Characterization of epitopes on the 25 kD protein of the macrogametes/zygotes of Plasmodium falciparum

Abstract

A sexual stage-specific protein of Plasmodium falciparum with a Mr of 25,000 is one of the target antigens of transmission-blocking antibodies. The contributions of tertiary structure and post-translational modifications (glycosylation and acylation) to the structure of the epitopes on this protein were the subject of detailed investigations. After modification of the three-dimensional structure and modification or cleavage of carbohydrate groups and linked fatty acids, the immunological reactivity was investigated by three different techniques: (i) immunoprecipitation of radiolabelled proteins, (ii) enzyme-linked immunosorbent assay (ELISA), and (iii) Western blotting. The results of the experiments indicate that the immunological reactivity of the major epitopes on the 25 kD protein, including the epitope involved in transmission-blocking immunity, are dependent on the tertiary structure of the protein and on the presence of linked fatty acids, but not on the presence or absence of carbohydrate groups.

Ookinete destruction within the mosquito midgut lumen explains Anopheles albimanus refractoriness to Plasmodium falciparum (3D7A) oocyst infection

“…The anti-Pfs25 mAb 32F71 used is apparently unable to recognise paraformaldehyde-fixed ookinetes (data not shown), consistent with previous reports that the epitope recognised by this mAb is conformation-dependent (Will Roeffen, Department of Medical Microbiology, Nijmegen Center for Molecular Life Science, the Netherlands, personal communication; Fries et al, 1989). …”

Recombinant Pfs25 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in experimental animals.

“…We chemically synthesized a gene for Pfs25 that encoded amino acids 22-190 of the natural 217-amino acid precursor protein (10), and thereby deleted the proposed NH2-terminal secretory signal sequence. Also deleted was the hydrophobic COOH-terminal region that has been proposed to serve as a signal for attachment of a glycosylphosphatidyl-inositol membrane anchor (10,11). Expression and secretion from yeast of this Pfs25 extracellular domain was achieved by fusion of the synthetic gene to DNA sequences encoding the yeast ol factor pheromone secretory signal/leader sequence (8).…”

Potent Malaria Transmission-Blocking Antibody Responses Elicited by Plasmodium falciparum Pfs25 Expressed in Escherichia coli after Successful Protein Refolding

“…First, we opted to employ alternate ways to solubilize inclusion bodies using brief exposure to high pH (29), and the second consideration was protein refolding to obtain Pfs25 in the appropriate conformation (11,22,38). It is well known that the correct pairing of cysteine residues in the protein has important consequences for protein folding, protein stability, and biological function.…”