The Disulfide Bond Structure of Plasmodium Apical Membrane Antigen-1

Abstract

Apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is one of the leading asexual blood stage antigens being considered for inclusion in a malaria vaccine. The ability of this molecule to induce a protective immune response has been shown to be dependent upon a conformation stabilized by disulfide bonds. In this study we have utilized the reversed-phase high performance liquid chromatography of dithiothreitol-reduced and nonreduced tryptic digests of Plasmodium chabaudi AMA-1 secreted from baculovirus-infected insect cells, in conjunction with N-terminal sequencing and electrospray-ionization mass spectrometry, to identify and assign disulfide-linked peptides. All 16 cysteine residues that are conserved in all known sequences of AMA-1 are incorporated into intramolecular disulfide bonds. Six of the eight bonds have been assigned unequivocally, whereas the two unassigned disulfide bonds connect two Cys-Xaa-Cys sequences separated by 14 residues. The eight disulfide bonds fall into three nonoverlapping groups that define three possible subdomains within the AMA-1 ectodomain. Although the pattern of disulfide bonds within subdomain III has not been fully elucidated, one 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 clustered 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 recognized by protective immune responses.

Fine Mapping of an Epitope Recognized by an Invasion-inhibitory Monoclonal Antibody on the Malaria Vaccine Candidate Apical Membrane Antigen 1

“…Using a conditional expression system in T. gondii, Mital et al (28) confirmed that AMA1 plays a role in the later stages of host cell invasion and obtained evidence that it may mediate regulation of rhoptry discharge. Determination of the x-ray crystal structure of AMA1 of P. vivax (PvAMA1) (29) and P. falciparum (PfAMA1) (30) confirmed previous indications (31) that the AMA1 ectodomain comprises three disulfide-constrained domains and showed that two of these (domains I and II) form PAN modules, often implicated in protein-protein and protein-carbohydrate interactions. Consistent with this, two reports have suggested an erythrocyte-binding role for AMA1 (32,33).…”

Safety and immunogenicity of multi-antigen AMA1-based vaccines formulated with CoVaccine HT™ and Montanide ISA 51 in rhesus macaques

“…The AMA1 ectodomain, which is the vaccine target, is shed as 44 and 48 kDa alternate antigens before the parasite enters the red cell [5,10]. The ectodomain has 16 cysteine residues that form disulphide bonds to divide the antigen's tertiary structure into three different but interactive domains [11]. …”

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

“…This created both a 5Ј truncation and a 142-base pair internal deletion that resulted in the loss of multiple conserved cysteines thought to be important for protein folding (Hodder et al, 1996). AMA1/AMA1-myc parasites were transfected with the knockout construct.…”

“…Since its initial discovery Ͼ15 years ago, AMA1 has received considerable attention as a malaria vaccine candidate (Deans et al, 1988;Collins et al, 1994;Anders et al, 1998;Kennedy et al, 2002;Stowers et al, 2002). AMA1 proteins are type I transmembrane proteins, with a short C-terminal cytoplasmic tail and a large N-terminal extracellular domain (ectodomain) containing 12-16 conserved cysteine residues (Waters et al, 1990;Hodder et al, 1996;Donahue et al, 2000;Hehl et al, 2000;Gaffar et al, 2004). Like other microneme proteins, AMA1 is secreted onto the parasite surface, where its ectodomain is proteolytically cleaved and shed (Narum and Thomas, 1994;Donahue et al, 2000;Hehl et al, 2000;Howell et al, 2003).…”