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

• Published in: Molecular and biochemical parasitology Vol. 98; no. 2; pp. 163 - 173
• Main Authors: Alejo Blanco, A.Richard, Paez, Andres, Gerold, Peter, Dearsly, A.Louise, Margos, Gabriele, Schwarz, Ralph T., Barker, Guy, Rodriguez, Maria C., Sinden, Robert E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 25-01-1999
• Subjects: Acylation; Animals; Antigen; Baculoviridae - genetics; Baculovirus; Biological Transport; Brefeldin A; Cell Compartmentation; Gametogenesis; Glycosylation; Glycosylphosphatidylinositol; Glycosylphosphatidylinositols; Membrane Proteins - biosynthesis; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Ookinete; Plasmodium berghei; Plasmodium berghei - cytology; Plasmodium berghei - metabolism; Protein Processing, Post-Translational; Protozoan Proteins - biosynthesis; Protozoan Proteins - genetics; Protozoan Proteins - isolation & purification; Recombinant Proteins - biosynthesis; Spodoptera - cytology; Ookinete; Pfs25, Plasmodium falciparum sexual stage protein of 25 kDa; MSP, merozoite surface protein; Brefeldin A; TX-114, Triton X-114; GPI, glycosylphosphatidylinositol; IFAT, indirect fluorescent antibody test; Baculovirus; PI-PLC, phosphatidylinositol phospholipase C; Antigen; Plasmodium berghei; GPI-PLD, GPI dependent phospholipase D; MAb, monoclonal antibody; Pbs21, Plasmodium berghei sexual stage protein of 21 kDa; PCR, polymerase chain reaction; Glycosylphosphatidylinositol; aa, amino acids; ER, endoplasmic reticulum
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(98)00162-5

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 induction of gametogenesis; synthesis was triggered at gametogenesis, not by fertilisation. 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 glycosylphosphatidylinositol-dependent phospholipase D (GPI-PLD) and HNO
2.
3H-myristic and palmitic acid,
3H-glucosamine and mannose incorporation indicated Pbs21 was acylated and glycosylated. Linkage of the acyl group was sensitive to HNO
2, which released an acyl-phosphatidylinositol more hydrophobic than that released from P3 of
Trypanosoma brucei. All these properties are consistent with the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. In contrast recombinant Pbs21 (rPbs21), expressed in
Spodoptera frugiperda cells, was sensitive to both PI-PLC and GPI-PLD, consistent with the protein being modified by a different (
S. frugiperda) GPI anchor. Brefeldin A blocked secretion of rPbs21 within a cytoplasmic reticular compartment. Following deletion of the putative GPI anchor addition site (amino acids 189–213), the protein was transported to the cell surface and secreted directly into the aqueous phase of the culture medium. Deletion of amino 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 the GPI anchor in the ER-Golgi network is essential for the successful secretion of the recombinant protein, which is additionally dependent upon an N-terminal secretory signal sequence.