Abstract
Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a malaria merozoite integral membrane protein that plays an essential but poorly understood role in invasion of host erythrocytes. The PfAMA-1 ectodomain comprises three disulfide-constrained domains, the first of which (domain I) is preceded by an N-terminal prosequence. PfAMA-1 is initially routed to secretory organelles at the apical end of the merozoite, where the 83-kDa precursor (PfAMA-1(83)) is converted to a 66-kDa form (PfAMA-1(66)). At about the time of erythrocyte invasion, PfAMA-1(66) selectively translocates onto the merozoite surface. Here we use direct microsequencing and mass spectrometric peptide mass fingerprinting to characterize in detail the primary structure and proteolytic processing of PfAMA-1. We have determined the site at which processing takes place to convert PfAMA-1(83) to PfAMA-1(66) and have shown that both species possess a completely intact and unmodified transmembrane and cytoplasmic domain. Following relocation to the merozoite surface, PfAMA-1(66) is further proteolytically cleaved at one of two alternative sites, either between domains II and III, or at a membrane-proximal site following domain III. As a result, the bulk of the ectodomain is shed from the parasite surface in the form of two soluble fragments of 44 and 48 kDa. PfAMA-1 is not detectably modified by the addition of N-linked oligosaccharides.
Highlights
Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a malaria merozoite integral membrane protein that plays an essential but poorly understood role in invasion of host erythrocytes
On analysis by Western blot, all but the last two species were recognized by a polyclonal anti-PfAMA-1 antiserum; the 55- and 28-kDa bands were reactive with a horseradish peroxidase-conjugated anti-mouse IgG antibody alone, suggesting that they represent IgG heavy and light chain probably derived from slight leaching of the affinity column
PfAMA-1-derived proteins, each was subjected to five cycles of Edman degradation
Summary
Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a malaria merozoite integral membrane protein that plays an essential but poorly understood role in invasion of host erythrocytes. N-terminal Amino Acid Sequencing of AMA-1 Processing Products Affinity-purified from Schizont Extracts—SDS-PAGE fractionation of proteins eluted from the mAb 4G2dc1 affinity column revealed the presence of just two major components of apparent mass of 83 and 66 kDa, as well as a less abundant doublet of 46 kDa and minor bands of 55 and 28 kDa (Fig. 1A).
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