A Plasmodium falciparum protein located in Maurer's clefts underneath knobs and protein localization in association with Rhop-3 and SERA in the intracellular network of infected erythrocytes

We report on the characterization of monoclonal antibodies against Plasmodium falciparum schizonts, which recognize parasite proteins of 130 kDa and 20 kDa. The 130-kDa protein was released by alkaline sodium carbonate treatment, suggesting that the protein is a peripheral membrane protein, while the 20-kDa protein remained associated with the membranes following alkali treatment, suggesting it may be an integral membrane protein. Both proteins were localized to large cytoplasmic vesicles within the cytoplasm of trophozoite and schizont-infected erythrocytes by immunofluorescence assay and confocal microscopy. Both proteins colocalized with Bodipy-ceramide in trophozoite and immature schizont-infected erythrocytes, but not in segmenters. The 130-kDa protein was localized by immunoelectron microscopy (IEM) to Maurer's clefts underneath knobs in a knobby and cytoadherent (K+/C+) P. falciparum strain. No IEM reactivity was obtained in a knobless and non-cytoadherent (K-/C-) parasite strain. We investigated stage-specific protein expression and protein localization by indirect immunofluorescence assay. Bodipy-ceramide colocalization assays with Rhop-3 and serine-rich antigen (SERA)-specific antibodies were performed. A similar colocalization in trophozoites and schizonts was obtained using the rhoptry-specific antibody 1B9 reactive with the 110-kDa Rhop-3 protein. In segmenters, unlike trophozoites and immature schizonts, there was no Bodipy-ceramide colocalization with antibody 1B9. A difference in protein colocalization was seen using specific antibody 152.3F7.1.1, reactive with SERA. Antibodies to SERA colocalized with Bodipy-ceramide in schizonts, including segmenters. Collectively the data suggest that Rhop-3 transits through the intracellular network en route to the rhoptries and both vesicle-specific proteins may function in the intracellular network.