Function and mechanism of O-fucosylation of malaria parasite TSR-domain proteins

描述（由申请人提供）：血小板反应蛋白1型重复序列（TSR）结构域在疟疾寄生虫恶性疟原虫的整个生命周期中在滑行运动、宿主细胞识别和入侵中发挥重要作用。这些结构域存在于蛋白质中，这些蛋白质在寄生虫从人类传播到蚊子和返回期间特别重要。本项目的目的是探索和表征恶性疟原虫关键分子TSR结构域的O-岩藻糖基化。正如在不同生物体中所描述的，TSR结构域通常被蛋白质-O-岩藻糖基转移酶2（PoFUT 2）岩藻糖基化，这种修饰是含TSR蛋白质的最佳折叠和分泌所必需的。此外，TSR结构域上的O-岩藻糖基化共有序列与其配体结合基序一致，表明O-岩藻糖可以改变TSR结构域的配体结合亲和力。PoFUT 2同源物由恶性疟原虫保守并表达，并且GDP-岩藻糖（O-岩藻糖基化反应的底物供体）由寄生虫主动合成并掺入。连同通过蛋白质组学分析检测唾液腺子孢子中的O-岩藻糖基化机制，证据强烈指向恶性疟原虫中PoFUT 2介导的O-岩藻糖基化机制的保守性。我们建议（1）通过表征两种内源性表达的和必需的含TSR的蛋白质（分别为环子孢子和TRAP相关蛋白，CTRP;环子孢子蛋白，CS）来探索这些推定的翻译后修饰;和（2）通过表型分析O-岩藻糖基化无效突变体在动合子和子孢子阶段来评估TSR修饰的生物学意义。TSR结构域是疟疾中宿主-寄生虫相互作用所必需的。对恶性疟原虫TSR翻译后修饰机制的深入了解将最终为未来探索糖基化在疟疾寄生虫生物学中的基本作用奠定基础。

DESCRIPTION (provided by applicant): Thrombospondin type-1 repeat (TSR) domains play essential roles in gliding motility, host-cell recognition and invasion throughout the life cycle o the malaria parasite, Plasmodium falciparum. These domains are present in proteins that are particularly important during parasite transmission from humans to mosquitoes and back. The aim of this project is to explore and characterize the O- fucosylation of TSR domains of critical P. falciparum molecules. As it has been described across diverse organisms, TSR domains are commonly fucosylated by the protein-O-fucosyltransferase 2 (PoFUT2) and this modification is required for optimal folding and secretion of TSR-containing proteins. Furthermore, the O-fucosylation consensus sequence on TSR domains coincides with its ligand-binding motif, suggesting that O-fucose may alter ligand-binding affinities of TSR-domains. A PoFUT2 homolog is conserved and expressed by P. falciparum, and GDP-fucose, the substrate donor of O-fucosylation reactions, is actively synthesized and incorporated by the parasite. Together with the detection of the O-fucosylation machinery in salivary gland sporozoites by proteomic analyses, the evidence strongly point to the conservation of a PoFUT2 mediated O-fucosylation mechanism in P. falciparum. We propose to (1) explore these putative posttranslational modifications by characterizing two endogenously expressed and essential TSR-containing proteins in the ookinete and sporozoite stages (the Circumsporozoite and TRAP-related protein, CTRP; and the Circumsporozoite protein, CS, respectively) and (2) evaluate the biological significance of TSR modification by phenotyping O- fucosylation null mutants in the ookinete and sporozoite stages. TSR domains are essential for host- parasite interactions in malaria. A deeper insight into a mechanism of posttranslational modification of P. falciparum TSR will ultimately lay the foundation for future exploration into the fundamental role of glycosylation in malaria parasite biology.