The biosynthesis and post-translational modification of Pbs21 an ookinete-surface protein of Plasmodium berghei

Radiolabelled methionine incorporation into synchronised Plasmodium berghei gametocytes or ookinete cultures, showed that Pbs21 is not synthesised in bloodstage parasites; synthesis was detected within three hours of inductio n of gametogenesis; synthesis was triggered at gametogenesis, not by fertil isation. We show native Pbs21 to be a hydrophobic membrane protein that was insensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC), but sensitive to alkaline hydroxylamine, and partially sensitive to glycosy lphosphatidylinositol-dependent phospholipase D (GPI-PLD) and HNO2. H-3-myr istic and palmitic acid, H-3-glucosamine and mannose incorporation indicate d Pbs21 was acylated and glycosylated. Linkage of the acyl group was sensit ive to HNO2, which released an acyl-phosphatidylinositol more hydrophobic t han that released from P3 of Trypanosoma brucei. All these properties are c onsistent with the presence of a malaria-specific glycosylphosphatidylinosi tol (GPI) anchor. In contrast recombinant Pbs21 (rPbs21), expressed in Spod optera frugiperda cells, was sensitive to both PI-PLC and GPI-PLD, consiste nt with the protein being modified by a different (S. frugiperda) GPI ancho r. Brefeldin A blocked secretion of rPbs21 within a cytoplasmic reticular c ompartment. Following deletion of the putative GPI anchor addition site (am ino acids 189-213), the protein was transported to the cell surface and sec reted directly into the aqueous phase of the culture medium. Deletion of am ino acids 205-213 disrupted Pbs21 processing, transport through the ER and distribution onto the cell, surface. Deletion of amino acids 1-28 prevented transport of Pbs21 into the ER. This suggests that correct processing of t he GPI anchor in the ER-Golgi network is essential for the successful secre tion of the recombinant protein, which is additionally dependent upon an N- terminal secretory signal sequence. (C) 1999 Elsevier Science B.V. All righ ts reserved.