Figure 1.
Both the PfRh2a and PfRh2b proteins are processed to release an 85 kDa product into culture supernatants.
(A) Schematic representation of the antibodies used in this study and the approximate location of the processing event leading to a common 85 kDa fragment and unique 285 kDa PfRh2a and 297 kDa PfRh2b products. The C-terminal transmembrane domains of both the PfRh2a and PfRh2b proteins are shown. Antibodies prefaced with the letter ‘R’ are Rabbit antibodies while the other antibodies are Mouse monoclonals. (B) Culture supernatants contain unprocessed and large processed fragments for both PfRh2a and 2b proteins. Western blots probed with six antibodies recognising a PfRh2 common region (3A2, R1171), Rh2a-specific (8F9, R1088) and PfRh2b-specific (4B7, R1090) regions are shown. The 3A2 and R1171 antibodies detect all four PfRh2a and PfRh2b processed and unprocessed proteins in 3D7 supernatants. The 4B7 and R1090 antibodies detect the processed 297 and unprocessed 382 kDa PfRh2b proteins. The 8F9 and R1088 antibodies detect the processed 285 and unprocessed 370 kDa PfRh2a proteins. Apart from weak reactivity with both unprocessed and processed PfRh2b proteins with the 4B7 and R1090 antibodies, no proteins from FCR3 supernatants were detected. As a loading control, EBA-175 antibodies were used to probe the same blots. (C) Only N-terminal antibodies detect the 85 kDa processed product both in culture supernatants and schizonts. Four different N-terminal antibodies detect the 85 kDa processed product in 3D7-derived supernatants and schizonts but not in FCR3 supernatants. The same blots were reprobed with EBA-175 antibodies as a loading control. (D) Four different N-terminal antibodies immunoprecipitate the 85 kDa product. 3D7 culture supernatants were immunoprecipitated with all four N-terminal antibodies. Immunoprecipitates derived from rabbit antibodies were probed with mouse antibody 6F12 and immunoprecipitates derived from mouse antibodies were probed with both rabbit antibodies.
Figure 2.
Processing of the 85 kDa fragment is brefeldin A-sensitive.
(A) Sensitivity of the production of the 85 kDa fragment to BFA and E64. Synchronised 3D7 schizont cultures were treated with BFA at 5 ug/ml (methanol as control) or E64 at 10 µM (water as control) for 1 h before resuspending in methionine/cysteine-free medium and [35S]methionine/cysteine for a further 1 h with inhibitors as before. Labelled parasites were then lysed and normal rabbit serum [(−) control] or R1170 anti-85 kDa fragment antibody used for immunoprecipitations. The 85 kDa band is arrowed while two cross-reacting bands at ∼140 and ∼220 kDa are asterisked. (B) The level of the 85 kDa fragment peaks at a 15 min chase time. 3D7 schizonts were labelled with [35S]methionine/cysteine for 10 min, then chased for 0, 5, 15, 30 and 60 min. Samples were immunoprecipitated with normal rabbit serum or R1170. The right panel shows 0 min samples probed with an antibody to the 85 kDa fragment (6F12).
Figure 3.
The 85 kDa processed form of PfRh2a and PfRh2b colocalises with the corresponding C-terminal regions in both merozoite and schizont stages.
(A) The 85 kDa product co-localises with the C-terminal PfRh2a product. Free merozoites or schizonts of the 3D7 parasite were dual stained with 8F9 anti-PfRh2a and R1170 anti-85 kDa antibodies. Both merozoites and schizonts give an apical staining pattern with both antibodies, indicating co-localisation. Nuclei were stained with DAPI. (B) The 85 kDa product co-localises with the C-terminal PfRh2b product. Parasites were dual stained with 4B7 anti-PfRh2b and R1170 anti-85 kDa antibodies. As for (A), both antibodies give a co-localised apical staining pattern. (C) The 85 kDa product co-localises with both the PfRh2a and PfRh2b C-terminal products. Parasites were dual stained with 3A2 anti-PfRh2a/2b and R1170 anti-85 kDa antibodies. As for (A), both antibodies give a co-localised apical staining pattern.
Figure 4.
Both PfRh2a and PfRh2b proteins form a complex with the 85 kDa processed product.
(A) Proteins from synchronised 3D7 schizont stage parasites were immunoprecipitated with either R2A9 Abs or normal rabbit serum [(−) control]. Immunoprecipitated proteins were separated by SDS-PAGE and probed with either 6F12 or 3A2 antibodies. The left panel shows that the R2A9 immunoprecipitate also brings down the 85 kDa processed product as detected with the 6F12 antibody, indicating that in 3D7 schizonts, the 85 kDa product is complexed with both the 285 kDa PfRh2a protein and the 297 kDa PfRh2b protein, as shown in the Right panel. (B) Proteins from 3D7 culture supernatants were immunoprecipitated, separated by SDS-PAGE and probed as in (A) above. The result shows that the 285/85 kDa PfRh2a and the 297/85 kDa PfRh2b complexes still remain in supernatant following invasion. (C–D) In order to confirm that both PfRh2a and PfRh2b were capable of complex formation, proteins from culture supernatants of both 3D7Δ2a and 3D7Δ2b parasites were immunoprecipitated as before. The results show that both PfRh2b (C) and PfRh2a (D) can form a complex with the 85 kDa processed product.
Figure 5.
C-terminal tagging of the PfRh2a protein in P. falciparum by transfection.
(A) The plasmid for tagging PfRh2a and PfRh2b with 3×HA epitopes by 3′ crossover recombination is shown. The human dihydrofolate reductase-thymidylate synthase (hdhfr) gene encodes resistance to WR99210. The whole cassette that includes a promoter and terminator is labelled as hdhfr. The P.berghei dhfr 3′ terminator region (PbDT 3′) served as the terminator for the gene being tagged. (B) Pfrh2a is tagged with 3HA. Proteins from schizont stages of the W2mef parasite or the line expressing tagged PfRh2a (Rh2aHA) were separated by SDS-PAGE then probed with either anti-HA Abs (Left panel) or the 8F9 antibody to the PfRh2a protein (Right panel). The anti-HA antibody detect two high molecular weight proteins specifically in the tagged line, a processed protein at ∼295 kDa and the unprocessed protein at ∼380 kDa. The protein detected at ∼150 kDa is a cross-reacting protein found also in the untagged wild-type parasite. (C) PfRh2a is C-terminally cleaved at invasion and the resulting stub carried into the Ring. The schematic diagram shows the C-terminal cleavage, most likely due to a rhomboid protease, occurring within the transmembrane (TM) region of Rh2aHA parasites. The HA-tagged cytoplasmic tail (CT) is also shown. A synchronised mixed schizont/Ring-stage culture, containing Rings <4 h old, was sorbitol-synchronised to remove schizonts. The young Rings were then lysed either with saponin or TNET, spun down, and both supernatant and pellet fractions collected. HA-containing proteins from both supernatant fractions were immunoprecipitated with anti-HA Abs. Immunoprecipitated proteins together with proteins from the saponin and TNET pellets were separated by SDS-PAGE, blotted, then probed with anti-HA antibodies. The C-terminally processed tail of ∼18 kDa was detected in both the saponin and TNET extracted ring fraction but not in the saponin-lysed pellet fraction. A cross-reacting protein at ∼25 kDa was seen only in the saponin fractions. (D) The HA-tagged stub is carried into ring stages. Young rings less than 4 h old and schizonts of the Rh2aHA line were dual stained with 12CA5 anti-HA (Roche, Germany) and a common region antibody (R2A11, Figure 1). The PfRh2a/b ecto-domain is not carried into rings, while the HA-tagged stub is carried into rings. Nuclei were stained with DAPI.
Figure 6.
Immuno-electron microscopy localisation of PfRh2a and PfRh2b during merozoite invasion of human erythrocytes.
(A) The PfRh2a/2b proteins localise to the rhoptry neck and moving junction of merozoites. Transmission electron micrographs of pre- and mid-invasion merozoites showing PfRh2a/b-specific immunogold labelling (white arrows). At the pre-invasion stage, PfRh2a/b localises to the rhoptry neck (inset), while during mid-invasion, PfRh2a/b localises both to the moving junction and to the merozoite surface within the invasion pit. Scale bar = 0.2 µm. (B) The EBA-175 protein localises to the micronemes and apical tip of merozoites. At the pre-invasion stage, EBA-175 is located within micronemes (inset), while during mid-invasion, EBA-175 can be located either at the apical tip or within the invasion pit. Scale bar = 0.2 µm.
Figure 7.
The PfRh2a/b 85 kDa processed product binds erythrocytes and binding is inhibited by antibodies to the binding domain.
(A) Culture supernatant from both 3D7 and W2mef parasites was bound to untreated (Unt.), Low trypsin (0.067 mg/ml), High Trypsin (1 mg/ml), neuraminidase (N) or chymotrypsin-treated (C) erythrocytes. Bound proteins were eluted with 1.5 M NaCl, separated on SDS-PAGE gels, Western blotted and probed with an antibody to the 85 kDa product (6F12) and an antibody to EBA-175. PfRh2a/b binding to erythrocytes was sensitive to neuraminidase and chymotrypsin, but resistant to both Low and High Trypsin concentrations. The input culture supernatant is labelled as ‘supn’. The lower panels probed with anti-EBA-175 Abs were included as a control for erythrocyte enzyme treatments as it is known that EBA-175 binding is neuraminidase sensitive, partially trypsin sensitive, but chymotrypsin resistant. (B) R1170 antibodies block native PfRh2a/b binding. Protein G-purified R1070, R1170 or normal rabbit serum antibodies at final concentrations from 0.1 to 1.0 µg/µl or the 1F10 monoclonal antibody at final concentrations from 0.1 to 0.5 µg/µl were preincubated with 3D7 culture supernatant before adding Untreated erythrocytes. Bound proteins were eluted with 1.5 M NaCl, separated on SDS-PAGE gels, blotted and probed with an antibody (6F12) to the 85 kDa PfRh2a/b binding domain.
Figure 8.
Recombinant rRh215 binds erythrocytes.
(A) Schematic diagram of the PfRh2a/b protein showing the location of the rRh215 and 2b1 fusion proteins. The rRh215 is located within the 85 kDa binding domain of PfRh2a/b. The processing event leading to the 85 kDa product is indicated by the arrow. The 2b1 fusion protein is from a PfRh2b unique region at the C-terminus of the protein. The regions of the protein in black at the N and C-termini represent the signal sequence and transmembrane domains respectively. (B) 1 µg rRh215 (native) or 1 µg denatured rRh215 (boiled) were bound to untreated erythrocytes. Bound proteins were eluted with 1.5 M NaCl, separated on SDS-PAGE gels, blotted and probed with an antibody (R1170) to the rRh215 fusion protein. Unbound proteins removed from the Untreated erythrocytes are also shown. (C) 1 µg rRh215 was bound to untreated (Unt.), Low trypsin (0.067 mg/ml), High Trypsin (1 mg/ml), neuraminidase (N) or chymotrypsin-treated (C) erythrocytes. Bound proteins were eluted, separated and probed as in B above. In the lower panel, both enzyme-treated and untreated erythrocytes were also probed with an antibody to EBA-175 to ensure the enzyme treatments had been successful. Recombinant rRh215 binding to erythrocytes was partially sensitive to neuraminidase and chymotrypsin, but resistant to both Low and High Trypsin concentrations. Unbound proteins removed from the Untreated erythrocytes are also shown. The 2b1 fusion protein was bound to untreated erythrocytes. Bound proteins were eluted with 1.5 M NaCl, separated on SDS-PAGE gels, Western blotted and probed with the 4B7 antibody raised to the 2b1 fusion protein. The 2b1 fusion protein showed no binding to Untreated erythrocytes but was clearly present in the Unbound fraction. (D) R1170 antibodies block binding of rRh215 to erythrocytes. Protein G-purified R1170 antibodies at final concentrations from 0.03 to 0.5 µg/µl were preincubated with 0.5 µg rRh215 fusion protein before adding Untreated erythrocytes. Bound proteins were eluted with 1.5 M NaCl, separated on SDS-PAGE gels, Western blotted and probed with Protein G-purified R1170.
Figure 9.
Antibodies to rRh215 block merozoite invasion.
(A) R1170 antibodies to rRh215 block invasion of both untreated and Low trypsin-treated erythrocytes. Protein G-purified IgG at 2 mg/ml final concentration from both R1070 and R1170 prebleeds and kill bleed sera were added to 3D7 parasites at the trophozoite stage together with target erythrocytes that were untreated or Low trypsin (0.067 mg/ml)-treated. Following reinvasion in the presence of antibodies, cultures were continued to the trophozoite stage, when parasite numbers were determined in order to see the effect of antibodies on invasion. Percent invasion in the absence of antibodies was adjusted to 100% invasion. Experiments were done at least twice in triplicate. Error bars show the standard error of the mean. P-values were calculated using the Students t-test. (B) Antibodies to rRh215 block invasion mediated by PfRh2b but not PfRh2a in 3D7 parasites. Protein G-purified IgG from R1170 serum at 2 mg/ml final concentration was added to 3D7Δ2a (express PfRh2b only), 3D7Δ2b (express PfRh2a only) and FCR3 (express neither PfRh2a nor PfRh2b) parasites at the trophozoite stage together with target erythrocytes that were untreated or treated with 0.03 mg/ml Trypsin. Other details of the experiments were the same as in (A) above.
Figure 10.
Schematic diagram illustrating post-Golgi and rhomboid processing of PfRh2a/2b proteins.
(A) Full-length PfRh2a/2b proteins are initially processed in a BFA-sensitive post-Golgi compartment to produce the 85 kDa fragment. The proteins are then transported to the apical tip, where following invasion, processing within the transmembrane domain, most likely by PfROM4, occurs. (B) Schematic diagram illustrating complex formation and the fate of various PfRh2a/2b processed products. Full-length PfRh2a and PfRh2b proteins are predicted to be 370 and 382 kDa respectively. Cleavage to produce the 85 kDa product and its subsequent complexing with the transmembrane-containing PfRh2a and PfRh2b products, which occurs at the schizont stage, is shown. Following invasion, C-terminal rhomboid processing results in both complexed and uncomplexed forms being shed into culture supernatant while the small processed stub of ∼7 kDa in wild-type untagged parasites is carried into the ring stage. A small amount of full-length unprocessed proteins (370/382 kDa) found both in schizonts and in supernatants following invasion, is shown in faint outline.