恶性疟原虫SURFIN蛋白色氨酸富含区在蛋白转运和致病性的功能分析

The human malaria parasite, Plasmodium falciparum, modifies the red blood cells (RBCs) that it infects by exporting proteins to the host cell. Exported proteins which interact with the skeleton structure of iRBCs membrane are responsible for the functional and morphology changes of iRBCs, and are also major virulence factors of malaria parasites. Plasmodium falciparum SURFIN is a type I transmembrane protein encoded by a surf gene family, which consists of ten genes with two exon structures. SURFIN protein shows a sequence similarity with known parasite proteins that are exported to the surface of the parasite-infected red blood cell (iRBC). The extracellular region of SURFIN share similar structure with the extracellular cysteine-rich domain (CRD) with P. vivax VIR protein and the intracellular tryptophan-rich (WR) domain are related in sequence with P. knowlesi SICAvar, and P. falciparum Pf332 and PfEMP-1. Two members of this family, SURFIN4.2 and SURFIN4.1 were shown to be located on the surface of iRBC and Maurer's clefts, and after releasing of the merozoites, they were on apical end and surface of merozoites respectively. In our previous research work, we found that the transmembrane region of SURFINs is essential for the initial recruitment of the protein to the ER, and the following sorting step to the parasitophorous vacuole is determined by two independent signals located in the N-terminus 50 amino acids. TM region with the adjacent cytoplasmic region also contain information for the efficient recruitment to the ER and/or for the efficient translocation across the parasitophorous vacuole membrane. Notably, when a recombinant protein contains a WR region, the signal seems diffused in the iRBC cytosol and surface compared with that does not contain this region, which only located at Maurer's clefts. In a previous report, 260 aa within the intracellular region of Pf332 was identified to interact with actin. The intracellular region of SURFINs shared conserved motif WR with this region of Pf332. Thus the intracellular WR region of SURFINs may mediate iRBCs membrane interaction, which may highlight the pathogenesis function of SURFINs during P. falciparum infection. Thus, in current study, we initiated to analyze the function of the cytoplasmic tryptphan-rich (WR) domain of SURFIN. By generating a panel of recombinant protein using wheat-germ cell-free system, Plasmodium transfection, and gene disruption, we plan to identify the function of WR region of SURFINs in trafficking and intracellular stage pathogenesis.

恶性疟原虫通过大量原虫蛋白改变红细胞结构和功能。与红细胞骨架作用的原虫蛋白为其主要致病因子。恶性疟原虫SURFIN蛋白为I型跨膜蛋白，由surf多基因家族编码，含10个家族成员。其胞外段与间日疟原虫VIR蛋白的CRD结构相似；胞内段的WRD与诺氏疟原虫SICAvar，恶性疟原虫Pf332和PfEMP-1的WRD具有同源序列。SURFIN4.1和SURFIN4.2表达于感染红细胞茂氏点和其表面，在裂殖子阶段则位于表面和顶端复合体。SURFINs N端50个氨基酸，跨膜区和胞内尾可介导其茂氏点定位。WRD区域同时存在时，荧光信号分布于红细胞膜表面。此WRD区域与Pf332的WRD区域内的肌动蛋白连接序列高度保守。因此，SURFIN的WRD可能通过介导此蛋白与红细胞膜骨架蛋白相互作用在蛋白转运和红内期致病性过程中发挥作用。本研究聚焦于SURFIN的WRD区域，通过分子生物学方法探讨其功能作用。

恶性疟原虫感染红细胞表面的输出蛋白是疟疾致病性和致死性的病理基础。因此，亦是红内期发病阻断疫苗的理想候选抗原。然而，迄今为止，恶性疟原虫输出蛋白的转运机制的研究多集中于PEXEL阳性蛋白，而对于恶性疟原虫蛋白质谱中大量存在的PNEPs（PEXEL negative proteins）输出蛋白的研究尚处于起步阶段。本项目聚焦于表达在感染红细胞膜表面的PNEPs蛋白 — SURFINs蛋白家族，首次对SURFIN蛋白在茂氏点 — 感染红细胞胞浆/膜转位过程中的相互作用蛋白进行了研究。研究显示，SURFIN4.1蛋白的N端在转运过程中不断裂，且SURFIN4.1N-T-Cyt-GFP重组蛋白需线性结构通过PTEX转运子。含WRD2的SURFIN4.12MycN-T-Cyt-4.2WRD2-GFP可结合更多的红细胞膜骨架蛋白，如肌动蛋白（actin）和血影蛋白（spectrin）。体外重组蛋白表达实验明确了SUFRIN4.2/PvSTP2的色氨酸富含区（Tryptophan rich domains, WRDs）均可与红细胞外翻形膜（Inside-out vesicles, IOVs）相互作用，其解离常数值（Kd）为0.26 ~ 0.68 μM。深入研究发现，SURFIN4.2蛋白的第二个色氨酸富含区（WRD2）参与红细胞膜骨架成员：actin和spectrin特异性结合，其解离常数值（Kd）分别为5.16 μM和0.51 μM，最大结合率值（Bmax）分别为1.92 μM 和1.34 µM。同时，对SURFINs家族成员SURFIN1.1蛋白的研究显示，含胞外段（N-ter，CRD和Var区域），跨膜区（TM）和部分胞内段（Cyt）的SURFIN1.1重组蛋白（SURFIN1.1NCVT-Cyt-GFP）定位于茂氏点，提示内源性SURFIN1.1为原虫输出蛋白。深入研究显示，SURFIN1.1蛋白编码基因surf1.1敲除后不影响恶性疟原虫感染红细胞的粘附性和其它恶性疟原虫致病性相关基因的转录和表达。SURFINs蛋白结构与多种原虫主要致病抗原，如：PfEMP1和Pf332相似，探讨其WRDs在SURFINs蛋白转运和致病性过程中的作用将为有效红内期发病阻断疫苗的研制、开发提供新的依据。