Distinct Protein Classes Including Novel Merozoite Surface Antigens in Raft-like Membranes of Plasmodium falciparum

Abstract

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature bloodstage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPIanchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys 6 ) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys 6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.Developing a vaccine to control human malaria is a global health priority. The recent availability of the genome sequence of the protozoan parasite Plasmodium falciparum, the major cause of malaria, allows the use of genomic technologies such as microarray and proteomics to identify novel vaccine and drug targets (1-5). Most membrane proteins that coat the surface of the erythrocyte-invasive merozoite form of the parasite are attached to the plasma membrane via a C-terminal glycosylphosphatidylinositol (GPI) 5 anchor. To date, four GPI-anchored merozoite surface proteins (MSP-1, -2, -4, and -5) have been identified, and two others (MSP-10 and rhoptry-associated membrane antigen (RAMA)) appear to reside at least in part in organelles at the apical end of the parasite (6 -10). Another protein originally localized to the merozoite surface, MSP-8, appears to instead reside in the ring stage (11). Most other merozoite surface proteins (e.g. MSP-3/6 family members, MSP-7, and acidic basic repeat antigen (ABRA)) are not directly membrane-associated but are indirectly linked to the surface, probably in most cases via interactions with GPI-anchored proteins (12-15). In contrast to the apical and peripheral classes of bloodstage antigens, the GPI-anchored proteins appear to be essential to blood-stage growth with repeated attempts to genetically disrupt six GPI-anchored merozoite proteins resulting in only one "knock-out," that of the msp-5 gene (16). 6 This, together with considerable data highlighting their potential as targets of protective antibodies, place...

Structure of thePlasmodium6-cysteine s48/45 domain

“…Pf12, Pf38, and Pf41 are readily recognized by immune sera from infected patients from endemic areas (10,25,26); however, no function has been ascribed to any of them yet. The first step in invasion of merozoites into red blood cells is the binding of any part of the merozoite to the red blood cell surface.…”

“…Because Pf41 has no GPI anchor signal or transmembrane domain for attachment to the membrane, it is likely to bind another merozoite surface protein. Pf41, Pf38, and Pf12 are strongly recognized by immune sera from naturally infected patients, and Pf38 and Pf92 are under balancing selection (10,(25)(26)(27). Attempts to delete Pf92 have been reportedly unsuccessful; however, no information for the deletion of the other genes is available (28).…”

“…Each member has a signal sequence and three of them are GPI-anchored to the merozoite surface (10,14). Because Pf41 has no GPI anchor signal or transmembrane domain for attachment to the membrane, it is likely to bind another merozoite surface protein.…”

“…Conserved homologs are present in the genomes of other Plasmodium species. Most of the proteins containing the s48/45 domain are expressed on the surface of the parasite (8)(9)(10)(11). Pfs48/45 and Pfs230 are current targets of malaria transmissionblocking vaccine development (12,13).…”

Diversity of extracellular proteins during the transition from the ‘proto-apicomplexan’ alveolates to the apicomplexan obligate parasites

“…The 6-cys domain proteins are related to the Toxoplasma and Eimeria GPI-linked surface coat SAG proteins, and have been proposed to have originated via lateral transfer of metazoan ephrin proteins (Gerloff et al 2005;Arredondo et al 2012;Reid et al 2014). Ten members of the family are present in Plasmodium and appear to have roles in multiple stages of the lifecycle, including hepatocyte and intraerythrocytic stages (Ishino et al 2005;Sanders et al 2005;Annoura et al 2014).…”