The roles of the glycosylphosphatidylinositol anchor on the production and immunogenicity of recombinant ookinete surface antigen Pbs21 of Plasmodium berghei when prepared in a baculovirus expression system

Abstract

Malarial ookinetes express an immunodominant surface protein (P28) that is a priority candidate for the development of transmission-blocking vaccines. The full length P28 gene from Plasmodium berghei [Pbs21(1-213)] and a deletion construct [Pbs21(1-188)] encoding a protein that lacks the 25 C-terminal amino acids, including the glycosylphosphatidylinositol (GPI) anchor signal, were expressed in insect cells using baculovirus vectors. Pbs21(1-213) protein is strongly hydrophobic, found in the cytoplasm and on the surface of Spodoptera Sf21 cells, and in the culture medium. Pbs21(1-188) protein was largely found in the aqueous phase of the medium and in the cytoplasm of Sf21 cells, but was not detected on the cell surface. The presence of 25 C-terminal amino acids is therefore critical to the attachment of recombinant Pbs21 to the parasite plasma membrane. Mice were immunized subcutaneously or intramuscularly with affinity purified recombinant Pbs21(1-213), Pbs21(1-188) or native Pbs21 proteins. Following two immunizations, native Pbs21 induces higher titres when administered by either route, than the recombinant protein bearing an insect GPI anchor, which in turn is markedly more immunogenic than the recombinant polypeptide lacking a GPI anchor. When specific anti Pbs21 antibody titres exceeded 1 mg/ml all three antigens were capable of inducing transmission blockade > or = 90%, below 1 mg/ml blockade did not correlate with antibody concentration.

Application of Synthetic 8-Kd and Recombinant Gp50 Antigens in the Diagnosis of Neurocysticercosis by Enzyme-Linked Immunosorbent Assay

“…33,34 For native GP50, a glycosyl-phosphatidylinositolanchored protein, the anchor may play a role in its antigenicity. [35][36][37][38][39] The recombinant protein was expressed without the anchor attachment sequence. For sTS18var1, which is chemically synthesized, it may be the lack of glycosylation, or another post-translational modification, that is impacting upon its sensitivity compared with the native 8-kD proteins.…”

Effect of GPI anchor moiety on the immunogenicity of DNA plasmids encoding the 19‐kDa C‐terminal portion of Plasmodium falciparum MSP‐1

“…These observations agree with the results of previous studies. Martinez et al have shown that recombinant P. berghei sexual-stage protein, Pbs21, expressed in a baculovirus system as a GPI anchored protein, was markedly more immunogenic than the recombinant protein lacking GPI-moiety (25). Wang et al demonstrated that plasmids encoding P. yoelii MSP-4/5 as GPI-anchored protein induced higher level of antibodies compared to the plasmid expressing MSP4/5 as transmembrane protein (19).…”

Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites

“…The gametocytes are known to harbor many transcripts that while in the human host are rapidly translated into proteins following gametogenesis in the mosquito, which occurs within a 10–15 min time period, followed by macrogamete fertilization to form the mosquito-infective ookinete stage. Fertilized zygotes and motile ookinetes express two major GPI-anchored proteins, P25 and P28 [37]. We hypothesize that sugar nucleotide pools are ramped-up and stored in preparation for gametogenesis inside the mosquito vector.…”