THE DISULFIDE BOND STRUCTURE OF PLASMODIUM APICAL MEMBRANE ANTIGEN-1

Apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is one of t he leading asexual blood stage antigens being considered for inclusion in a malaria vaccine. The ability of this molecule to induce a protec tive immune response has been shown to be dependent upon a conformatio n stabilized by disulfide bonds. In this study we have utilized the re versed-phase high performance liquid chromatography of dithiothreitol- reduced and nonreduced tryptic digests of Plasmodium chabaudi AMA-1 se creted from baculovirus-infected insect cells, in conjunction with N-t erminal sequencing and electrospray-ionization mass spectrometry, to i dentify and assign disulfide-linked peptides. All 16 cysteine residues that are conserved in all known sequences of AMA-1 are incorporated i nto intramolecular disulfide bonds. Six of the eight bonds have been a ssigned unequivocally, whereas the two unassigned disulfide bonds conn ect two Cys-Xaa-Cys sequences separated by 14 residues. The eight disu lfide bonds fall into three nonoverlapping groups that define three po ssible subdomains within the AMA-1 ectodomain. Although the pattern of disulfide bonds within subdomain III has not been fully elucidated, o ne of only two possible linkage patterns closely resembles the cystine knot motif found in growth factors. Sites of amino acid substitutions in AMA-1 that are well separated in the primary sequence are clustere d by the disulfide bonds in subdomains II and III. These findings are consistent with the conclusion that these amino acid substitutions are defining conformational disulfide bond-dependent epitopes that are re cognized by protective immune responses.