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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Biochem Parasitol. Author manuscript; available in PMC 2013 May 1.
Published in final edited form as:
PMCID: PMC3307866
NIHMSID: NIHMS350556

Identification of a specific region of Plasmodium falciparum EBL-1 that binds to host receptor glycophorin B and inhibits merozoite invasion in human red blood cells

Abstract

The malaria parasite Plasmodium falciparum invades human erythrocytes through multiple pathways utilizing several ligand-receptor interactions. These interactions are broadly classified in two groups according to their dependency on sialic acid residues. Here, we focus on the sialic acid-dependent pathway by using purified glycophorins and red blood cells (RBCs) to screen a cDNA phage display library derived from Plasmodium falciparum FCR3 strain, a sialic acid-dependent strain. This screen identified several parasite proteins including the erythrocyte-binding ligand-1, EBL-1. The phage cDNA insert encoded the 69-amino acid peptide, termed F2i, which is located within the F2 region of the DBL domain, designated here as D2, of EBL-1. Recombinant D2 and F2i polypeptides bound to purified glycophorins and RBCs, and the F2i peptide was found to interfere with binding of D2 domain to its receptor. Both D2 and F2i polypeptides bound to trypsin-treated but not neuraminidase or chymotrypsin-treated erythrocytes, consistent with known glycophorin B resistance to trypsin, and neither the D2 nor F2i polypeptide bound to glycophorin B-deficient erythrocytes. Importantly, purified D2 and F2i polypeptides partially inhibited merozoite reinvasion in human erythrocytes. Our results show that the host erythrocyte receptor glycophorin B directly interacts with the DBL domain of parasite EBL-1, and the core binding site is contained within the 69 amino acid F2i region (residues 601–669) of the DBL domain. Together, these findings suggest that a recombinant F2i peptide with stabilized structure could provide a protective function at blood stage infection and represents a valuable addition to a multi-subunit vaccine against malaria.

Keywords: Malaria, Phage display, Plasmodium falciparum, Erythrocytes, Glycophorin B, EBL-1

1. Introduction

Plasmodium falciparum merozoites are known to use multiple pathways to invade human red blood cells (RBCs) [1, 2]. These pathways are grouped into two classes depending upon the presence of sialic acid on host receptors. Some parasite strains use ligands that preferentially bind to host receptors lacking sialic acid, whereas others, such as FCR3, are restricted to the sialic acid-dependent pathways [3]. Neuraminidase treated RBCs lack sialic acid residues, and are known to be highly resistant to certain strains of P. falciparum including FCR3, Camp, Dd2, and FVO [3]. The P. falciparum FCR3 strain is completely dependent upon the presence of sialic acid residues for invasion, since it is unable to propagate in neuraminidase-treated RBCs [3]. The major sialoglycoproteins in the RBC membrane are glycophorins and thus are implicated in merozoite invasion [46]. Soluble glycophorins as well as antibodies against glycophorins block parasite invasion in vitro [7, 8]. Moreover, RBCs genetically deficient in specific glycophorins are known to confer partial resistance to invasion by P. falciparum [9, 10]. The four glycophorins in human erythrocytes, glycophorin-A (GPA), glycophorin-B (GPB), glycophorin-C (GPC), and glycophorin-D (GPD) constitute ~2% of the total membrane protein mass. GPA and GPB are encoded by two distinct genes, with GPA constituting the dominant glycophorin in human erythrocytes. In contrast, GPC and GPD are encoded by a single gene. GPD contains a truncated amino terminal cytoplasmic domain, so that the carboxyl-terminal amino acids (residues 21–128) are identical between GPD and GPC [11]. Thus, human erythrocytes carry three glycophorins with distinct extracellular domains, and these proteins serve as the main receptors involved in the sialic acid-dependent pathways.

The first P. falciparum ligand identified that mediates RBC invasion through a sialic acid-dependent pathway was the erythrocyte-binding antigen-175 (EBA-175). EBL-175 binds to GPA, the most abundant glycophorin in human RBCs, and this biochemical interaction has been extensively characterized [1215]. EBA-175 belongs to the Duffy binding-like (DBL) protein family, which includes EBA-140 (BAEBL), EBA-165 (PEBL), EBA-181 (JESEBL), and EBL-1 [16]. EBA-165 is unlikely to play any direct role in invasion since it does not express a functional protein due to a frame shift mutation in the coding region [17]. EBA-140 specifically binds to GPC, binding is sialic acid-dependent, and it is unable to bind to Gerbich negative human erythrocytes lacking exon 3 in the glycophorin C gene [18, 19]. Similarly, the Leach phenotype erythrocytes, lacking GPC, are partially resistant to invasion by P. falciparum [20]. The molecular identity of the erythrocyte receptor for EBA-181 is unknown at present. EBA-181 binds to RBCs in a sialic acid-dependent manner. The receptor is trypsin-resistant and chymotrypsin-sensitive, but the receptor is not GPB. Moreover, the EBA-181 knockout parasites showed no change in invasion efficiency, implying possible redundancy of this interaction [21, 22]. A phage display screen identified the 10 kDa domain of erythrocyte membrane protein 4.1R as a binding motif for parasite EBA-181, suggesting a cytoskeletal function of EBA-181 under certain conditions [23]. Recently, Mayer and colleagues reported the identification of GPB as a host receptor for parasite EBL-1 [24]. Their finding originated from the highly polymorphic nature of the GPB gene, particularly the unusually high occurrence of the GPB-null phenotype in the Efe pygmies of the Democratic Re public of Congo [25].

In this study, we utilized purified glycophorins and human RBCs to screen a phage display cDNA library from FCR3, a sialic acid-dependent Plasmodium falciparum strain. Several parasite proteins, including EBL-1, were identified from the library screen as they specifically bind to both glycophorins and RBCs. Here, we provide evidence that the major binding site of EBL-1 to GPB is located within the 69-amino acid segment, termed F2i, of the D2 domain of EBL-1. Importantly, both recombinant F2i peptide and D2 domain recognizing the GPB partially inhibited the merozoite invasion of human erythrocytes.

2. Materials and Methods

2.1. Parasite culture

P. falciparum (FCR3 strain) was maintained continuously in a culture using standard conditions [26]. The parasite culture was maintained in 75 cm2 flasks at 37°C under 5% CO2, 5% O2, and 90% N2 at 5% hematocrit of fresh type O+ human erythrocytes in a complete medium containing RPMI-1640 medium with L-glutamine and 25 mM HEPES (Invitrogen) supplemented with 10 mg/ml gentamycin, 200 µM Hypoxanthine, 0.2% NaHCO3, and 0.5% Albumax II (Invitrogen), and adjusted to a pH of 7.4. Ring-stage parasites were synchronized using 5% sorbitol and the late stage parasites were enriched to greater than 95% parasitemia by centrifugation through a gradient of 65% (v/v) Percoll (GE Healthcare).

2.2. P. falciparum phage display library preparation

P. falciparum (FCR3 strain, asynchronous) T7 phage display cDNA library was generated and amplified by the liquid lysate amplification method as described before [27]. The typical size of the P. falciparum cDNA inserts was more than 722 bp [27]. The parasite phage library was propagated using a log-phase culture of E. coli BLT5403 and amplified by shaking for 2–3 hours at 37°C in LB medium containing carbenicillin (100 µg/ml) until cell lysis occurred. The phage particles were clarified by centrifuging the cell lysate at 12,000 × g for 20 minutes. The amplified-library lysates were stored at 4°C for short term or at −70°C with 15% glycerol for long term storage. The library titer was determined by the plaque assay.

2.3. Phage display library screen using purified human glycophorins or intact RBCs

The biopanning technique was used to screen the P. falciparum phage library against purified human MN-glycophorins (Sigma, #G5017) or freshly isolated RBCs [28]. Four rounds of biopanning were performed on immobilized glycophorins on the ELISA plate or on RBCs. The final phage solution was titrated, and the plaques were grown in LB medium containing carbenicillin (100 µg/ml) for 3h at 37°C. The phage DNA was then extracted from individual amplified phage plaques using the QIAprep Spin M13 kit (Qiagen). Partial sequencing of the phage DNA was carried out with T7 select down primer 5′- AACCCCTCAAGACCCGTTTA-3′ using the BigDye Terminator version 3.0 Cycle Sequencing Kit (Applied Biosystems).

2.4. Recombinant protein expression and purification in E. coli

The Duffy-binding-like (DBL) domain (D2) of EBL-1 gene (GenBank accession number: AY769519) was amplified by PCR from P. falciparum (FCR3) genomic DNA and cloned into pET32a (Novagen) to generate recombinant plasmid pET32a-D2. The PCR primers used were 5′-GCCGGATCCTGTGGGAAGAAAATAAAGG-3′ (sense, BamHI) and 5′-GCCGTCGACACATACCATACAAGCCTCT-3′ (antisense, SalI). Recombinant D2 of EBL-1 was expressed in E. coli BL21(DE3) as a fusion to the thioredoxin, Trx, and His6 tag. The thioredoxin tag was chosen to enhance solubility of the recombinant protein. The soluble Trx-D2 fusion protein was affinity-purified using Ni-NTA beads. A 207-bp region of F2 in EBL-1 domain 2 (named here as F2i), which corresponded to the phage insert cDNA sequence of EBL-1 recognized in the screen using RBCs and glycophorins, was PCR amplified from pET32a-D2 and cloned into pGEX-6P-2 (GE Healthcare) using the forward primer 5′-GCCGGATCCTGCAATGCTATATTGGGAAG-3′ (sense, BamHI) and reverse primer 5′-GCCGTCGACTACCATACTAGACCATATTA-3′ (antisense, SalI). The recombinant F2i was expressed in E. coli DH5α as a fusion to GST. The soluble GST-F2i fusion protein was affinity-purified using GSH beads. Expression of recombinant proteins was induced by 0.4 mM IPTG at 25°C for 3 hours, cells were pelleted at 3,750 rpm for 15 minutes in a Sorvall 5C RC Plus at 4°C, and cell lysis was carried out in the presence of lysozyme (1.0 mg/ml), DNase II (5.0 ug/ml), and RNase (10.0 ug/ml) with protease inhibitors for 30 minutes on ice, followed by sonication and centrifugation at 3,750 rpm for 20 minutes to recover the soluble fractions for affinity purification. The identity of each fusion protein was confirmed by Western blotting using HRP-conjugated anti-His6 monoclonal antibodies (Santa Cruz Biotechnology; Invitrogen) and anti-GST polyclonal antibody (Santa Cruz Biotechnology). It is to be noted that due to the instability and storage conditions of various fusion proteins, purification of recombinant proteins was repeated several times with consistent results across different protein preparations. For example, data shown in Figures 3 A–C and Figure 4 A–B were confirmed with multiple preparations of TRX-D2.

Figure 3
Plasmodium falciparum EBL-1 binds to purified glycophorins and human RBCs. (A) A concentration-dependent binding of Trx-D2 and GST-F2i to purified glycophorins. Binding of Trx-D2 (filled circles), GST-F2i (filled squares), Trx (empty circles), and GST ...
Figure 4
(A) Interaction of malaria parasite EBL-1 D2 domain to human erythrocytes. The Trx-D2 fusion protein binds to trypsin-treated RBCs but not to neuraminidase, chymotrypsin, and glycophorin B-null RBCs. Western blots using anti-His6 mAb to detect Trx-D2: ...

For the circular dichroism (CD) measurements, the F2i fragment was subcloned into pET32a using BamHI and SalI sites and expressed in E. coli BL21(DE3). The expression of Trx-F2i was induced by 0.4 mM IPTG at 25°C for 4h. The cell lysis was carried out in the presence of 20 mM sodium phosphate, pH 7.8, 500 mM NaCl, 1.0 mg/ml lysozyme, 10% glycerol, and multiple protease inhibitors. After incubation for 30 min on ice, the cell pellet was sonicated, treated with 5.0 ug/ml DNAase II and 10 ug/ml RNase A, and centrifuged to recover the soluble fraction. The soluble Trx-F2i protein was affinity-purified using Ni-NTA beads in the presence of 10% glycerol, and dialyzed to a final buffer concentration of 4.0 mM sodium phosphate, pH 7.8, and 5% glycerol for CD analysis.

2.5. Circular dichroism spectroscopy

The CD spectra were collected using an Aviv 62DS spectrometer. Both Trx-F2i and Trx proteins were measured in buffer consisting of 4.0 mM sodium phosphate, 5% glycerol, pH 7.8, and the CD spectra were generated using a 2 mm path length cuvette containing 200 µL of 5.0 mM sample at 20°C. The scans were performed with five repeats at an average of 1.0 nm increment recordings f rom 190 to 250 nm. The sample concentrations were determined using absorbance at 280 nm.

2.6. ELISA

Purified glycophorins (1–2 µg) in 100 µl of 100 mM NaHCO3 buffer (pH 9.5) were added to the wells of a 96-well ELISA plate (Corning). The plate was incubated at 4°C overnight. The wells were blocked for at least 2 h at room temperature with 300 µl of the blocking buffer PBST (3% BSA in 0.1% Tween-20, 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4). Trx-D2 and GST-F2i protein were added to the coated wells in varying concentrations (0–10 µM) and incubated at room temperature for 3 h. The wells were washed with PBST, and each sample was incubated with HRP-conjugated anti-His6 and anti-GST antibodies, respectively, for 2 h at room temperature. The wells were washed with PBST extensively, and bound proteins were detected using a solution of TMB substrate (Pierce). The reaction was stopped after 15 min by adding 100 µl of 2.0 M H2SO4, and the color was measured at 450 nm. Trx and GST tag proteins were used as negative controls and subtracted for each Trx-D2 and GST-F2i reading, respectively. The dissociation constants (means ± S.E.) were determined from duplicate experiments.

2.7. Binding of recombinant D2 domain and F2i peptide of EBL-1 to RBCs

For the binding assays, the Trx-D2 or GST-F2i fusion proteins were incubated with 50 µl of packed RBCs in RPMI-1640 medium to a final volume of 800 µl for 2h at room temperature with gentle rotation. The mixture was then placed on top of 500 µl of sillicon oil and centrifuged at 14,000 g for 1 min. The supernatant and silicon oil were removed and bound proteins were eluted by incubating with 20 µl of 1.5 M NaCl for 10 min at room temperature, followed by centrifugation at 12,000 g for 1 min. The eluted proteins were separated by SDS-PAGE and identified by immunoblotting.

The RBCs were treated with enzymes as described before [4]. Briefly, RBCs were incubated with neuraminidase (66 mU/ml), trypsin (1.0 mg/ml), and chymotrypsin (1.0 mg/ml) at 37°C for 1h, treated with soybean trypsin/chymotrypsin inhibitor, and extensively washed prior to binding assays. The efficiency of enzyme treatment was confirmed by Western blotting using anti-GPA, anti-GPC, and anti-Band 3 antibodies as previously described [29]. Glycophorin B-deficient (S-s-U) RBCs were obtained from a donor from the Virginia Blood Services (Richmond, VA) where the cells were tested to confirm the absence of cell surface GPB. The Trx-D2 fusion protein was incubated with 50 µl of packed GPB-deficient cells in RPMI-1640 medium and treated as described before. Similarly, for the binding inhibition assays, the GST-F2i fusion protein (1 µM or 10 µM) was incubated with RBCs in RPMI-1640 medium for 1 h at room temperature, then, the Trx-D2 protein (1 µM) was added and further incubated for 2 h at room temperature. The erythrocytes were sedimented and the bound proteins were eluted as above and analyzed by Western blotting. Equal amount of Trx (1 µM) was used as a negative control.

2.8. Erythrocyte invasion assay

Trx-D2 and GST-F2i proteins were tested for their capacity to inhibit invasion of P. falciparum FCR3 merozoites to RBCs. Parasite cultures were synchronized using 5% sorbitol treatment, and schizont-stage infected-RBCs were purified by centrifugation on a 63% Percoll gradient. Parasites were washed with RPMI-1640 and mixed with uninfected RBCs in malaria culture medium. The mixture of infected and uninfected RBCs was seeded into a 96-well plate containing varying concentrations of Trx-D2 or GST-F2i protein (10 µM, 5 µM, 2.5 µM, 1.2 µM, 0.6 µM, 0.3 µM) to a final volume of 200 µl, 2% hematocrit, and 2% parasitemia. Purified GST and Trx protein tags served as negative controls. Parasites were incubated for 18 to 20 h at 37°C to allow parasite reinvasion. Following the incubation period, thin smears of each well were made, and stained with Giemsa. Approximately 1,000 RBCs were counted for each sample to quantify parasitemia, and the percent inhibition of invasion was calculated relative to the control sample as follows: (parasitemia for GST or Trx control – parasitemia for sample containing GST-F2i or Trx-D2) / parasitemia for GST or Trx control. Experiments were carried out in triplicate, and each condition was performed at least twice. Data are presented as mean ± S.D.

3. Results

3.1. Plasmodium falciparum phage display cDNA library screen

To identify P. falciparum proteins interacting directly with glycophorins, purified glycophorins were used to screen a sialic acid-dependent strain of P. falciparum (FCR3) phage display cDNA library. After 4 rounds of biopanning against immobilized glycophorins, the phage DNA was extracted and sequenced from individual positive plaques. Sequence analysis of the plaques and subsequent BLAST searches using the databases at NCBI and PlasmoDB revealed several P. falciparum genes including EBL-1, EBA-175, and hypothetical proteins PFA0420w and MAL13P1.249. When RBCs were used as bait, nucleotide sequence analysis of the positive phage clones revealed that the cDNA inserts encoded proteins EBL-1, EBA-175, MSP1, hypothetical proteins Pf07_0024, PFL1130c, PF13_0167, and PFF0220w. An identical 207-bp nucleotide sequence of EBL-1 was found in multiple phage clones using glycophorins and RBCs as bait. This cDNA insert segment, designated here as F2i, was in the correct open reading frame encoding 69 amino acids. The 69 amino acid insert spans from residues 601 to 669 of the coding sequence of EBL-1. Subsequent sequence alignment analysis placed the 69 amino acid insert, F2i, within the F2 segment of the DBL domain, D2, of EBL-1 (Fig.1).

Figure 1
Schematic representation of the primary structure of Plasmodium falciparum EBL-1. (A) EBL-1 consists of a putative signal peptide (SP) sequence, Duffy-binding-like (DBL) ligand domain consisting of two cysteine-rich regions F1 and F2, transmembrane (TM) ...

3.2. Binding specificity of EBL-1 domains to glycophorins

Based on the results of phage display cDNA library screen, we tested direct binding of recombinant EBL-1 domains D2 and F2i to glycophorins using an ELISA. The D2 and F2i segments were expressed in E. coli and purified as Trx-D2 and GST-F2i fusion proteins, respectively. Both fusion constructs expressed as soluble proteins in bacteria (Fig. 2). Purified Trx-D2 protein expressed with the correct predicted molecular mass of 92 kDa (Fig. 2, lanes 1–3). Purified GST-F2i protein expressed as a band with the predicted molecular mass of 34 kDa, with additional truncated products including a dominant band running at the GST tag alone (Fig. 2, lanes 4–6). The expression of Trx-D2 and GST-F2i was confirmed by Western blotting using antibodies against His6 and GST, respectively (Fig. 2).

Figure 2
Expression and purification of recombinant Plasmodium falciparum EBL-1 segments in E. coli. Lane 1, Coomassie blue stained SDS-PAGE of purified Trx-D2; lane 2, Western blot using anti-His6 mAb of purified Trx-D2 and Trx (lane 3). Coomassie blue stained ...

To quantify direct interaction between EBL-1 domains and glycophorins, a serial dilution ELISA was performed using recombinant Trx-D2 and GST-F2i. Glycophorins were coated on the ELISA plate, followed by the incubation of serially diluted Trx-D2 or GST-F2i, establishing a concentration-dependent interaction between EBL-1 fusion proteins with the immobilized glycophorins (Fig. 3A). As negative controls, Trx and GST proteins showed little interaction with the immobilized glycophorins (Fig. 3A). Similarly, no signal was observed without any coating of glycophorins. Dissociation constants for direct binding of soluble Trx-D2 and GST-F2i to immobilized glycophorins were calculated to be 692±41 nM and 836±57 nM, respectively. Notwithstanding the potential modulatory effects of other glycophorins present in the mixture of glycophorins used in the ELISA, these results suggest that the F2i peptide in the context of the full length D2 domain of EBL-1 showed similar binding affinity for the immobilized glycophorin-B.

3.3. F2i segment mediates EBL-1 domain 2 interaction with human RBCs

To determine direct binding of EBL-1 domains with intact erythrocytes, the Trx-D2 and GST-F2i fusion proteins were individually incubated with freshly isolated erythrocytes. Both Trx-D2 and GST-F2i fusion proteins bound specifically to erythrocytes, whereas Trx and GST proteins in an equivalent concentration did not interact with erythrocytes (Fig. 3B, 3C). To investigate if the F2i segment interferes with the binding of the D2 domain, RBCs were pre-treated with GST-F2i fusion protein and then incubated with Trx-D2 protein. Binding of Trx-D2 protein to the RBCs was reduced with an increasing concentration of GST-F2i protein (Fig. 3B). The GST protein alone did not affect Trx-D2 binding to erythrocytes. Although a low-affinity interaction of erythrocytes with other regions of the D2 domain of EBL-1 cannot be ruled out at this stage, our data suggest that the D2 domain of EBL-1 interacts directly with the surface of intact erythrocytes, and the major binding site of the D2 domain is likely to be contained within the F2i segment.

3.4. Sialic acid-dependent interaction of EBL-1 D2 domain with erythrocytes

To further characterize the binding properties of Trx-D2 domain with intact erythrocytes, binding assays were performed using erythrocytes treated individually with neuraminidase, trypsin, and chymotrypsin. Bound proteins were eluted and analyzed by SDS-PAGE and Western blotting. The Trx-D2 domain fusion protein did not bind to either neuraminidase or chymotrypsin-treated erythrocytes indicating that the D2 domain of EBL-1 specifically interacts with erythrocytes in a sialic acid-dependent manner (Fig. 4A). In contrast, the Trx-D2 domain bound to trypsin-treated erythrocytes with the same efficiency as normal untreated erythrocytes (Fig. 4A). These results indicate that the D2 domain of EBL-1 binds to human erythrocytes via a receptor that is trypsin-resistant but chymotrypsin-sensitive. Prior studies have established that glycophorins A, C, and D are sensitive to trypsin treatment of intact erythrocytes [12, 18, 30, 31]. These observations, combined with direct interaction of D2 and F2i fusion proteins with purified glycophorins (Fig. 3A), suggest that glycophorin B is the receptor for EBL-1. This conclusion is consistent with the known characteristics of erythrocyte glycophorin B that is sensitive to neuraminidase and chymotrypsin and resistant to trypsin. Our findings are also in agreement with the recently published evidence that glycophorin B is the receptor for EBL-1 [24]

3.5. The D2 domain of EBL-1 does not bind to glycophorin B null human erythrocytes

To further confirm the identity of glycophorin B as a receptor for the D2 domain of EBL-1, we utilized human erythrocytes with complete deficiency of glycophorin B (S-s-U-). Glycophorin B deficient (S-s-U-) erythrocytes were obtained from the Virginia Blood Services where they had been tested to confirm the absence of cell surface glycophorin B [24]. The hematological properties of glycophorin B deficient (S-s-U-) erythrocytes are nearly identical to normal erythrocytes except for the lack of glycophorin B [32]. This phenotype occurs with very low frequency, and previous studies have suggested that glycophorin B plays a role in merozoite invasion as individuals lacking glycophorin B are relatively less susceptible to malaria parasite infection [33, 34]. We performed binding assays using recombinant Trx-D2 domain of EBL-1 and glycophorin B deficient human erythrocytes. Our data demonstrate that the Trx-D2 domain does not bind to erythrocytes lacking glycophorin B (S-s-U-) as compared to normal glycophorin B positive (+) erythrocytes (Fig. 4A, lane 7).

3.6. Inhibition of merozoite invasion by recombinant D2 and F2i fusions of EBL-1

To investigate the biological significance of molecular interactions between parasite EBL1 and erythrocyte glycophorin B, erythrocyte reinvasion inhibition assays were performed using the sialic-acid dependent FCR3 strain of P. falciparum. Purified schizont stage parasite-infected erythrocytes were incubated with an increasing concentration of soluble Trx-D2 and GST-F2i fusion proteins. In vitro erythrocyte invasion assays were performed, and the results show a dose-dependent inhibition of parasite invasion in erythrocytes by both Trx-D2 and GST-F2i (Fig. 4B). No trophozoite or schizont accumulation was observed during the invasion inhibition assay, indicating that the Trx-D2 and GST-F2i fusions added to the culture medium did not affect the intracellular maturation and release of the merozoites from erythrocytes. The Trx-D2 and GST-F2i fusions at a concentration of 10 µM inhibited merozoite invasion in human erythrocytes by ~31% and ~27%, respectively. Due to the limitations of invasion assays, specifically volume restrictions and relatively low molar fusion protein concentrations, higher concentrations of fusions could not be tested in the invasion assays under these conditions. All invasion inhibition experiments were performed in triplicate and minimum variation was observed within each experiment. The Trx and GST proteins were used as negative controls under the same conditions, resulting in baseline inhibition of erythrocyte invasion (Fig. 4B). These data suggest that the D2 and F2i segments of EBL-1 inhibit merozoite invasion presumably by competing with merozoites for specific binding to glycophorin B receptor on the erythrocyte cell surface. These findings provide additional evidence for a functional role of EBL-1 in the parasite invasion process. Our results suggest that a direct interaction between F2i segment of parasite EBL-1 and host glycophorin B plays an important functional role in the malaria parasite invasion process in human erythrocytes

3.7. Circular dichroism (CD) analysis of F2i segment of EBL-1

Because of the relatively small size of the F2i segment, consisting of 69 amino acids, it was necessary to assess the secondary structural integrity of F2i by circular dichroism spectroscopy. The F2i fragment was subcloned into pET32a vector and expressed as Trx-F2i fusion protein (Fig. 2, lanes 7–9). This cloning and expression step was necessary because of the presence of multiple bands in the GST-F2i fusion (Fig. 2, lanes 4–6) that complicated the interpretation of CD data. Although the purified Trx-F2i protein showed minimal degradation, the fusion protein was expressed predominantly in the inclusion bodies of transformed bacteria. Soluble Trx-F2i fusion protein was recovered by extracting the bacterial pellet with high salts in the presence of glycerol. Affinity-purified Trx-F2i required the presence of 5–10% glycerol for solubility. Using qualitative sedimentation assays, we confirmed the binding of Trx-F2i fusion protein to RBCs and trypsin-treated RBCs, but not to neuraminidase or chymotrypsin-treated RBCs (data not shown). It is noteworthy that the presence of glycerol at higher concentrations of Trx-F2i interfered with RBCs thus precluding the quantification of binding parameters and invasion assays.

Affinity-purified Trx and Trx-F2i proteins were subjected to CD analysis under identical conditions. The CD spectra and spectral difference are shown in Fig. 5. Based on the sequence and structural homology of known EBL-175 domain, the expected secondary structure of F2i peptide was predicted as three helices [35]. The spectral difference showed the largest negative intensity at 222 nm and 208 nm, which is characteristic of helices. While a small contribution from beta sheet structure (intensity at ~217 nm) cannot be ruled out, it is clear that there is little random coil structure (intensity at 190–200 nm). These data suggest that the F2i segment is likely to form a helical folded conformation in native EBL-1 protein.

Figure 5
Circular dichroism (CD) analysis. The difference between the CD spectra of Trx-F2i and Trx in millidegrees as a function of the wavelength in nm. Insert: CD signal of Trx-F2i (filled circles) and Trx (empty circles), and the difference (triangles).

4. Discussion

Plasmodium falciparum utilizes a repertoire of ligand-receptor interactions to invade human erythrocytes. These interactions are broadly grouped into two invasion pathways. The first group engages multiple parasite proteins to erythrocyte surface receptors, and these interactions are dependent upon the sialic acid content of host receptors. The second group of invasion pathways follows a similar strategy except that the parasite protein interactions with host receptors are sialic acid-independent [36]. The erythrocyte glycophorins are considered as the principal mediators of the sialic acid-dependent pathways (Fig. 6), whereas the host anion transporter band 3 and complement receptor 1 have recently been shown to mediate components of the sialic acid-independent pathways [29, 3739]. These two seemingly distinct yet inter-dependent invasion routes ensure the malaria parasite to enter human erythrocytes efficiently and swiftly.

Figure 6
A sialic acid-dependent parasite invasion pathway model. Cartoon shows the erythrocyte glycophorin receptors and malaria parasite EBL ligand interactions during parasite invasion in human red blood cells. EBL-1 is involved in erythrocyte receptor GPB ...

Intense interest in elucidating the components of the ligand-receptor interactions of two invasion pathways is driven by the need to identify novel parasite antigenic epitopes required for the development of malaria vaccine. Our previous studies have identified components of the sialic acid-independent invasion pathway. We identified parasite MSP1-MSP9 complex binding to host band 3 as one potential ligand-receptor interaction for the sialic acid-independent pathway [37]. Moreover, our earlier observations with band 3 null mouse erythrocytes [40], and by others on human erythrocytes [41], have shown a close association of host band 3 with glycophorin A in the erythrocyte membrane. Since the retention of glycophorin A on the erythrocyte surface is not feasible without band 3 [42], we proposed a model whereby the band 3-glycophorin A complex serves as a major receptor for P. falciparum invasion in human erythrocytes [37]. This model provides a molecular framework for integrating both sialic acid-dependent and independent pathways of merozoite invasion in human erythrocytes.

To test this model, we screened a phage display cDNA library to identify parasite proteins that bind to erythrocyte glycophorins. Phage display technique has been successfully used in the malaria field for identifying new epitopes of peptides and antibodies using parasite antigens such as AMA-1, MSP1, CSP, and Pfs48/25 [28, 4346]. Our strategy was to screen a phage display cDNA library made from the sialic acid-dependent strain of P. falciparum (FCR3) using a mixture of purified glycophorins as bait. In addition, we performed a similar phage display screen using intact erythrocytes as bait. The details of the phage display library used in the present study have been published before [27]. Since glycophorin A is the major glycophorin present in human erythrocytes, purified preparations of glycophorins isolated from human RBCs generally consists of 85% glycophorin A, 10% glycophorin B, 4% glycophorin C, and 1% glycophorin D [15]. Indeed, both phage display screens identified EBA-175 as a parasite ligand binding to purified glycophorins as well as intact erythrocytes. Since EBA-175 is a well-known parasite ligand for glycophorin A, its identification further supports the feasibility of our experimental strategy for identifying novel parasite ligands for other glycophorins (Fig. 6).

Both screens identified multiple phage clones encoding a 69 amino acid insert of EBL-1 (Fig. 1). It is to be noted that the two screens pulled out dozens of specific phage clones; however, we amplified and sequenced only about 25 phage clones for further analysis. Among the successfully sequenced clones, five encoded EBA-175, three encoded EBL-1, and several clones encoded hypothetical proteins. We did not find any phage clone encoding the EBA-140 in our limited number of sequenced clones. Like EBA-175, the EBL-1 belongs to a family of Duffy-binding-like proteins. EBL-1 is the largest member of the ebl family of genes; the single copy gene is located on chromosome 13, and encodes a polypeptide of 2,647 amino acids with a predicted mass of 304.5 kDa. The full length EBL-1 protein contains two Cys-rich domains within an amino-terminal DBL domain (D2), which includes F1 and F2 sub-domains, and a c-Cys domain adjacent to the C-terminal transmembrane domain [47]. The c-Cys domain is relatively better conserved than the DBL domains in the EBL family of proteins. While other EBL family members contain eight conserved cysteine residues, the EBL-1 contains four cysteine residues [47]. The precise function of the c-Cys domains has not been determined. In the EBL family of P. falciparum, the DBL domains generally mediate the erythrocyte binding function. Interestingly, a recent study examined the binding of several EBL-1-derived peptides with human erythrocytes, and found that several binding peptides w ere located within the F1 and F2 sub-domains of EBL-1 [48]. These observations are consistent with our findings that the phage-encoded 69 amino acid peptide termed F2i (residues 601–669), bearing the binding site for glycophorin B, is located within the F2 sub-domain of DBL domain in EBL-1.

The interactions between parasite ligands and erythrocyte surface receptors are often characterized by their enzyme sensitivity to neuraminidase, trypsin, and chymotrypsin [3, 12, 18, 21, 24]. Using a similar approach, our data indicate that both D2 and F2i segments of EBL-1 bind to human RBCs in a trypsin-resistant manner, and these interactions are sensitive to neuraminidase and chymotrypsin (Fig. 4A). Among the four glycophorins, only glycophorin B is resistant to trypsin but sensitive to chymotrypsin, and the sialic acid residues of all glycophorins can be removed by neuraminidase. These observations suggested that glycophorin B is the likely receptor for EBL-1. This prediction was further confirmed by the lack of EBL-1 D2 domain binding to glycophorin B-null human erythrocytes (S-s-U-) (Fig. 4A).

A recent study has identified glycophorin B as a receptor for EBL-1 [24]. In this study, the region 2 (DBL domain) of EBL-1 was expressed in CHO-K1 cells, and its binding to glycophorin B positive RBCs, but not with glycophorin B-null RBCs, was demonstrated [24]. Individual F1 and F2 sub-domains of EBL-1 were also expressed in CHO-K1 cells, but these constructs did not support binding to human RBCs [24]. Our phage display screens revealed that the glycophorin B binding peptide is located within the F2 subdomain of EBL-1 (Fig. 1A). In addition, our study provides the first direct evidence that both D2 and F2i segments of EBL-1 inhibit merozoite invasion in human erythrocytes (Fig. 4B). This result is consistent with previous findings showing reduced merozoite invasion in glycophorin B-deficient erythrocytes by several parasite strains [49]. Similarly, a previous study showed that the 7G8 strain of P. falciparum cannot invade glycophorin B-deficient erythrocytes, whereas other strains could invade these cells at 72–79% efficiency as compared to normal erythrocytes [50]. In contrast, a subsequent study showed that P. falciparum 7G8 strain can invade glycophorin B-deficient erythrocytes with ~50% efficiency [49]. It is also noteworthy that several Plasmodium falciparum strains do not express apparently functional forms of EBL-1, and may be under selective genetic pressure that is unique to this gene, particularly within the F2 segment of the DBL/D2 domain [51, 52]. Irrespective of the extent of receptor utilization by the invading merozoites, our findings identify a relatively short F2i segment of EBL-1 as the cognate parasite ligand for glycophorin B on human erythrocytes. We suggest that an optimized 69-amino acid synthetic peptide of EBL-1 could be considered for inclusion in the multi-subunit malaria vaccine.

Acknowledgements

We are very grateful to Dr. Ghislaine Mayer of the Virginia Commonwealth University, Richmond, VA, for kindly providing us glycophorin B-null human erythrocytes. We are also indebted to Dr. Louis Mi ller of the NIH for advice and guidance in obtaining the glycophorin B-null erythrocytes. We are grateful to Dr. Brian Kay of the University of Illinois at Chicago for his invaluable advice about the phage display library screens, and thank Rolf Fendel, Ravi Ranjan, Changling Ma, Danielle Donzal, and Donna-Marie Mironchuk for suggestions, proofreading, and graphic arts of the manuscript. This work was supported by the NIH Grants HL60961 and HL89517 (A.C), and the National Research Foundation of South Africa GUN 2069449 and the Medical Research Council of South Africa (T.C.). XL designed and performed majority of the experiments, and participated in manuscript writing. MM and CR designed and performed the recombinant protein expression experiments. MM managed the resubmission of the original manuscript. JM performed the circular dichroism experiments. TC made the phage display library and participated in manuscript editing. AC designed and supervised the research project, and participated in manuscript writing. Dr. Li’s current address is Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.

Footnotes

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