_____ Thrombospondin type-1 repeats (TSRs) are small, adhesive protein domains that are found in phyla as diverse as chordates, nematodes, and apicomplexans. TSR-bearing proteins, which are typically localized to the cell surface or extracellular matrix, serve a wide variety of functions, including cell attachment and motility. TSRs are usually glycosylated at highly conserved motifs wherein tryptophans may be modified with a C-linked mannose and a conserved serine or threonine residue may be modified with an O-linked fucose. Parasites of the genus Plasmodium, the causative agent of the disease malaria, express ten conserved TSR- bearing proteins at various stages throughout their life cycle. Disrupting any one of them severely disrupts or halts parasite development. The most extensively studied of the TSR-bearing proteins are the sporozoite surface proteins CSP and TRAP. It has been shown that the TSRs in these proteins contain motifs that bind to proteoglycans and thus play a role in parasite invasion of host cells. We have recently used mass spectrometry to demonstrate for the first time that the TSRs of CSP and TRAP are glycosylated in vivo. This discovery revealed a gap in the understanding of these otherwise well-studied invasins: TSR-bearing proteins are critical to the Plasmodium life cycle, yet nothing is known about the role of glycosylation in maintaining their function or virulence. We hypothesize that the O-fucosylation and C-mannosylation of TSRs in Plasmodium is required for the proper function of the glycosylated proteins, and we predict that preventing glycosylation of these proteins will inhibit their function and disrupt the Plasmodium life cycle. In order to test this hypothesis, we will first characterize the glycosylation status of all TSR-bearing proteins expressed in P. falciparum. We will then generate transgenic parasite lines with mutations that prevent modification of the TSRs of specific proteins in order to determine the role of glycosylation in the function of these virulence factors.
_____ Malaria remains one of the great global health problems today, with nearly half a million deaths and millions of new infections occurring annually. This research will determine how the malaria parasite specially modifies a small number of its most important proteins in order to enhance their function. Understanding these modifications will help to improve the design of vaccines that target these proteins in order to arrest parasite development and prevent disease.
