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Fundación Instituto de Inmunología de Colombia and Universidad Nacional de Colombia, Bogotá, Colombia
Reprint requests to: Hernando Curtidor, Fundación Instituto de Inmunología de Colombia, Carrera 50 No. 26-00, Bogotá, Colombia, 020304 Zona CAN; e-mail: hernando_curtidor{at}fidic.org.co; fax: +57-1-3244672/73 x108.
(RECEIVED August 30, 2004; FINAL REVISION October 4, 2004; ACCEPTED October 9, 2004)
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Keywords: malaria protein; erythrocyte binding ligand-1; peptides; Plasmodium falciparum
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041084305.
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Introduction |
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Members of the erythrocyte binding-like (ebl) family represent some of those merozoite proteins involved in the merozoite’s invasion of the erythrocyte; they bind with high affinity to glycoproteins on the surface of the erythrocyte. Erythrocyte binding antigen-175 (EBA-175) binds to glycophorin A and mediates an invasion pathway for merozoite entry into erythrocytes (Camus and Hadley 1985; Orlandi et al. 1992; Sim et al. 1994; Duraisingh et al. 2003). EBA-140 (BAEBL) binds to glycophorin C and functions in a pathway for merozoite invasion (Mayer et al. 2001; Lobo et al. 2003; Maier et al. 2003); EBA-181 (JESEBL) binds to the surface of erythrocytes in a sialic acid-dependent manner to a trypsin-resistant/chymotrypsin-sensitive receptor (Gilberger et al. 2003a).
However, in the case of EBL-1, its interaction with the erythrocyte has not been studied; its receptor on the erythrocyte surface also remains unknown. EBL-1 is a putative erythrocyte binding protein which is encoded by the ebl-1 gene (Peterson et al. 1995; Peterson and Willems 2000).
ebl-1 has been identified as a second ebl family member in P. falciparum on the basis of consensus family characteristics: a single-copy gene encoding two Cys-rich domains, one Duffy-binding-like (DBL) domain, and a C-Cys domain (Peterson and Willems 2000; Adams et al. 2001). EBL-1 has only four conserved cysteine residues, compared to the other ebl products, which have eight. The DBL domain mediates erythrocyte binding activity in ebl products (Smith et al. 2000). ebl-1 is also transcribed in late schizonts and is linked to a rapid proliferation phenotype (Adams et al. 2001).
The ebl-1 gene presents characteristics similar to those of P. falciparum eba-175 and Plasmodium vivax DAP genes (Peterson and Willems 2000; Michon et al. 2002). It has been determined that these genes do participate in the merozoite invasion of erythrocytes (Chitnis et al. 1996; Gilberger 2003b), and it has thus been suggested that EBL-1 is probably involved in erythrocyte receptor recognition, playing a synergistic or an alternative role in the invasion process (Peterson et al. 1995; Peterson and Willems 2000; Adams et al. 2001).
In the present study, we attempted to delineate specific erythrocyte binding capacity and biological activity of P. falciparum-derived EBL-1 peptides. The results show that five peptides bound specifically and not sialic acid-dependently to erythrocytes: 29895 (41HKKKSGELNNNKS GILRSTY60) toward the N-terminal region, 29903 (201LY ECGKKIKEMKWICTDNQF220) located in the DBL/F1 region, 29923 (601CNAILGSYADIGDIVRGLDV620), 29924 (621WRDINTNKLSEKFQKIFMGGY640) located in the DBL/F2 region, and 30018 (2481LEDIINLSKKKKK SINDTSFY2500) located toward the C-terminal region of EBL-1. Interestingly, all five of these high-activity binding peptides (HABPs) specifically bound to a 36-kDa protein on erythrocyte membrane and inhibited in vitro merozoite invasion depending on the peptide concentration used.
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Results |
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2%, according to criteria established earlier (Urquiza et al. 1996; Rodriguez et al. 2000). The specific erythrocyte binding activity for 133 synthetic peptides covering the total length of the P. falciparum 3D7 strain EBL-1 protein (GenBank accession no. CAD52344
[GenBank]
was determined by using binding assays. Five erythrocyte HABPs were found in EBL-1-peptides: 29895 (41HKKKSGELN NNKSGILRSTY60), 29903 (201LYECGKKIKEMKWIC TDNQF220), 29923 (601CNAILGSYADIGDIVRGLDV620), 29924 (621WRDINTNKLSEKFQKIFMGGY640), and 30018 (2481LEDIINLSKKKKKSINDTSFY2500).
The results show that the HABPS were located in different EBL-1 protein regions: peptide 29895 toward the N-terminal region, peptide 29903 was located in the DBL/F1 region; peptides 29923 and 29924 were located in the DBL/F2 region, and peptide 30018 toward the EBL-1 C-terminal region (Fig. 1
). No HABPs were found in the central region.
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Figure 2 shows that the jumbled peptides presented specific binding activity lower than that for native HABPs. HABP 29923 analog jumbled peptide 32257 presented the highest specific binding activity (1.2); HABP 29923 happened to be the peptide having the highest specific binding activity (2.8). The results thus indicated that HABP binding activity was due to their specific sequences.
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) allowed calculation of affinity constants (Kd) and Hill coefficients (nH) and the approximate number of binding sites per cell (Attie and Raines 1995).
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Cross-competition assays
Cross-competition assays were carried out for each of the HABPs by inhibiting them with unlabeled native peptide or other unlabeled HABPs to determine whether they were able to displace the radiolabeled peptide. The cross-competition assays showed that 125I-labeled HABPs were inhibited, in some cases, by other nonlabeled HABPs (Table 1). For example, it can be seen that radiolabeled peptide 29895 was inhibited by nonlabeled 29903 and 30018 peptides. Radiolabeled peptide 29924 was inhibited by all the other nonradiolabeled HABPs, mainly for peptide 29903 and 29923 peptides; in contrast, radiolabeled peptide 29923 was not inhibited by any of the other nonradiolabeled HABPs. The majority of HABPs were not mutually inhibiting.
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).
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shows that enzymatic treatment of erythrocytes similarly affected the binding of peptides 29924 and 30018, becoming lessened when they were treated with chymotrypsin or trypsin and increasing when this was done with neuraminidase. Similarly, the binding of peptides 29903 and 29923 lessened when erythrocytes were treated with chymotrypsin, but this was not affected when they were treated with trypsin or neuraminidase. Peptide 29895 presented behavior different from that of the other HABPs—binding to erythrocytes treated with trypsin diminished, and was not affected by treatment with neuraminidase; this was the only HABP whose binding was not affected when erythrocytes were treated with chymotrypsin (on the contrary, it increased it).
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) show that all peptides inhibited merozoite invasion by more than 70%. It can also be seen that invasion inhibition depended on peptide concentration.
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Discussion |
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Among those merozoite ligands involved in the invasion are products belongs to a family of genes (ebl) encoding proteins involved in the specific recognition of host cell receptors, including the P. vivax and P. knowlesi Duffy-binding proteins (Adams et al. 2001). Each ebl appears as a single-copy gene not having cross-hybridization to any other locus in the P. falciparum genome, and all have similar exon-intron structure with conserved splicing boundaries, indicating a common evolutionary origin (Adams et al. 1992, 2001; Michon et al. 2002).
To date, six ebl proteins have been identified in the P. falciparum genome: EBA-175 (Camus and Hadley 1985; Orlandi et al. 1992; Sim et al. 1994; Duraisingh et al. 2003), EBA-140 (Thompson et al. 2001; Mayer et al. 2003; Lobo et al. 2003; Maier et al. 2003), EBA-165 (Triglia et al. 2001), EBA-181 (Gilberger et al. 2003a), MAEBL (Ghai et al. 2002), and EBL-1 (Peterson et al. 1995; Peterson and Willems 2000). The receptor has been identified for some of these proteins which bind to the RBC membrane; its participation in the invasion process has been determined (Gilberger et al. 2003a,b; Lobo et al. 2003). The interaction between EBL-1 protein and the erythrocyte has not been characterized to date, meaning that it remains unknown whether EBL-1 binds to erythrocytes and thus which is its receptor.
However, the similarity of the ebl-1 gene’s characteristics to those of the rest of the members of the ebl family, their transcription in late schizonts, their relationship with a rapid proliferation phenotype, and the EBL-1 protein’s high homology with other ebl products suggests that EBL-1 is probably involved in some of the merozoite-erythrocyte interactions in the invasion process (Peterson et al. 1995; Peterson and Willems 2000; Adams et al. 2001).
This work focused on analyzing EBL-1 erythrocyte binding sequences as EBL-1 (or a region derived from it) could be directly acting on erythrocyte receptor recognition, merozoite attachment, or synergistic invasion of erythrocytes in an alternative role in the invasion process.
The binding assays showed that five P. falciparum EBL-1 derived peptides specifically bound to erythrocytes (Fig. 1
) and that HABP binding depends on each specific sequence in particular and not on amino acid composition (Fig. 2). The high affinity (Kd values ranging from 245–513 nM) and positive cooperativity (nH values of 1.0–1.5) indicate strong HABP interaction with erythrocytes (Fig. 3). Lesser or similar Kd values have been reported for HABPs derived from EBA-175 (Rodriguez et al. 2000) and EBA-140 (Rodriguez et al 2003) proteins in the ebl family.
The cross-competition assays showed that nonlabeled HABPs inhibited radiolabeled HABP binding to different degrees (Table 1). However, radiolabeled peptide 29923 was not inhibited by any of the other nonradiolabeled HABPs. It was also seen that some HABPs were mutually inhibiting. The results suggest that HABPs bind to different receptors on erythrocyte membrane or to a single receptor in different binding sites. The latter would be the most feasible, bearing in mind that all EBL-1 HABPs bound to a protein having an apparent 36 kDa molecular weight on erythrocyte membrane (Fig. 4).
When the binding assays were performed with enzyme-treated human erythrocytes, it was also observed that neuraminidase (cleaving terminal sialic acids from glycoproteins) did not affect the binding of any of the HABPs; on the contrary, the binding of HABPs 29924 and 30018 increased (Table 2). Chymotrypsin treatment lessened the binding of all HABPs except HABP 29895, whose binding increased. Trypsin treatment diminished the binding of HABPs 29895, 29924, and 30018. These preliminary results indicate that peptides bind to different receptor sites on the same receptor-molecule on the erythrocyte membrane, such binding not being sialic acid-dependent. This is interesting, since it has been reported that P. falciparum can invade erythrocytes independently of sialic acid (Narum et al. 2000; Duraisingh et al. 2003).
It was possible to define three EBL-1 erythrocyte specific binding regions from the binding assay results. A binding region was located in the N-terminal region, comprised of HABPs 29895 and 29903. In turn, HABP 29903 was located in the DBL-F1 domain N-terminal (Fig. 1).
The second binding region corresponds to HABP 30018, located in the EBL-1 protein’s C-terminal, just before the start of the C-Cys region (Figs. 1, 5B). To date, no function has been identified for the C-Cys domain, even though this is more conserved than the DBL domains (Adams et al. 2001). However, the EBA-175 protein also presents an HABP just before the start of the C-Cys region (Rodriguez et al. 2000), and EBA-140 protein also presents an HABP at the start of the C-Cys region (Fig. 5A; Rodriguez et al. 2003). Although it is not indispensable for invasion, this cysteine-rich region could be a participant in the merozoite’s initial recognition of erythrocyte.
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, 5A); this fact is interesting since it was reported that DBL domains mediate erythrocyte binding activity in the ebl products and that the EBA-175 DBL/F2 domain binds to glycophorin-A on erythrocyte membrane (Orlandi et al. 1992; Sim et al. 1994; Sim 1998; Michon et al. 2002). Some HABPS corresponding to EBA-175 and EBA-140 proteins have been described which are included within the proteins’ DBL/F2 region (Rodriguez et al. 2000, 2003).
Sequence alignment and comparison of the erythrocyte binding profiles and the EBL-1, EBA-175, and EBA-140 protein DBL/F2 domains (Fig. 5B) lead to the observation that the HABPs cover almost all of the DBL/F2 domain’s central region. This is interesting since it was noted that P. knowlesi and P. vivax invasion depend on recognition of a single receptor (e.g., Duffy blood group antigen) (Miller et al. 1979; Adams et al. 2001). On the contrary, P. falciparum does not depend on a single receptor for invasion; it can use alternative invasion pathways or different receptors on the erythrocyte membrane (Hadley et al. 1987; Orlandi et al. 1992; Dolan et al. 1994; Goel et al. 2003; Lobo et al. 2003; Maier et al. 2003).
The partial disruption of the eba-175 in two different P. falciparum clones is associated with a switch toward a sialic acid-independent invasion pathway, showing that alternative parasite ligands exist (Kaneko et al. 2000; Reed et al. 2000). Additionally, distinct pathways depending on glycophorin-A, -B, and -C, and an unknown receptor (the X pathway) have been identified (Dolan et al. 1994).
It has thus been suggested that some of the receptors involved in invasion and the use of alternative invasion pathways are probably related to EBA-175, and associated with expression of different ebl products (Chitnis and Blackman 2000; Adams et al. 2001). The idea that EBL-1 protein or regions deriving from it presenting erythrocyte binding activity (in this case HABPs belonging to the DBL/F2 region) are involved in the invasion process thus cannot be discarded.
In fact, when we tested the HABPs on in vitro P. falciparum cultures, we observed that all HABPs significantly inhibited merozoite invasion (Table 3). These results suggest that the HABPS were blocking the merozoite-erythrocyte interaction, inhibiting invasion.
Further studies are still necessary to clarify EBL-1 HABPs’ specific role at the time of merozoite invasion of erythrocytes. The possibility of using HABP sequences from P. falciparum EBL-1 protein for designing tools for the specific inhibition of P. falciparum merozoite interaction with erythrocytes also needs deeper study. Taken together, the results reported in this work suggest that P. falciparum EBL-1 protein regions could be participating in parasite recognition or invasion of erythrocyte, in the same way as other ebl family members.
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Materials and methods |
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in one-letter code.
Radiolabeling
The peptides were labeled with 125I according to previously described methodology (Urquiza et al. 1996). Briefly, 3.2 µL Na125I (100 mCi/mL) was oxidized with 12.5 µL chloramine-T (2.25 µg/µL) and added to 5 µg peptide for 5 min at room temperature. The reaction was stopped by adding 15 µL sodium bisulfite (2.25 µg/µL) and 50 µL NaI (0.16 M). The radiolabeled peptide was then separated on a Sephadex G-10 column (Pharmacia).
Binding assay
Human erythrocytes (2 x 108 cells/µL) obtained from healthy donors were washed in PBS buffer until the buffy coat was removed and then incubated with different radiolabeled-peptide concentrations (10–200 nM), in the absence (total binding) or presence (nonspecific binding) of 40 µM unlabeled peptide. The sample reached 200 µL final volume with PBS and was incubated for 90 min at room temperature (Urquiza et al. 1996; Curtidor et al. 2001). The cells were then washed five times with PBS, and cell-bound radiolabeled peptide was quantified in an automatic gamma counter (4/200 plus ICN Biomedicals). The binding assays were performed in triplicate.
Jumbled-peptide binding assay
The sequences of HABPs determined in the binding assay described above were used in synthesizing the same peptides but now in a jumbled order (i.e., same amino acid composition as HABPs but having random sequence) and then tested in binding assays. The assays were carried out in triplicate in conditions identical to those described above in the "Binding assay" section. Synthesized peptides are shown in Figure 2 in one-letter code.
Saturation assays
An erythrocyte binding assay was used to ascertain saturation with all HABPs; the following modifications were introduced: 1.5 x 108 cells were used at 255 µL final volume; radiolabeled peptide concentrations were between 0 and 1000 nM. The unlabeled peptide concentration was 40 µM. Cells were washed with PBS, and a gamma counter was used to measure cell-bound radiolabeled peptide (Weiland and Molinoff 1981; Enna 1984).
Cross-competition assays
The binding to erythrocytes for each HABP was inhibited by all the other HABPs in cross-competition assays. Radiolabeled HABPs (200 nM) were incubated with 2 x 108 erythrocytes for 90 min at room temperature, in the presence or the absence of the same or other unlabeled HABPs (20 µM). After incubation, unbound peptide was removed with three 5-mL PBS washes, and cell-bound radiopeptide was measured. The cross-competition assays were done in triplicate in the same conditions.
Cross-linking assays
Radiolabeled HABPs were cross-linked to erythrocyte membranes in the presence or absence of unlabeled peptide for identifying specific erythrocyte binding sites. The cross-linking binding test was performed by using a final 1% cell concentration and following incubation with the radiolabeled peptide in the presence or absence of 40 µM unlabeled peptide for 90 min at room temperature. After incubation, cells were washed with PBS, and the bound peptide was cross-linked with 10 µM BS3, Bis (sulfosuccinimidyl suberate) (Pierce) for 20 min at 4°C. The reaction was stopped with 20 mM Tris-HCl (pH 7.4) and washed again with PBS. Then cells were then treated with lysis buffer (5 mM Tris-HCl, 7 mM NaCl, 1 mM EDTA, 0.1 mM PMSF). The obtained membrane proteins were solubilized in Laemmli buffer and separated by SDS/PAGE (12% w/v polyacrylamide gels). The gels were exposed on BioRad Imaging Screen K (BioRad Molecular Imager FX; BioRad Quantity One, Quantitation Software) for 2 d to determine which proteins had become cross-linked to the radiolabeled peptides. The apparent molecular weight was determined using molecular weight markers (NEB).
Enzymatic treatment
Erythrocytes (5%) suspended in PBS buffer were treated with 150 µU/mL neuraminidase (ICN 9001-67-6) at 37°C for 1 h, washed five times with PBS buffer, and centrifuged at 1000g for 5 min. In the same way, erythrocytes (5%) were treated with trypsin (Sigma T-1005) or chymotrypsin (Sigma C-4129) in TBS buffer (5 mM Tris-HCl, 140 mM NaCl [pH 7.4]), at a final 0.75 g/mL concentration. After incubation at 37°C for 1 h, the samples were washed five times with PBS buffer to which 0.1 mM PMSF had been added. After enzyme-treatment, these erythrocytes were tested in a binding assay with HABPs as described (Camus and Hadley 1985; Curtidor et al. 2001).
Merozoite invasion inhibition assay
The in vitro cultures of the FCB-2 strain of P. falciparum were synchronized at the ring stage with sorbitol solution, and incubated until the late schizont stage. The cultures were grown in RPMI 1640 medium supplemented with 10% human plasma. (Trager and Jensen 1976; Lambros and Vanderberg 1979). The culture (0.5% final parasitemia and 5% hematocrit) was seeded in 96-well cell culture plates (Nunc) in the presence of test peptides at 200, 100 and 50 µM concentrations. Each peptide was tested in triplicate. After incubation for 18 h at 37°C in a 5% O2/5%CO2/90% N2 atmosphere, the supernatant was recovered and the cells stained with 15 µg/mL hydroethydine, incubated at 37°C for 30 min, and washed three times with PBS. The suspensions were analyzed using a FACsort in Log FL2 data mode using CellQuest software (Becton Dickinson Immunocytometry System) (Wyatt et al. 1991). Infected and uninfected erythrocytes treated with EGTA and chloroquine were used as controls.
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Acknowledgments |
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References |
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