Identification of the binding domain of Plasmodium falciparum erythrocyte binding protein.

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Invasive form of Plasmodium falciparum malaria parasite possesses microorganelle, called Rhoptry, at its apex, from which erythrocyte binding protein RhopH complex (comprising RhopH1, RhopH2, and RhopH3) is discharged and considered to have essential role, because of the failure of attempt to disrupt gene locus. We showed that this complex binds erythrocyte surface via protein and GPI-anchor dependent manner, but the precise mechanism is still unknown. Thus, in this project, we generated genetically manipulated Plasmodium falciparum, which express a part of RhopH complex fused with GFP and asked if each chimeric protein have erythrocyte binding ability. To this end, we generated 4 parasite lines possessing plasmids which would express 1/3 of RhopH1, 2/3 of RhopH1, full length of RhopH1 fused with GFP, or GFP alone under the rhoptry protein promoter. However, we could observe GFP signal only from parasite for 1/3 of RhopH1 and GFP only. The reason why GFP signal is not detected from parasites with 2/3 or full length of RhopH1 is unknown, but we speculate that this maybe because of the relatively large plasmid size. Interestingly, parasite expressing GFP fused with only signal peptide of the RhopH2 showed rhoptry localization of GFP. This indicates that the default destination of the protein with signal peptide could be rhoptry among multipel microorganelle. Immunoprecipitation against RhopH2 did not co-purify 1/3 of RhopH1 plus GFP, indicating this part is not responsible for the RhopH complex formation. Also, we could not observe erythrocyte binding ability for this 1/3 of RhopH1 chimeric protein, suggesting that this region may not be the binding domain.