Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum erythrocyte binding ligand 1 (EBL-1)-derived peptides

Abstract

Erythrocyte binding ligand 1 (EBL-1) is a member of the ebl multigene family involved in Plasmodium falciparum invasion of erythrocytes. We found that five EBL-1 high-activity binding peptides (HABPs) bound specifically to erythrocytes: 29895 ((41)HKKKSGELNNNKSGILRSTY(60)), 29903 ((201)LYECGK-KIKEMKWICTDNQF(220)), 29923 ((601)CNAILGSYADIGDIVRGLDV(620)), 29924((621)WRDINTNKLSEK-FQKIFMGGY(640)), and 30018 ((2481)LEDIINLSKKKKKSINDTSFY(2500)). We also show that binding was saturable, not sialic acid-dependent, and that all peptides specifically bound to a 36-kDa protein on the erythrocyte membrane. The five HABPs inhibited in vitro merozoite invasion depending on the peptide concentration used, suggesting their possible role in the invasion process.

Figure 1.

Erythrocyte binding assays using EBL-1 peptides. Each one of the black bars represents the slope of the specific binding graph, which is called the specific binding activity. Peptides having specific binding activity ≥2% were considered as having high specific erythrocyte binding (HABPs). A schematic representation of the EBL-1 protein is at the right, showing SP (signal peptide), F1 and F2 (DBL domains), TR (transmembrane region), and Cys (cysteine-rich region) (Adams et al. 2001; Michon et al. 2002).

Figure 2.

HABP jumbled-peptide erythrocyte binding profile. The jumbled peptides’ binding activities were lower than those for native HABPs.

Figure 3.

Saturation curves for (top) 29895, 29903, (middle) 29923, 29924, and (bottom) 30018 HABPs. Increasing quantities of labeled peptide were added in the presence or absence of unlabeled peptide. The curve represents the specific binding. The affinity constants (Kd) were 513, 481, 415, 450, and 245 nM; Hill coefficients were 1.1, 1.4, 1.0, 1.5, and 1.0; and the number of binding sites per cell was 5500, 5700, 10,500, 7000, and 8000, respectively. 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 4.

Autoradiograph from HABP cross-linking assays. Erythrocyte membrane proteins were cross-linked with all radiolabeled peptide HABPs. Only HABP 29903 (lanes 1,2) and 29923 (lanes 3,4) autoradiographs are shown. Lanes 1 and 3 show total binding (i.e., cross-linking in the absence of unlabeled peptide), and lanes 2 and 4 show inhibited binding (i.e., cross-linking in the presence of unlabeled peptide).

Figure 5.

(A) Schematic representation of EBA-175, EBA-140, and EBL-1 protein DBL/F2 domains. The position of each HABP is shown for each protein. The arrows show the position of conserved cysteine residues. (B) EBA-175, EBA-140, and EBL-1 protein C-Cys domain sequence alignment. Boxed amino acids indicate the HABPs for each protein: peptide 1818 in EBA-175, peptide 26170 in EBA-140, and 30018 in EBL-1 (Rodriguez et al. 2000, 2003). The arrows indicate the start of cysteine-rich regions in each protein (Adams et al. 2001).