcete Z.
      • Oldfield L.
      • Roth R.
      • Shonhai A.
      • Stephens L.
      • Louw A.I.
      Heterologous expression of plasmodial proteins for structural studies and functional annotation.
      ), much of the biochemical characterization of the PfEBA175-GYPA interaction has been performed using just the RII fragment.
      In the study reported here, we expressed the full-length ectodomain of PfEBA175 (PfEBA175 FL) in a soluble recombinant form using a mammalian expression system. We confirmed that the recombinant protein is biochemically active and exhibited binding properties similar to those of native PfEBA175 isolated from parasite cultures. Using this protein, we investigated the biophysical parameters of the EBA175-GYPA interaction and compared them with those of the RII fragment to demonstrate that regions outside the DBL domains contributed to GYPA binding. These data have important implications for the rational design of an effective PfEBA175-based malaria vaccine.

      DISCUSSION

      P. falciparum EBA175 has long been considered an attractive anti-malarial vaccine target because of its important role in erythrocyte invasion mediated through its interactions with GYPA expressed on the surface of host erythrocytes. The technical difficulties associated with expressing Plasmodium proteins recombinantly have meant that most biochemical and vaccine research has relied on expressing subfragments of the EBA175 ectodomain, most commonly the tandem DBL domains known as region II. In this study, we successfully expressed the entire full-length ectodomain of PfEBA175 as a functionally active soluble recombinant protein that enabled us to perform a detailed biochemical analysis of its interaction with native GYPA, and we directly compared this with the region II subfragment.
      One interesting finding from a systematic interaction screen against a panel of glycans was that the full-length PfEBA175 bound Neu5Acα2–3Gal-containing glycans as expected but also interacted with Neu5Gc. These results are consistent with the observations of Orlandi et al. (
      • Orlandi P.A.
      • Klotz F.W.
      • Haynes J.D.
      A malaria invasion receptor, the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum recognizes the terminal Neu5Ac(α2–3)Gal-sequences of glycophorin A.
      ) who reported that the binding of native PfEBA175 to erythrocytes was potently inhibited by Neu5Gc and oligosaccharides containing Neu5Acα2–3Gal. Neu5Gc is not present on human erythrocytes, due to the absence of the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase, which is required for the conversion of Neu5Ac to Neu5Gc; however, it is the predominant form of sialic acid on the erythrocytes of other apes (
      • Muchmore E.A.
      • Diaz S.
      • Varki A.
      A structural difference between the cell surfaces of humans and the great apes.
      ). This difference in sialic acid composition has been proposed to be responsible for the restriction of P. falciparum and the related parasite Plasmodium reichenowi to their respective human and chimpanzee hosts. Martin et al. (
      • Martin M.J.
      • Rayner J.C.
      • Gagneux P.
      • Barnwell J.W.
      • Varki A.
      Evolution of human-chimpanzee differences in malaria susceptibility: relationship to human genetic loss of N-glycolylneuraminic acid.
      ) recombinantly expressed PfEBA175 RII and P. reichenowi EBA175 RII (PrEBA175 RII) on the surface of monkey COS cells and observed binding only to human and chimpanzee erythrocytes, respectively, leading to the proposal that PfEBA175 RII recognizes Neu5Ac-carrying GYPA, whereas PrEBA175 RII binds Neu5Gc-containing GYPA. The findings in our study do not support this hypothesis. Further investigation of the contribution of the EBA175-GYPA interaction to the restriction of Plasmodium spp. parasites to their natural hosts is therefore warranted, and this will be facilitated by the ability to express the entire ectodomain of EBA175 in an active form. This is of topical interest due to the recent discovery of new Plasmodium species that infect gorillas and that are the closest known relatives of P. falciparum (
      • Liu W.
      • Li Y.
      • Learn G.H.
      • Rudicell R.S.
      • Robertson J.D.
      • Keele B.F.
      • Ndjango J.B.
      • Sanz C.M.
      • Morgan D.B.
      • Locatelli S.
      • Gonder M.K.
      • Kranzusch P.J.
      • Walsh P.D.
      • Delaporte E.
      • Mpoudi-Ngole E.
      • Georgiev A.V.
      • Muller M.N.
      • Shaw G.M.
      • Peeters M.
      • Sharp P.M.
      • Rayner J.C.
      • Hahn B.H.
      Origin of the human malaria parasite Plasmodium falciparum in gorillas.
      ,
      • Rayner J.C.
      • Liu W.
      • Peeters M.
      • Sharp P.M.
      • Hahn B.H.
      A plethora of Plasmodium species in wild apes: a source of human infection?.
      ).
      From the six regions of homology that constitute the extracellular domain of PfEBA175, only RII is known to be essential and sufficient for binding to human erythrocytes (
      • Sim B.K.
      • Chitnis C.E.
      • Wasniowska K.
      • Hadley T.J.
      • Miller L.H.
      Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum.
      ). Several previous studies using short chemically synthesized peptides (
      • Jakobsen P.H.
      • Heegaard P.M.
      • Koch C.
      • Wasniowska K.
      • Lemnge M.M.
      • Jensen J.B.
      • Sim B.K.
      Identification of an erythrocyte binding peptide from the erythrocyte binding antigen, EBA-175, which blocks parasite multiplication and induces peptide-blocking antibodies.
      ,
      • Kain K.C.
      • Orlandi P.A.
      • Haynes J.D.
      • Sim K.L.
      • Lanar D.E.
      Evidence for two-stage binding by the 175-kDa erythrocyte binding antigen of Plasmodium falciparum.
      ,
      • Rodriguez L.E.
      • Urquiza M.
      • Ocampo M.
      • Suarez J.
      • Curtidor H.
      • Guzman F.
      • Vargas L.E.
      • Triviños M.
      • Rosas M.
      • Patarroyo M.E.
      Plasmodium falciparum EBA-175-kDa protein peptides which bind to human red blood cells.
      ) or in vitro translated EBA175 fragments (
      • Kain K.C.
      • Orlandi P.A.
      • Haynes J.D.
      • Sim K.L.
      • Lanar D.E.
      Evidence for two-stage binding by the 175-kDa erythrocyte binding antigen of Plasmodium falciparum.
      ) have suggested additional erythrocyte binding determinants outside of region II, although a recent study did not support this (
      • Jiang L.
      • Gaur D.
      • Mu J.
      • Zhou H.
      • Long C.A.
      • Miller L.H.
      Evidence for erythrocyte-binding antigen 175 as a component of a ligand-blocking blood-stage malaria vaccine.
      ). These findings have led to the proposition of two-step binding models that invoke conformational changes in the EBA175 protein (
      • Jakobsen P.H.
      • Heegaard P.M.
      • Koch C.
      • Wasniowska K.
      • Lemnge M.M.
      • Jensen J.B.
      • Sim B.K.
      Identification of an erythrocyte binding peptide from the erythrocyte binding antigen, EBA-175, which blocks parasite multiplication and induces peptide-blocking antibodies.
      ,
      • Kain K.C.
      • Orlandi P.A.
      • Haynes J.D.
      • Sim K.L.
      • Lanar D.E.
      Evidence for two-stage binding by the 175-kDa erythrocyte binding antigen of Plasmodium falciparum.
      ). Our quantitative binding analysis did not find any evidence to support a conformational change in EBA175 (at least a relatively slow one that could be detected using our SPR method) but did clearly show that the full-length ectodomain of EBA175 bound native GYPA with an affinity one order of magnitude higher than the region II fragment alone suggesting that extracellular regions of PfEBA175 outside of RII do play some role in the interaction with GYPA.
      The region II fragment of EBA175 crystallized as an anti-parallel dimer with the putative glycan-binding sites being formed at the dimer interface, leading to the suggestion that GYPA, which forms a dimeric complex at the erythrocyte surface, induces EBA175 dimerization upon binding (
      • Tolia N.H.
      • Enemark E.J.
      • Sim B.K.
      • Joshua-Tor L.
      Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum.
      ). The SPR data we obtained for the interaction of PfEBA175 with GYPA fitted better to a bivalent analyte model than to a simple 1:1 binding model, which is consistent with the dimerization of EBA175 playing a role in its interaction with GYPA. Although we showed that both PfEBA175 RII and PfEBA175 FL were primarily monomeric in solution, both were capable of self-association, and the full-length ectodomain of EBA175 could bind itself with ∼70-fold higher affinity than the RII fragment. Extracellular regions outside of RII may therefore facilitate the interaction with GYPA by promoting homodimerization of EBA175.
      Our binding analysis also revealed that soluble PfEBA175 FL has a 2-fold lower affinity for Neu5Acα2–3Gal than for GYPA, but PfEBA175 RII bound both with similar affinities; therefore, RII probably interacts primarily with the glycan moieties of GYPA, whereas PfEBA175 FL forms contacts with both the oligosaccharides and the polypeptide backbone. We attempted to further investigate this by expressing GYPA as a recombinant protein so that it could be purified in large quantities and biochemically manipulated. Although we were able to express and purify the ectodomain of GYPA in a soluble form (rGYPA) and show that it was antigenically active by monoclonal antibody binding, it was unable to bind PfEBA175 FL (data not shown). We attributed the inability of rGYPA to bind PfEBA175 to under-sialylation because glycan profiling of rGYPA using a panel of lectins showed significantly lower levels of sialylation relative to native GYPA. Despite increasing the level of rGYPA sialylation by co-transfecting our GYPA expression construct with plasmids encoding an α-2,3-sialyltransferase and/or CMP-sialic acid transporter, this increase was not sufficient to confer binding to PfEBA175 (data not shown). In addition, we were unable to detect any binding to native GYPA using a fragment consisting of regions III to VI of EBA175 by AVEXIS (data not shown); among other possibilities, the interaction of EBA175 with the GYPA polypeptide backbone could therefore be dependent on the binding of RII to the glycan moieties on the receptor.
      In conclusion, the 10-fold higher binding affinity of PfEBA175 FL for GYPA in comparison with the PfEBA175 RII fragment is likely due to the participation of the extracellular regions outside of RII in the homodimerization of EBA175 and the formation of additional contacts with GYPA.
      The ability to express the entire ectodomain of P. falciparum EBA175 as a biochemically active recombinant protein and the finding that regions of EBA175 outside of the tandem DBL domains are important for GYPA binding suggested that PfEBA175 FL could be a better vaccine candidate than PfEBA175 RII alone. Consistent with these observations, when normalized for immunoreactivity to RII, the antisera to PfEBA175 FL was ∼5-fold more potent than the antisera to PfEBA175 RII. This suggests that antibodies directed against extracellular regions of PfEBA175 outside of RII also contribute to inhibiting erythrocyte invasion. We envisage that the findings reported here will contribute to a more complete understanding of the molecular basis of erythrocyte invasion by P. falciparum and eventually lead to the development of an effective vaccine against this infectious disease.

      Acknowledgments

      We thank Leyla Bustamante and Michel Theron for support in performing and analyzing P. falciparum invasion assays, Laura Romanelli for technical assistance with GYPA expression trials, and Susan Lea for MALS expertise.

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