Plasmodium falciparum: hetero-oligomeric complexes of rhoptry polypeptides

We have previously described several monoclonal antibodies (McAbs) which specifically recognize antigens in the rhoptries of Plasmodium falciparum and which immunoprecipitate polypeptides of 82, 70, 67, 39, and 37 kDa. We now show that only p82, p70, p67, and a 86-kDa precursor (Pr86) of p82 possessed epitopes for these McAbs. These four proteins were not synthesized until schizogony. These results and proteolysis experiments indicated that Pr86, p82, p70, and p67 were the products of the same gene, whereas the dissimilar digestion patterns of p39 and p37 suggested that p39 was encoded by a second gene and p37 by yet another. Complexes of these proteins (termed RI complexes) are maintained by noncovalent interactions since the ionic detergent SDS was sufficient to dissociate them into individual polypeptides. Sucrose gradient centrifugation demonstrated that RI complex formation was not dependent on the presence of antibody and that these complexes had higher sedimentation rates than the 185-kDa P. falciparum merozoite glycoprotein. Covalent crosslinking with the reversible, homobifunctional, primary amine-specific reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by RI McAb immunoprecipitation resulted in purification of intact complexes which were not dissociable by SDS alone. Immunodepletion experiments with a subtype of RI McAb which does not immunoprecipitate p37 suggested that the binding of p39 and p37 to the other RI proteins was mutually exclusive. Therefore, the minimal composition of the RI complexes is one molecule of Pr86, p82, p70, or p67 and one of p39 or p37. The epitopes of Pr86, p82, p70, and p67 for the RI McAbs were sensitive to disulfide bond reduction. Surprisingly, reduction increased their electrophoretic mobilities. This enhanced mobility could not be accounted for by post-translational glycosylation, phosphorylation, or acylation, or by covalent attachment via the sulfhydryl moiety of cysteine residues to additional parasite proteins. We suggest that, due to an asymmetric distribution of amino acids in the Pr86-class molecules, SDS binding results in a lower charge to mass ratio in the native folded polypeptides and a higher charge to mass ratio upon disulfide bond reduction and unfolding of the polypeptides.