A protective glycosylphosphatidylinositol-anchored membrane protein of Plasmodium yoelii trophozoites and merozoites contains two epidermal growth factor-like domains

Using sera from mice immunized and protected against Plasmodium yoelii mala ria, we identified a novel blood-stage antigen gene, pypag-2. The 2.1-kb py pag-2 cDNA contains a single open reading frame that encodes a 409-amino-ac id protein with a predicted molecular mass of 46.8 kDa. Unlike many charact erized plasmodial antigens, blocks of tandemly repeated amino acids are lac king in the pypAg-2 protein sequence. Recombinant pypAg-2, comprising the f ull-length protein minus the predicted N-terminal signal and C-terminal anc hor sequences, was produced and used to raise a high-titer polyclonal rabbi t antiserum. This antiserum was used to identify and characterize the nativ e protein through immunoblotting, immunoprecipitation and immunofluorescenc e assays. Consistent with the presence of a glycosylphosphatidylinositol an chor, pypAg-2 fractionated with the detergent phase of Triton X-114-solubil ized proteins and could be metabolically labeled with [H-3]palmitic acid. B y immunofluorescence, pypAg-2 expression was localized to both the trophozo ite and merozoite membranes. Similar to Plasmodium falciparum merozoite sur face protein 1, pypAg-2 contains two C-terminal epidermal growth factor (EG F)-like domains. Most importantly, immunization with recombinant pypAg-2 pr otected mice against lethal P. yoelii malaria. Thus, pypAg-2 is a target of protective immune responses and represents a novel addition to the family of merozoite surface proteins that contain one or more EGF-like domains.