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

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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.