Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

Abstract

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.

Figure 1.

Erythrocyte binding assays using PfMSP-3 peptides. (A) Each of the black bars represents the slope of the specific binding graph, which is named specific binding activity. Peptides with 2% were considered as having high specific erythrocyte binding (HABPs). (B) Specific binding activity for HABP peptide analogs; original and jumbled peptide sequences are shown to the right, and the bars on the left represent specific binding. The numbers in the first column represent FIDIC’s internal code number for those peptides synthesized.

Figure 2.

Saturation curves for 31193, 31202, and 31209 HABPs. Increasing quantities of labeled peptide were added in the presence or the absence of unlabeled peptide. The curve represents the specific binding. In the Hill plot (inset), the abscissa is log F and the ordinate is log (B/Bmax − B), where F is free peptide, B is bound peptide, and Bmax is the maximum amount of bound peptide.

Figure 3.

MSP-3 peptide binding to enzyme-treated erythrocytes. Peptide binding was compared between enzyme-treated RBC and untreated RBC. The bars represent the percentage of specific binding activity obtained when radiolabeled HABPs were incubated with erythrocytes treated with chymotrypsin, trypsin, and neuraminidase with respect to untreated erythrocytes.

Figure 4.

Autoradiograph from HABP cross-linking assays. Erythrocyte membrane proteins were cross-linked with radiolabeled peptides 31193 (lanes 1,2), 31202 (lanes 3,4), and 31209 (lanes 5,6). (Lanes 1,3,5) Total binding (i.e., cross-linking in the absence of unlabeled peptide). (Lanes 2,4,6) Inhibited binding (i.e., cross-linking in the presence of unlabeled peptide). (Lane 7) Total binding of scrambled peptide 33010.

Figure 5.

Circular dichroism analysis of MSP-3 HABPs. α-Helix-like structural elements were present in all HABPs; as maximum value was presented at 190 nm and minimum values at 208 and 222 nm, this suggested helical structural elements.