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 Plasmodi um falciparum schizonts, which recognize parasite proteins of 130 kDa and 2 0 kDa. The 130-kDa protein was released by alkaline sodium carbonate treatm ent, suggesting that the protein is a peripheral membrane protein, while th e 20-kDa protein remained associated with the membranes following alkali tr eatment, suggesting it may be an integral membrane protein. Both proteins w ere localized to large cytoplasmic vesicles within the cytoplasm of trophoz oite and schizont-infected erythrocytes by immunofluorescence assay and con focal microscopy, Both proteins colocalized with Bodipy-ceramide in trophoz oite and immature schizont-infected erythrocytes, but not in segmenters. Th e 130-kDa protein was localized by immunoelectron microscopy (IEM) to Maure r's clefts underneath knobs in a knobby and cytoadherent (K+/ C+) P. falcip arum strain. No IEM reactivity was obtained in a knobless and non-cytoadher ent (K-/C-) parasite strain. We investigated stage-specific protein express ion and protein localization by indirect immunofluorescence assay. Bodipy-c eramide colocalization assays with Rhop-3 and serine-rich antigen (SERA)spe cific antibodies were performed. A similar colocalization in trophozoites a nd schizonts was obtained using the rhoptry-specific antibody 1B9 reactive with the 110-kDa Rhop-3 protein. In segmenters, unlike trophozoites and imm ature schizonts, there was no Bodipy-ceramide colocalization with antibody 1B9. A difference in protein colocalization was seen using specific antibod y 152.3F7.1.1, reactive with SERA. Antibodies to SERA colocalized with Bodi py-ceramide in schizonts, including segmenters. Collectively the data sugge st that Rhop-3 transits through the intracellular network en route to the r hoptries and both vesicle-specific proteins may function in the intracellul ar network.