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. 2025 Nov 7;124(11):125. doi: 10.1007/s00436-025-08582-1

P. falciparum EBA-181 ligand - searching for the receptor on human erythrocytes

Marlena Jodłowska 1,#, Patrycja Burzyńska 1,#, Agata Zerka 1, Ewa Jaśkiewicz 1,2,✉
PMCID: PMC12592255  PMID: 41198968

Abstract

Malaria caused by Plasmodium is responsible for approximately 250 million clinical cases and 600,000 deaths per year, mostly in Africa. Our understanding of Plasmodium parasite biology remains incomplete. The key step of Plasmodium invasion is the blood stage, which is mediated by Erythrocyte binding-like (EBL) and Reticulocyte binding-like (RBL) proteins. Three P. falciparum EBL proteins and their receptors are functional: EBA-175 – Glycophorin A (GPA), EBL-1 - Glycophorin B (GPB), and EBA-140 - Glycophorin C (GPC). Because the fourth EBA-181 ligand recognizes the mysterious erythrocyte receptor Z, which remains unknown, we aimed to characterize its specificity and search for its receptor. The Surface Plasmon Resonance method was employed to evaluate the interaction of the recombinant EBA-181 ligand with sugar moieties and the Rh2b ligand. We have demonstrated that the EBA-181 ligand binds to Neu5Ac and Neu5Gc sialic acids, interacting with the Rh2b protein with a dissociation constant of approximately 3 µM. Moreover, the EBA-181 protein binds to about 100 kDa erythrocyte membrane protein. The P. falciparum EBA-181 merozoite ligand binding was shown to be sialic acid-dependent. It seems that the Rh2b merozoite protein might be the co-ligand cooperating with EBA-181 in erythrocyte invasion. Finally, our studies suggested that erythrocyte Band 3 protein may be a putative receptor for the EBA-181 ligand.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00436-025-08582-1.

Keywords: Plasmodium falciparum, The EBA-181 ligand specificity, Rh2b ligand, Erythrocyte receptor

Introduction

Malaria remains a major global health problem that causes severe mortality and morbidity (World Health Organization 2024). This disease is caused by Apicomplexan pathogens of the Plasmodium genus, the most deadly of which is P. falciparum (Miller et al. 2013). Despite considerable efforts, the molecular mechanisms responsible for Plasmodium host invasion remain incompletely understood. The blood stage of infection is of particular interest since blocking parasite mediators of invasion can protect from the merozoite RBC attachment (Gaur et al. 2004).

Erythrocyte binding-like (EBL) and Reticulocyte binding-like (RBL) proteins were shown to play a crucial role in the binding of merozoites to human erythrocytes (RBC) by binding to specific receptors on the RBC surface (Gaur et al. 2004; Cowman and Crabb 2006; Tham et al. 2012). Four P. falciparum EBL proteins have been identified: the erythrocyte binding ligand EBL-1, which binds glycophorin B (GPB) (Mayer et al. 2009); the erythrocyte binding antigens EBA-140 (Narum et al. 2002; Lin et al. 2012) and EBA-175 (Orlandi et al. 1990; Tolia et al. 2005) that target the glycophorin C (GPC) and A (GPA) receptors, respectively (Orlandi et al. 1992; Sim et al. 1994; Maier et al. 2003; Lobo et al. 2003; Rydzak et al. 2015), and EBA-181 (Gilberger et al. 2003) which receptor is unknown. The EBL ligands are transmembrane proteins and share a similar domain organization. The polypeptide chain of EBA proteins can be divided into six regions ranging from RI to RVI. Region RII (RII) is the receptor-binding domain containing two cysteine-rich domains (F1 and F2) that are homologs of Plasmodium vivax domains of the Duffy binding proteins (DBL) (Adams et al. 1992, 2001). The EBA-181 ligand shares 25.3 and 24.6% amino acid homology with EBA-175 and EBA-140 ligands, respectively, particularly in the F1/F2 domains, so not as high as it is in the case of EBA-175 and EBA-140 (37% of homology among their F1/F2 domains) (Gilberger et al. 2003).

The P. falciparum Rh family comprises Rh1, PfRh2a, PfRh2b, Rh4, and Rh5 proteins (Tham et al. 2012). The complement receptor 1 (CR1) is the receptor for the Rh4 ligand (Tham et al. 2010). The Rh5 ligand recognizes basigin on RBCs, mediates the invasion process as a part of the multiprotein complex Rh5/Ripr/Cyrpa/113 (Healer et al. 2022). The Rh5-basigin interaction was suggested as the key interaction for Plasmodium host-specificity (Zenonos et al. 2015). PfRh2a and PfRh2 b proteins, which differ in the C-terminal sequence, do not bind to RBCs. However, the disruption of the Rh2b coding gene and inhibition of merozoite invasion using anti-Rh2b antibodies indicated that it plays an important role in the RBC invasion (Lopaticki et al. 2011). Thus, the EBL and Rh proteins target different receptors on the RBC surface, consistent with multiple invasion pathways, and can function cooperatively, which is believed to be the invasion strategy of the malaria parasite (Pasvol 2003; Tham et al. 2012).

Although the identity of the EBA-181 receptor on the RBCs remains unknown, EBA-181 ligand interaction with its receptor is sensitive to RBC treatment with chymotrypsin and neuraminidase but resistant to trypsin. This enzyme sensitivity profile pointed to GPB, which can be cleaved from the RBC surface by chymotrypsin treatment. However, the EBA-181 binding to S-s-U-variant erythrocytes, which lack GPB, ruled out this possibility (Gilberger et al. 2003).

Sequencing the EBA-181 binding region RII of 20 P. falciparum clones from various malaria-endemic areas indicated five polymorphic amino acids within the F1 and F2 domains (Mayer et al. 2004; Maier et al. 2009). Using a transient expression of the recombinant polymorphic forms of EBA-181 RII on CHO-K1 and COS7 cells, four RBC binding patterns were found based on RBC enzyme treatment. It was suggested that EBA-181 may bind four distinct receptors; thus, it was concluded that amino acid polymorphism mediates changes in receptor specificity (Mayer et al. 2004). However, another study suggested that amino acid polymorphism in RII does not affect its specificity but only its receptor binding affinity. It was confirmed that the EBA-181 RII binding was sensitive to neuraminidase and chymotrypsin RBC treatment (Maier et al. 2009).

Moreover, a cross-linking assay, using seventy-eight 20-mer synthetic peptides covering the EBA-181 protein sequence, showed that five peptides, located in RII, bind to RBCs and recognize two single membrane proteins (bands) having apparent 53 and 33 kDa molecular weight, which have not been identified (Vera-Bravo et al. 2005).

Another report revealed an interaction between the recombinant fragment of EBA-181 (aa 945–1097), located within Region III-V, and the 10 kDa domain of human erythrocyte skeleton protein 4.1 (Lanzillotti and Coetzer 2006). This conserved 10 kDa domain of 4.1 protein interacts with several proteins and represents a pivotal point in the control of RBC membrane integrity (Conboy 1993). Since protein 4.1 is localized at the inner RBC membrane, it seems unlikely to serve as a receptor for EBA-181 during the initial stages of parasite invasion when the RBC membrane is still integral.

In this study, we evaluated the specificity of the P. falciparum EBA-181 ligand and searched for its RBC receptor by expressing its recombinant, whole ectodomain in HEK293 cells. We show that the EBA-181 recognizes N-Acetylneuraminic (Neu5Ac) and N-Glycolylneuraminic (Neu5Gc) sialic acids and binds the Rh2b merozoite protein. Finally, our studies suggest that glycosylated Band 3 protein might be the putative receptor for the EBA-181 ligand.

Materials and methods

Cells

  • HEK293F (Human Embryonic Kidney) Expi293F™ Cells cells (Thermo Fisher Sci.)

  • human red blood cells (RBCs) (human erythrocytes were freshly donated and collected on EDTA).

Materials

  • biotinylated polyacrylamide (PAA) polymers coupled to sugars:

    Neu5Ac and Neu5Gc, Galactose (Gal), and Fucose (Fuc) (GlycoTech)

  • RH2b-bio was a gift from Gavin Wright (Addgene plasmid #47778; http://n2t.net/addgene:47778; RRID: Addgene_47778)

  • EBA181-bio was a gift from Gavin Wright (Addgene plasmid #47744; http://n2t.net/addgene:47744; RRID: Addgene_47744)

Methods

Expression of the recombinant whole ectodomain of P. falciparum merozoite ligands EBA-181 and Rh2 b (non-biotinylated and biotinylated) was performed in HEK293 cells and Expi293 Expression System according to manufacturer instructions (ThermoFisher Sci.). After 5 days of cell culture, the presence of the recombinant protein in cells and culture supernatant was evaluated by Western blotting using an anti-His monoclonal antibody (HIS.H8, Invitrogen) or ExtrAvidin conjugate with alkaline phosphatase (Sigma-Aldrich). The RH2b-bio plasmid from the Addgene repository with a 6xHis tag added on the C-terminus was used for the expression.

The bacterially expressed recombinant EBA-181 binding Region II (RII) from the P. falciparum 3D7 strain was ordered from Genescript and used for studies due to the low expression yield of the recombinant whole ectodomain of EBA-181 ligand. Two tags, 6xHis and c-myc, were added to its sequence on the C-terminus.

NiNTA affinity chromatography (on Qiagen resin) was used for one-step purification of the recombinant ectodomain of Rh2 b from a culture medium. Elution was performed using imidazole: 20, 50, and 200 mM concentrations. Fractions containing recombinant protein were collected based on dot-blotting assay. The concentration was measured using NanoDrop (ThermoFisher Sci.).

SDS-PAGE, Western blotting, and Western far-blotting (overlay) were used to evaluate the purity of the recombinant ligands or RBC membrane proteins binding, respectively. The nitrocellulose (NC) membrane transferred by SDS-PAGE separated proteins of frozen RBC membranes or RBC glycophorins (in-home) was incubated overnight at 4 °C with the recombinant Region II of EBA-181 ligand. Monoclonal antibodies: anti-his (HIS.H8, Invitrogen), anti-myc (clone 9E10), anti-glycophorins A and B (in-house), anti-Band 3 (antibody 3B3.136, in-house), and anti-band 4.1 (EPB41 Polyclonal Antibody, Invitrogen) or ExtrAvidin conjugate with alkaline phosphatase (Sigma-Aldrich) were used to visualize proteins on NC membrane. Protein standards (PageRuler Plus Prestained Protein Ladder, ThermoFisher Sci.) were purchased.

Flow cytometry (FACS) analysis was used to investigate the binding of recombinant Region II of EBA-181 to RBCs. Region II was incubated with 3 × 105 human red blood cells (RBC) in phosphate-buffered saline (PBS), pH 7.4, for 2 h at 4 °C. After three washings with PBS, RBCs were incubated for 1 h at 4 °C with mouse anti-his MoAb, and then incubated for 30 min at 4 °C with FITC-conjugated rabbit anti-mouse Ig antibody (DakoCytomation, Glostrup, Denmark). Erythrocytes were analyzed for fluorescence intensity using flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA).

Surface Plasmon Resonance (SPR) method was employed to study the interaction of EBA-181 recombinant RII and recombinant full ectodomain of EBA-140 with sugar moieties or protein Rh2b and EBA-181 RII. In the first case, monovalent biotinylated PA polymers coupled to sugars: Neu5Ac and Neu5Gc, Gal, and Fuc (GlycoTech) were used to coat the SA sensor chip, and the recombinant EBA-181 RII (0.2 µM) or EBA-140 ectodomain (0.05 µM) were used as an analyte. Similarly, the biotinylated recombinant Rh2b was bound to the sensor SA chip, and RII of EBA-181 served as the analyte.

Mass Spectrometry experiments were performed at the Mass Spectrometry Laboratory at the Institute of Biochemistry and Biophysics PAS. The fragmented pieces of the membrane were suspended in an ammonium bicarbonate buffer. First, the cysteines were reduced by 1 h incubation with 5 mM tris(2-carboxyethyl)phosphine (TCEP) in 100mM NH4HCO3 at 60 °C followed by 10 min incubation at room temperature with 20 mM methyl methanethiosulfonate (MMTS). Digestion was provided at 37 °C overnight with 1 µg of trypsin (Promega) in 100mM NH4HCO3. Peptide mixture was applied to nano-HPLC RP-18 column (nanoACQUITY BEH C18 - Waters 186003545) using an acetonitrile gradient (5% − 35% AcN in 70 min) in the presence of 0,05% formic acid with the flow rate of 250 nl/min. The column outlet was directly coupled to the ion source of the spectrometer, working in the regime of data-dependent MS to MS/MS switch. Other MS parameters were the following: m/z range: 300–2000 Th, MS1 resolution: 35 000, MS2 resolution: 15 000. A blank run (injection of Milli-Q water), ensuring cross-contamination monitoring from previous samples, preceded each analysis.

Raw data were processed by Mascot Distiller (v. 2.4.2) followed by Mascot Search (v. 2.4.1, Matrix Science, UK) against the users’ database (SwissProt). Search parameters for precursor and product ions mass tolerance were 5ppm and 0.01 KDa, respectively. Peptides with Mascot Score exceeding the threshold value corresponding to < 5% expectation value were considered to be positively identified.

Results

Expression and purification of the recombinant P. falciparum EBA-181 and Rh2b ligands

The recombinant whole ectodomain of merozoite ligand EBA-181 and its biotinylated form were expressed in HEK293 cells. Cell fractionation after expression revealed that the recombinant proteins were mostly present in the non-soluble cell fraction and very low amounts in the culture medium, not suitable for purification and further use (Fig. 1).

Fig. 1.

Fig. 1

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Western blotting of P. falciparum EBA-181 ectodomain (non-biotinylated and biotinylated) expressed in HEK293 cells; S-soluble cell fraction, NS-non-soluble cell fraction, Med 10x - cell medium concentrated 10 times; MoAb anti-his (HIS.H8) and ExtrAvidin conjugate were used, respectively

Similarly, the recombinant biotinylated ectodomain of merozoite ligand Rh2b was expressed in HEK293 cells and purified from cell culture medium. Degradation of the recombinant protein is observed (Crosnier et al. 2013) (Fig. 2).

Fig. 2.

Fig. 2

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Western blotting of P. falciparum Rh 2b and EBA-181 biotinylated ectodomains expressed in HEK293 cells; Extravidin conjugate was used; M - marker

The recombinant P. falciparum EBA-181 ligand binding specificity

The bacterial, recombinant EBA-181 Region II binding towards human RBCs was evaluated using flow cytometry (Fig. 3). Dose-dependent but weak binding was observed, perhaps due to low affinity of the RII fragment interaction or a cryptic nature of the RBC receptor.

Fig. 3.

Fig. 3

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FACS analysis of the recombinant EBA-181 and EBA-140 ligands RII region binding to human RBCs; color lines – different concentrations of RII, grey area – control RBCs; MoAb anti-myc 9e10 was used

The binding of the recombinant RII of EBA-181 ligand was then evaluated towards sugar moieties: sialic acids (Neu5Ac and Neu5Gc), galactose, and fucose using the SPR technique. Weak binding to galactose and no binding to fucose (not shown) sugar residues were observed. However, the EBA-181 binding region RII recognized both sialic acids (Fig. 4). Thus, EBA-181 exhibits similar specificity to other P. falciparum EBA ligands, which were proven to be sialic acid-dependent (Tolia et al. 2005; Rydzak et al. 2015; Ashline et al. 2015).

Fig. 4.

Fig. 4

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SPR analysis of the recombinant (a) RII region of EBA-181 ligand and (b) full ectodomain of EBA-140 ligand binding to sugar residues; blue line - galactose, grey line – N-acetylneuraminic acid, orange line – N-glycolylneuraminic acid

P. falciparum Rh2b protein as a co-ligand of P. falciparum EBA-181

Based on the previous suggestions about the cooperativity of EBA and Rh merozoite ligands in the RBC invasion, we have employed the SPR method to identify the direct interaction of the recombinant RII region of EBA-181 and Rh2b ligand. We have shown that the EBA-181 binding region RII interacts with the recombinant Rh2b protein (Fig. 5). The dissociation constant was about 2 µM, which is similar to the reported KD of CR1 and Rh4 homologous merozoite ligand interaction, which is 2,9 µM (Tham et al. 2010). These results suggest that the Rh2b protein might be the co-ligand cooperating with EBA-181 in the merozoite RBC invasion.

Fig. 5.

Fig. 5

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SPR analysis of the recombinant region RII of EBA-181 ligand binding kinetics to the biotinylated recombinant Rh2 b ligand

Searching for the human erythrocyte receptor of the P. falciparum EBA-181 ligand

The recombinant P. falciparum EBA-181 RII binding to human RBC membranes and crude glycophorins was performed using far-Western blotting (overlay). Human glycophorins were used since glycophorins A, B, and C/D are receptors for homologous EBAs ligands (EBA-175, EBL-1, and EBA-140, respectively). However, no glycophorin binding of RII EBA-181 was detected (Fig. 6). Moreover, the only bands in RBC membrane proteins interacting with R II EBA-181 ligand were about ≥ 100 kDa, which was visualized with anti-His or anti-myc antibody recognizing C-terminal tags added to the RII protein sequence. The molecular mass and diffuse character of this band suggested the Band 3 protein, which is the major RBC membrane glycoprotein.

Fig. 6.

Fig. 6

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Western-blotting (overlay) analysis of EBA-181 RII binding to erythrocyte membrane proteins: (1) RBC glycophorins, (2) lyophilized RBC membranes, (3) frozen RBC membranes, visualized with anti-His antibody; (4) RBC membranes visualized for band 4.1 (polyclonal antibody EPB41), (5) RBC membranes visualized for band 3 (Ab 3B3.136)

To confirm the EBA-181 interaction with the Band 3 receptor, the visualized band (≥ 100 kDa) was cut off for MS analysis. The protein sequences derived mostly from Band 3 were identified, of which only Band 3 is an N-glycosylated membrane protein that might ensure the sugar specificity and surface binding to the EBA-181 ligand.

Discussion

Plasmodium falciparum invasion into human erythrocytes involves multiple ligand-receptor interactions. It was shown that the EBL and Rh protein families are crucial to host RBC recognition and are responsible for mediating distinct invasion pathways (Gaur et al. 2004; Lopaticki et al. 2011).

It was shown that together with EBA-175 and EBA-140 ligands, the EBA-181 ligand also contributes to the immune response in individuals living in malaria-endemic areas (Lobo et al. 2004; Persson et al. 2013). The recombinant EBA-181 Region III-V can be recognized by naturally acquired antibodies obtained in individuals living in Brazil and Cameroon (Ford et al. 2007). Moreover, a fraction of IgG1 antibodies against the same fragment of EBA-181 was positively associated with decreased parasitemia in Peruvian individuals (Villasis et al. 2012). The role of the EBA-181 ligand in immunological response has also been studied by employing live parasites with the disruption of EBA-140, EBA-175, and EBA-181 ligands, which led to major changes in their susceptibility to inhibitory antibodies. This indicated that all three EBAs can contribute to immune evasion through the variation in their function (Persson et al. 2013).

Although the EBA-181 ligand has been proven as a significant protein engaged in the alternative pathway of P. falciparum invasion, its RBC receptor remains unknown. Studies on EBA-181 that have already been performed utilized native protein from the parasite culture supernatant (Maier et al. 2009), surface-expressed recombinant ligand on CHO and COS7 cells (Mayer et al. 2004), short synthetic peptides overlapping the whole sequence of EBA-181 RII (Vera-Bravo et al. 2005), or bacterially-expressed recombinant fragments of R III-R V domains of EBA-181 ligand (Lanzillotti and Coetzer 2006). For the first time, we have used the recombinant full ectodomain of the EBA-181 protein expressed in the mammalian system to study its specificity and to search for its receptor.

The previous studies (Lopaticki et al. 2011) suggested that EBA-181 can interact with other merozoite ligand Rh2b, which belongs to the RBL protein family, providing evidence that these protein families can act cooperatively in merozoite invasion. It was shown that when the eba181 and eba140 genes were disrupted, the Rh2b coding gene was deleted in the resulting parasites. Moreover, anti-Rh2b antibodies did not inhibit merozoite invasion when EBA-181 was not expressed, suggesting that these two proteins in some way cooperate and that the loss of one ablates function in the other. However, the authors were unable to coimmunoprecipitate Rh2b and EBA-181 from the P. falciparum 3D7 strain to demonstrate a direct interaction, proposing that Rh2b and EBA-181 may interact transiently during merozoite invasion. Indeed, the EBL proteins are located in micronemes, while Rh proteins are in the neck of the rhoptries so that they can associate only when the micronemal proteins are released (Duraisingh et al. 2003).

To evaluate the interaction of the recombinant EBA-181 and Rh2b ligands, we employed the sensitive, biophysical SPR method. We have shown RII – Rh2b interaction directly and estimated its KD constant, about 2 µM. The cooperative function of EBL and PfRh proteins in merozoite invasion has important implications since the combination of EBL and Rh proteins, including EBA-175 and Rh4, EBA-181 and Rh2b ligands, might be used in a combination vaccine efficiently blocking merozoite invasion.

Our attempt to identify the last unknown EBL protein receptor indicates the erythrocyte anion exchanger - Band 3 (Daniels 2013). This protein seems to be a reasonable candidate for the EBA-181 receptor for many reasons.

Firstly, it is an abundant RBC membrane protein, with about one million copies per RBC, known to engage in a myriad of interactions, including binding to P. falciparum merozoites.

Secondly, that is crucial in the context of EBA receptors, Band 3 is glycosylated, having one N-glycosidic chain attached to the Asn 642 residue of the external loop, which is accessible on the RBC surface (Tanner 1997). This glycan is close to the membrane surface and might be a cryptic antigen. It could explain why the recombinant EBA-181 RII and ectodomain did bind so weakly to the RBCs in flow cytometry (Fig. 3).

The Band-3 N-glycan structure was solved as a biantennary complex chain, which ends mostly with the poly-lactosamine units of various lengths that cause the diffuse character of the protein band in SDS-PAGE (Fukuda et al. 1984). However, it was shown that some oligosaccharide chains may be terminated with fucose or even sialic acid, which is required for EBA ligands binding. Moreover, a sequence of polypeptide chain, which forms with an attached oligosaccharide full glycotope seems important. Thus, homologous EBA-175, EBL-1, and EBA-140 ligands recognize sialic acids only in glycophorin A, B, and C contexts, respectively.

Finally, the Band 3-derived peptides appeared in MS analysis of the major band (100 kDa) identified in Western blotting by overlay with EBA-181 RII.

Supplementary Information

Below is the link to the electronic supplementary material.

436_2025_8582_MOESM1_ESM.pdf (393.9KB, pdf)

Supplementary Material 1 Supp. 1 MS analysis of 100 kDa erythrocyte membrane protein band.

Acknowledgements

All the authors would like to thank Marcin Czerwiński for his valuable comments and support.

Author contributions

E.J.- conceptualization, writing, funding; M.J. - investigation, data curation, visualization; P.B. -investigation, visualization, writing; A.Z.- funding.

Funding

This work was funded by the National Science Centre of Poland Preludium project (A.Z.) 2016/23/N/NZ6/01482 and the Harmonia project (E.J.) 2018/30/M/NZ6/00653.

Data availability

The datasets generated or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval

The study was approved by the Wrocław Medical University Bioethics Committee, Consent 641/2014, December 14, 2014. The procedures used in this study adhere to the tenets of the Declaration of Helsinki.

Consent to participate

Informed consent was obtained from a healthy subject (blood donor) involved in the study.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Marlena Jodłowska and Patrycja Burzyńska contributed equally to this work and are considered joint first authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

436_2025_8582_MOESM1_ESM.pdf (393.9KB, pdf)

Supplementary Material 1 Supp. 1 MS analysis of 100 kDa erythrocyte membrane protein band.

Data Availability Statement

The datasets generated or analyzed during the current study are available from the corresponding author on reasonable request.

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