Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

Abstract

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.

Figure 1.

Membrane and detergent-resistant lipid raft-like membrane-associated proteins (DRM). Molecule sizes are drawn at their approximate molecular weight. (Left panel) RAMA anchored to the membrane via a GPI tail and proteins noncovalently associated with RAMA and involved in merozoite invasion of erythrocytes (recognized in DRM proteomes). All high (RhopH) and low (RAP-1, -2, -3) molecular weight rhoptry protein members are also shown. (Top panel) Lateral view of the hypothetical organization of these proteins, (bottom panel) view from the top. (Right panel) DRM rafts formed by MSP-1 83 kDa, 30 kDa, and 38 kDa fragments (yellow) and noncovalently associated molecules such as MSP-6 (fuchsia) and MSP-7 (green). MSP-1 33 kDa (yellow) and the only 19 kDa (yellow) fragment anchored to the merozoite membrane via a GPI tail are also displayed. Other GPI tail (green twists traversing the pale green membrane) anchored membrane surface proteins such as MSP-2 (clear blue), MSP-3 (dark gray), MSP-4 (clear brown), MSP-5 (dark blue), MSP-8 (green), and Pf113 (dark gold), Pf92 (brown), Pf41 (red), and Pf12 (gray), recently identified in DRM proteome analysis, are also shown. Figure adapted from Pinzón et al. (2008) and reprinted with permission from Elsevier © 2008.

Figure 2.

(A) Erythrocyte binding assays using RhopH3 peptides. The length of the black bar indicates the slope of the specific binding graph of each peptide. Peptides are numbered according to investigator's numbering system. (ND) Not determined. (B) Schematic representation of conserved Cys residues in high molecular weight rhoptry complex. RhopH1/Clag 9 (C.G. Pinzon, H. Curtidor, and M.E. Patarroyo, unpubl.), RhopH1/Clag 3.2 (Ocampo et al. 2005), RhopH2 (protein in study), and RhopH3.

Figure 3.

Erythrocyte binding profile for two HABPs compared with that of their corresponding scramble peptide. Peptides are numbered according to the investigator's numbering system. Scramble peptides' binding activities were much lower than those of their native HABPs, or were even undetectable.

Figure 4.

Saturation curves for HABPs 33482, 33483, 33484, 33490, 33491, 33522, 33529, 33531, 33566, 33570, and 33580. Increasing amounts of radiolabeled peptide were added in the presence or absence of unlabeled peptide. The curve represents specific binding of labeled peptide to human erythrocytes. The abscissa of the Hill plot (inset graph) is log F and the ordinate is log (B/Bmax − B). (F) Free peptide, (B) amount of bound peptide, (Bmax) maximum amount of bound peptide.

Figure 5.

CD spectra of RhopH3 HABPs (30% TFE/water). All HABPs clearly showed a helical conformation.

Figure 6.

Effect of enzymatic treatment on HABPs' erythrocyte binding. HABPs are organized according to their erythrocyte-binding pattern to each of the assessed enzymes. (Top right panel) Those HABPs whose erythrocyte binding was susceptible to neuraminidase, chymotrypsin, and trypsin. (Top left panel) Those HABPs whose receptors were affected by chymotrypsin and trypsin. (Bottom panels) Trypsin- (left) and chymotrypsin (right)-susceptible HABPs. Those HABPs whose binding behavior was not affected by any enzymatic treatment are not shown.

Figure 7.

Cross-linking assays. Ligand–receptor complexes were obtained by using erythrocyte membrane proteins cross-linked with radiolabeled versions of each HABP. (Lanes 1,3,5,7 in A,B) Total binding for peptides 33482, 33483, 33491, 33521, 33522, 33529, 33570, and 33580, respectively. (Lanes 2,4,6,8 in A,B) Inhibited binding for peptides following the same order as above. The images were acquired by using BioRad Quantity One (Quantitation Software) and were not further manipulated.