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

恶性疟原虫疟疾寄生虫的侵入形式在其顶端具有称为Rhoptry的微细胞器，红细胞结合蛋白RhopH复合物（包括RhopH 1、RhopH 2和RhopH 3）从该微细胞器排出，并且由于破坏基因座的尝试失败而被认为具有重要作用。我们发现该复合物通过蛋白质和GPI锚定依赖的方式结合红细胞表面，但其确切机制尚不清楚。因此，在本项目中，我们产生了基因操纵的恶性疟原虫，其表达与GFP融合的RhopH复合物的一部分，并询问每个嵌合蛋白是否具有红细胞结合能力。为此，我们产生了4种具有质粒的寄生虫系，所述质粒在棒状蛋白启动子下表达1/3的RhopH 1、2/3的RhopH 1、与GFP融合的全长RhopH 1或单独的GFP。然而，我们只能观察到GFP信号从寄生虫的1/3的RhopH 1和GFP只有。在RhopH 1的2/3或全长的寄生虫中未检测到GFP信号的原因尚不清楚，但我们推测这可能是因为相对较大的质粒大小。有趣的是，表达仅与RhopH 2的信号肽融合的GFP的寄生虫显示GFP的棒状体定位。这表明，信号肽的蛋白质的默认目的地可能是多细胞器之间的棒状体。针对RhopH 2的免疫沉淀没有共纯化1/3的RhopH 1加GFP，表明该部分不负责RhopH复合物的形成。此外，我们无法观察到RhopH 1嵌合蛋白的这1/3的红细胞结合能力，这表明该区域可能不是结合结构域。

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.