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

Wanaguru, Madushi; Crosnier, Cécile; Johnson, Steven; Rayner, Julian C.; Wright, Gavin J.

doi:10.1074/jbc.m113.484840

Cited by 43 publications

(56 citation statements)

References 41 publications

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“…These results confirm that the acquisition of antibodies against placental parasites and then against VAR2CSA start developing as early as in first pregnancy (20,38,39) and highlight the importance of the N-terminal region of VAR2CSA (DBL1X-3X) in the development of the humoral response against PAM. This is borne out by other studies revealing the detailed mechanisms of other malaria-derived protein/ligand interactions (40)(41)(42)(43).…”

Section: Discussionmentioning

confidence: 92%

Parity-Dependent Recognition of DBL1X-3X Suggests an Important Role of the VAR2CSA High-Affinity CSA-Binding Region in the Development of the Humoral Response against Placental Malaria

et al. 2015

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f Plasmodium falciparum multidomain protein VAR2CSA stands today as the leading vaccine candidate against pregnancy-associated malaria (PAM). Most of the studies aiming to decrypt how naturally acquired immunity develops have assessed the immune recognition of individual VAR2CSA Duffy-binding-like (DBL) domains, thus overlooking the presence of conformational epitopes resulting from the overall folding of the full-length protein. In order to characterize the development of humoral immunity toward VAR2CSA, we made use of a large cohort of 293 Senegalese pregnant women to assess the level of recognition by plasma IgG of the full-length VAR2CSA protein of the 3D7 parasite strain (3D7-VAR2CSA), the CSA-binding multidomains 3D7-DBL1X to -DBL3X (3D7-DBL1X-3X), and the CSA nonbinding multidomains 3D7-DBL4 to -DBL6 (3D7-DBL4-6), as well as individual 3D7-DBL domains. Our results revealed a parity-dependent recognition of the full-length 3D7-VAR2CSA and of the CSA-binding region, 3D7-DBL1X-3X. Indeed, multigravid women possess significantly higher levels of antibodies directed against these constructs than primigravidae. Our results suggest an important role of antibodies targeting the CSA-binding region in the development of immunity against PAM, therefore providing new insights on how natural protection might be acquired and further information for the design of VAR2CSA-based vaccines. E ach year, pregnancy-associated malaria (PAM) is responsible for the deaths of as many as 363,000 neonates and for at least 10,000 maternal deaths worldwide (1). The massive accumulation of Plasmodium falciparum-infected erythrocytes (IEs) to the syncytiotrophoblast layer composing the maternal face of the placenta limits maternal-fetal exchanges, leading to clinical complications for both mother and child (2). This phenomenon is mediated by the VAR2CSA protein, a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins, that is preferentially expressed by placental parasites (3-5) and binds to the placental chondroitin-4-sulfate A (CSA) (6). After one or two pregnancies, women acquire antibodies targeting VAR2CSA that inhibit IE sequestration and decrease the negative outcomes of PAM (7,8). In vitro assays have shown that antibodies from multigravid women are able to block the interactions between CSA and IEs originating from different parts of the world (9), showing the existence of a cross-reactive antibody response and suggesting that a relative conservation of important inhibitory epitopes within VAR2CSA proteins may exit.VAR2CSA is a 350-kDa transmembrane protein composed of six Duffy-binding-like (DBL) domains. Previous works have described the CSA-binding properties of single DBL domains (3, 10, 11). Insight derived from structural studies of DBL domains revealed the importance of the DBL3X domain in VAR2CSA CSA binding but also suggested a higher-order structure organization of VAR2CSA where the positioning of different surfaces involved in chondroitin sulfate proteoglycan (CSPG) binding forms a specific bin...

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Section: Discussionmentioning

confidence: 92%

Parity-Dependent Recognition of DBL1X-3X Suggests an Important Role of the VAR2CSA High-Affinity CSA-Binding Region in the Development of the Humoral Response against Placental Malaria

et al. 2015

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“…However, this hypothesis is inconsistent with a much earlier study by Orlandi et al (24) in which the binding of native PfEBA175 to human erythrocytes was observed to be potently inhibited by soluble Neu5Gc and oligosaccharides containing Neu5Acα2-3Gal but not by Neu5Ac as a monosaccharide. This glycan-binding specificity was confirmed in a recent biochemical investigation using a recombinant protein consisting of the entire ectodomain of PfEBA175 (25). Furthermore, the sialic acid specificity of PfEBA175 orthologs cannot explain the restriction of ape Laverania parasites to their respective chimpanzee and gorilla hosts, both of which carry a functional CMAH gene (5).…”

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confidence: 78%

“…By using SPR, we have been able to address the host specificity of EBA175-GYPA interactions in a quantitative and directly comparable manner by measuring binding affinities. The proposed lack of PfEBA175 binding to Neu5Gc, which forms the basis of the EBA175-related host restriction hypothesis, also is at odds with the observation that soluble Neu5Gc as a monosaccharide inhibits the binding of native PfEB175 to human erythrocytes with a level of potency similar to that of oligosaccharides containing Neu5Acα-2,3-Gal (24) and our recent finding that a functionally active recombinant protein containing the full-length ectodomain of PfEBA175 binds Neu5Gc as well as Neu5Acα-2,3-Gal (25). We attempted to investigate the specificity of the EBA175-GYPA interaction from the perspective of the host receptor, but we were unable to express human GYPA in a functionally active, recombinant form using our mammalian expression system (25).…”

Section: Discussionmentioning

confidence: 99%

“…The proposed lack of PfEBA175 binding to Neu5Gc, which forms the basis of the EBA175-related host restriction hypothesis, also is at odds with the observation that soluble Neu5Gc as a monosaccharide inhibits the binding of native PfEB175 to human erythrocytes with a level of potency similar to that of oligosaccharides containing Neu5Acα-2,3-Gal (24) and our recent finding that a functionally active recombinant protein containing the full-length ectodomain of PfEBA175 binds Neu5Gc as well as Neu5Acα-2,3-Gal (25). We attempted to investigate the specificity of the EBA175-GYPA interaction from the perspective of the host receptor, but we were unable to express human GYPA in a functionally active, recombinant form using our mammalian expression system (25). Based on the available evidence, we therefore propose that the EBA175-GYPA interaction is unlikely to be the sole determinant of host tropism within Laverania parasites, including P. falciparum, but additional experiments using either native or biochemically active recombinant ape GYPA are required to dissect fully the host specificity of the EBA175-GYPA interaction.…”

Section: Discussionmentioning

confidence: 99%

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RH5–Basigin interaction plays a major role in the host tropism of Plasmodium falciparum

Wanaguru¹,

Liu²,

Hahn

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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137

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Plasmodium falciparum, the cause of almost all human malaria mortality, is a member of the Laverania subgenus which infects African great apes. Interestingly, Laverania parasites exhibit strict host specificity in their natural environment: P. reichenowi, P. billcollinsi, and P. gaboni infect only chimpanzees; P. praefalciparum, P. blacklocki, and P. adleri are restricted to gorillas, and P. falciparum is pandemic in humans. The molecular mechanism(s) responsible for these host restrictions are not understood, although the interaction between the parasite blood-stage invasion ligand EBA175 and the host erythrocyte receptor Glycophorin-A (GYPA) has been implicated previously. We reexamined the role of the EBA175-GYPA interaction in host tropism using recombinant proteins and biophysical assays and found that EBA175 orthologs from the chimpanzee-restricted parasites P. reichenowi and P. billcollinsi both bound to human GYPA with affinities similar to that of P. falciparum, suggesting that the EBA175-GYPA interaction is unlikely to be the sole determinant of Laverania host specificity. We next investigated the contribution of the recently discovered Reticulocyte-binding protein Homolog 5 (RH5)-Basigin (BSG) interaction in host-species selectivity and found that P. falciparum RH5 bound chimpanzee BSG with a significantly lower affinity than human BSG and did not bind gorilla BSG, mirroring the known host tropism of P. falciparum. Using site-directed mutagenesis, we identified residues in BSG that are responsible for the species specificity of PfRH5 binding. Consistent with the essential role of the PfRH5-BSG interaction in erythrocyte invasion, we conclude that species-specific differences in the BSG receptor provide a molecular explanation for the restriction of P. falciparum to its human host.surface plasmon resonance | protein interactions T he most deadly of the malaria parasites, Plasmodium falciparum, is highly divergent from the other species of Plasmodium known to infect humans (1-3), with its closest relatives comprising a group of chimpanzee and gorilla parasites from the subgenus Laverania (3-7). Despite the origin of P. falciparum as a zoonosis, and the continuing coexistence of humans and apes in West and Central Africa, extensive field studies have failed to detect P. falciparum in wild-living chimpanzees and gorillas (5). Although there are reports of P. falciparum infecting chimpanzees either in certain captive settings (2) or following splenectomy and deliberate transfer of P. falciparum-infected human blood (8-10), the resulting infections have low parasitemia and are not known to result in malignant tertian malaria, suggesting a host-specific barrier for replete infection. The existence of host-specific barriers within the Laverania subgenus is supported further by the strict host specificity exhibited by ape Laverania parasites in the wild: Plasmodium reichenowi, Plasmodium billcollinsi, and Plasmodium gaboni infect only chimpanzees, and Plasmodium praefalciparum, Plasmodium blacklocki, and Plasm...

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“…Figure 1D is reproduced from Wanaguru et al . with minor stylistic changes [8]. Bars represent mean ± standard deviation (SD), n = 3.…”

Section: Resultsmentioning

confidence: 99%

Quantification of Plasmodium-host protein interactions on intact, unmodified erythrocytes by back-scattering interferometry

Saetear

Perrin

Bartholdson

et al. 2015

Malar J

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BackgroundInvasion of host erythrocytes by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion involves recognition events between erythrocyte receptors and ligands on the merozoite, the invasive blood form of the parasite. Identifying and characterizing host-parasite interactions is impeded by the biochemical challenges of working with membrane-embedded glycoprotein receptors. For example, the interaction between P. falciparum erythrocyte binding antigen 175 (PfEBA175) and glycophorin A (GYPA) depends on post-translational modifications that are not easily added in recombinant expression systems, and the use of native GYPA is limited by the hydrophobic transmembrane region, making it difficult to biochemically manipulate. It would, therefore, be desirable to perform quantitative binding assays with receptors embedded within the membranes of intact human erythrocytes.MethodsThe extracellular region of GYPA was over-expressed as a soluble protein in HEK293E cells. This protein was characterized, sialylated and evaluated for binding to the PfEBA175 protein. The label-free and free-solution assay, backscattering interferometry (BSI), was used to perform binding assays of two well-characterized P. falciparum invasion ligands to intact unmodified human erythrocytes.ResultsFindings indicate that the post-translational modifications present on native GYPA are required for it to bind recombinant PfEBA175 and that these modifications cannot be recapitulated in vitro using mammalian overexpression methods. Here, BSI was used to obtain quantitative, high fidelity interaction determinations on intact, unmodified erythrocytes. Using BSI and purified recombinant proteins constituting the entire ectodomains of the P. falciparum merozoite ligands PfEBA175 and PfRH5, KDs of 1.1 μM and 50 nM were measured for the PfRH5-BSG and PfEBA175-GYPA interactions, respectively, in good agreement with previous biophysical measurements of these interactions.ConclusionsThese results demonstrate that BSI can be used to detect and quantify the interactions of two merozoite invasion ligands with their receptors on intact human erythrocytes. BSI assays were performed on unlabelled, free-solution proteins in their native environment, requiring only nanomoles of recombinant protein. This study suggests that BSI can be used to investigate host-parasite protein interactions without the limitations of other assay platforms, and therefore represents a valuable new method to investigate the molecular mechanisms involved in erythrocyte invasion by P. falciparum.

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

Cited by 43 publications

References 41 publications

Parity-Dependent Recognition of DBL1X-3X Suggests an Important Role of the VAR2CSA High-Affinity CSA-Binding Region in the Development of the Humoral Response against Placental Malaria

Parity-Dependent Recognition of DBL1X-3X Suggests an Important Role of the VAR2CSA High-Affinity CSA-Binding Region in the Development of the Humoral Response against Placental Malaria

RH5–Basigin interaction plays a major role in the host tropism of Plasmodium falciparum

Quantification of Plasmodium-host protein interactions on intact, unmodified erythrocytes by back-scattering interferometry

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