Structural and antigenic properties of merozoite surface protein 4 of Plasmodium falciparum

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylph osphatidylinositol-anchored integral membrane protein of 272 residues that possesses a single epidermal growth factor (EGF)-like domain near the carbo xyl terminus. We have expressed both full-length MSP4 and a number of fragm ents in Escherichia coli and have used these recombinant proteins to raise experimental antisera. All recombinant proteins elicited specific antibodie s that reacted with parasite-derived MSP4 by immunoblotting. Antibody react ivity was highly dependent on the protein conformation. For example, reduct ion and alkylation of MSP4 almost completely abolished the reactivity of se veral antibody preparations, including specificities directed to regions of the protein that do not contain cysteine residues and are far removed from the cysteine-containing EGF-like domain. This indicated the presence of co nformation-dependent epitopes in MSP4 and demonstrated that proper folding of the EGF-like domain influenced the antigenicity of the entire molecule. The recombinant proteins were used to map epitopes recognized by individual s living in areas where malaria is endemic, and at least four distinct regi ons are naturally antigenic during infection. Binding of human antibodies t o the EGF-like domain was essentially abrogated after reduction of the reco mbinant protein, indicating the recognition of conformational epitopes by t he human immune responses. This observation led us to examine the importanc e of conformation dependence in responses to other integral membrane protei ns of asexual stages. We analyzed the natural immune responses to a subset of these antigens and demonstrated that there is diminished reactivity to s everal antigens after reduction. These studies demonstrate the importance o f reduction-sensitive structures in the maintenance of the antigenicity of several asexual-stage antigens and in particular the importance of the EGF- like domain in the antigenicity of MSP4.