An EGF-like Protein Forms a Complex with PfRh5 and Is Required for Invasion of Human Erythrocytes by Plasmodium falciparum
Figure 2
PfRh5 and PfRipr form a complex.
(A) A triple Haemaglutinin (HA) tag was added to the C-terminus of PfRipr by 3′ single homologous crossover recombination. (B) HA-tagged PfRiprHA was detected in the parasite membrane pellet and in the culture supernatant of 3D7PfRiprHA line, confirming successful tagging. (C) PfRiprHA was analysed by SDS-PAGE under reducing and non-reducing conditions. Similar mobility, of the 65 kDa C-terminal fragment under both conditions, indicates that the N-terminal and C-terminal of PfRipr was not linked by disulphide bonds after processing. (D) Co-immunoprecitation of PfRh5 with PfRiprHA was performed using proteins solubilized from saponin pellets of 3D7-PfRiprHA schizont-infected erythrocytes. Immunoprecipitation of HA-tagged PfRipr by anti-HA antibody co-immunoprecipitated PfRh5. (E) Immunoprecitation with anti-PfRh5 antibody using the same schizont materials co-immunoprecipited PfRiprHA but not PfRh2a/b. (F) Co-immnuoprecipitation of PfRh5 with PfRiprHA from culture supernatants using anti-HA-Sepharose bead. Detection of PfRh5 but not PfRh2a or PfRh2b in the anti-HA bead bound material confirmed that PfRh5 was specifically co-immunoprecipitated with PfRiprHA. (G) Co-immunoprecipitation of PfRiprHA with PfRh5 from culture supernatants using monoclonal anti-PfRh5 antibody coupled to Mini-beads. Probing of the bound material with anti-HA antibody detected PfRiprHA only from the 3D7-PfRiprHA parasites and no PfRh2a/b was detected in the bound materials, indicating that PfRiprHA was specifically co-immunoprecipitated with PfRh5. (H) Immnunoprecipitation of culture supernatant from PfRiprHA parasites with rabbit anti-PfRh2a/b antibodies that recognize 85 kDa processed PfRh2a/b fragment did not pull-down PfRh5 or PfRipr. In all cases, the asterisk indicates a cross-hybridizing band corresponding to the heavy chain of IgG eluted from the antibody affinity beads. The arrows in Figure 2E, F and G indicate where 85 kDa PfRh2a/b band should appear.