PMC

Interactions between PfRH5 and primate BSG orthologs are species specific. (A) Binding of PfRH5 to human (HsBSG), chimpanzee (PtBSG), and gorilla (GgBSG) BSG. Biotinylated PfRH5 (bait) was immobilized on streptavidin-coated plates and probed with pentameric, β-lactamase–tagged (prey) forms of the indicated BSG orthologs. Biotinylated Cd4 was used as the negative control bait, and β-lactamase–tagged Cd4 was used as the negative control prey. (B) Kinetic analysis of the human (Upper) and chimpanzee (Lower) PfRH5–BSG interaction. Twofold serial dilutions of PfRH5 were injected at 100 µL/min over the indicated BSG orthologs immobilized in different flow cells of a streptavidin-coated sensor chip. The association and dissociation rate constants (k_a and k_d) were calculated by nonlinear curve fitting to the reference-subtracted sensorgrams. The equilibrium dissociation constant (K_D) was calculated from k_d/k_a and the half-life (t_1/2) from ln2/k_d. HsBSG-PfRH5: k_a = (3.0 ± 0.7) × 10⁵⋅M⁻¹⋅s⁻¹, k_d = 0.246 ± 0.002⋅s⁻¹; PtBSG-PfRH5: k_a = (1.40 ± 0.02) × 10⁵⋅M⁻¹⋅s⁻¹, k_d = 1.45 ± 0.08⋅s⁻¹. The k_a and k_d values are means ± SEM from two independent experiments.

Human GYPA binds EBA175 RII orthologs from P. falciparum, P. reichenowi, and P. billcollinsi with similar affinities. In each panel, the data corresponding to the EBA175 RII proteins from each species are color-coded: blue, P. reichenowi; green, P. billcollinsi; and red, P. falciparum (positive control). (A) Fluorescent beads coated with the EBA175 RII orthologs from chimpanzee-restricted parasites P. reichenowi and P. billcollinsi bind human erythrocytes in a sialic acid-dependent manner. Solid colored lines, untreated erythrocytes; dotted colored lines, neuraminidase-treated erythrocytes; black line, negative control (Cd4-coated beads). A representative experiment is shown. (B) The EBA175 RII orthologs from P. reichenowi and P. billcollinsi bind native human GYPA in a sialic acid-dependent manner using the AVEXIS assay. Native, sialylated (Left) and neuraminidase-treated, asialyl (Right) GYPA was biotinylated and immobilized on a streptavidin-coated plate. Binding of the EBA175 RII orthologs was assessed using β-lactamase–tagged pentamers using the AVEXIS assay. Data are shown as mean ± SD; n = 3. (C) Binding affinities at equilibrium of the EBA175 RII orthologs for native human GYPA were within the same order of magnitude, as determined by SPR. The reference-subtracted binding data obtained by injecting twofold dilutions of the purified EBA175 RII proteins at 20 µL/min over biotinylated GYPA immobilized on a streptavidin-coated sensor chip were plotted as a binding curve once equilibrium was reached, and the K_D was calculated by globally fitting a steady-state 1:1 interaction model. K_D is shown as mean ± SEM. Biotinylated β-d-glucose was used as the reference.

Identification of BSG residues that contribute to the species-specific recognition of PfRH5. (A) Chimpanzee BSG (PtBSG) and a panel of humanized mutants are presented as biotinylated baits and probed for binding with a β-lactamase–tagged PfRH5 pentamer prey using the AVEXIS assay. Data are shown as mean ± SD; n = 2. (B) Dissociation-phase SPR analysis of PfRH5 from human BSG (HsBSG, black squares), chimpanzee BSG (PtBSG, black triangles), and the PtBSG mutant E191K (red circles). PtBSG (E191K) bound PfRH5 with k_a = (2.8 ± 0.6) × 10⁵⋅M⁻¹⋅s⁻¹ and k_d = 0.089 ± 0.002⋅s⁻¹; results shown are mean ± SEM from two independent experiments. Data points are reference-subtracted dissociation phases averaged from three different concentrations of PfRH5. One representative experiment is shown (mean ± SD; n = 3). Only every other data point is displayed for ease of comparison. (C) Human BSG (HsBSG) and a panel of variants in which the equivalent residues from gorilla BSG were mutated in HsBSG were presented as baits and tested for binding with a PfRH5 prey using the AVEXIS assay. Data are shown as mean ± SD; n = 2. (D) The binding of human BSG (HsBSG, dark blue line) and three of its site-directed mutants (F27L, light blue; Q100K, purple; and +H103, green) to PfRH5 was analyzed by SPR. Measured parameters were HsBSG (F27L)-PfRH5: k_a = (1.4 ± 0.5) × 10⁵⋅M⁻¹⋅s⁻¹, k_d = 0.88 ± 0.01⋅s⁻¹; HsBSG (Q100K)-PfRH5: k_a = (2.19 ± 0.08) × 10⁵⋅M⁻¹⋅s⁻¹, k_d = 2.0 ± 0.4⋅s⁻¹; HsBSG (+H103)-PfRH5: k_a = (1.60 ± 0.03) × 10⁵⋅M⁻¹⋅s⁻¹, k_d = 1.30 ± 0.09⋅s⁻¹. For ease of comparison, only a single injected concentration of PfRH5 is shown. All values are mean ± SEM from two independent experiments. (E) X-ray crystal structure of human BSG (Protein Data Bank ID: 3B5H). The schematic shows the spatial organization of residues identified as important for species-specific discrimination of PfRH5 binding. The approximate location of the additional histidine (H103) in gorilla BSG is indicated by an arrow.