Function and Mechanism of O-Fucosylation of Malaria ParasiteTSR Domain Proteins

描述（由申请人提供）：血小板反应蛋白1型重复序列（TSR）结构域在恶性疟原虫（Plasmodium falciparum）整个生命周期的滑动运动、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，在寄生虫从人类传播到蚊子再传播到蚊子的过程中尤为重要。本项目的目的是探索和表征恶性疟原虫关键分子的TSR结构域的O-聚焦。正如在不同生物中所描述的那样，TSR结构域通常被蛋白- o -聚焦转移酶2 （PoFUT2）聚焦，这种修饰是含TSR蛋白的最佳折叠和分泌所必需的。此外，TSR结构域上的O-聚焦一致序列与其配体结合基序一致，表明O-聚焦可能改变TSR结构域的配体结合亲和力。PoFUT2同源物被疟原虫保守表达，而GDP- focus作为O-聚焦反应的底物供体，被疟原虫积极合成并结合。再加上通过蛋白质组学分析在唾液腺孢子体中检测到O-聚焦机制，这些证据有力地指出了PoFUT2介导的O-聚焦机制在恶性疟原虫中的保守性。我们建议：(1)通过表征卵母体和孢子子阶段的两种内源性表达和必需的含TSR的蛋白质（环孢子子和trp相关蛋白，CTRP和环孢子子蛋白，CS）来探索这些可能的翻译后修饰；(2)通过对卵母体和孢子子阶段的O-聚焦零突变体进行表型分析来评估TSR修饰的生物学意义。TSR结构域对疟疾中宿主-寄生虫相互作用至关重要。深入了解恶性疟原虫TSR翻译后修饰的机制，将最终为进一步探索糖基化在疟原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.