Figure 6: Ca²⁺-dependent permeabilization and membranolytic activity of PfPLP1

(a) Western blot of affinity-purified rPfPLP1 probed with anti-his antibody. (b) Dose-dependent membranolytic activity of recombinant PfPLP1 (rPfPLP1). Lysis of human RBCs was analyzed in the presence of different amount of rPfPLP1. The graph indicates per cent lysis of human RBCs by rPfPLP1 as compared with 100% hypotonic lysis of human RBCs in water. (c) Ca²⁺-dependent activity of rPfPLP1. The graph shows per cent lysis of human RBC by rPfPLP1 in the presence and absence of EGTA (extracellular Ca²⁺ chelator). Human RBC lysis in the presence of water is used as complete lysis control. (d) Western blot of affinity-purified rMACPF probed with anti-his antibody. (e) Dose-dependent membranolytic activity of rPfPLP1-MACPF. rMACPF domain shows concentration-dependent lytic activity of human RBCs. (f) Ca²⁺-dependent lytic activity of rPfPLP1-MACPF. rMACPF domain does not show Ca²⁺-dependent lytic activity. (g) Permeabilization activity of rPfPLP1 and rPfPLP1-MACPF. The human RBCs were incubated with Phalloidin Alexa 594 in the presence and absence of rPfPLP1 and rMACPF and visualized under confocal microscope. Phalloidin staining was detected in the human RBCs treated with rPfPLP1 or rMACPF but not in untreated human RBCs. (h) Western blot analysis of bound rPfPLP1 eluted by 1.5 M NaCl. Binding of rPfPLP1 was abolished in the absence of Ca²⁺. Recombinant PfF2, the receptor-binding domain of EBA-175, was used as a control for RBC binding. rPfF2 shows Ca²⁺ independent binding to human RBCs. (i) Oligomerization of rPfPLP1. rPfPLP1 is incubated with and without human RBCs at 37 °C for 30 min and analyzed for oligomeric rPfPLP1 by western blotting. Oligomeric PfPLP1 (>1,000 kDa) was detected only upon incubation with human RBC that runs much higher than monomeric PfPLP1, suggesting the presence of more than 15 molecules in the oligomer. Four high-molecular-weight bands were detected (marked by arrow) in the oligomerized fraction indicating heterogeneity of PfPLP1 oligomeric complexes.