The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane.

Glycosylphosphatidylinositol (GPI) anchor protein modification in species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of spp. has been primarily studied in the asexual blood stage of and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [ H]-palmitic acid and [ H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of -inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the -inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.