Domain III of Plasmodium falciparum apical membrane antigen 1 binds to the erythrocyte membrane protein Kx

Abstract

Plasmodium falciparum apical membrane antigen 1 (AMA1) is located in the merozoite micronemes, an organelle that contains receptors for invasion, suggesting that AMA1 may play a role in this process. However, direct evidence that P. falciparum AMA1 binds to human erythrocytes is lacking. In this study, we determined that domain III of AMA1 binds to the erythrocyte membrane protein, Kx, and that the rate of invasion of Kx null erythrocytes is reduced, indicating a significant but not unique role of AMA1 and Kx in parasite invasion of erythrocytes. Domains I͞II͞III, domains I͞II and domain III of AMA1 were expressed on the surface of CHO-K1 cells, and their ability to bind erythrocytes was determined. We observed that each of these domains failed to bind untreated human erythrocytes. In contrast, domain III, but not the other domains of AMA1, bound to trypsin-treated human erythrocytes. We tested the binding of AMA1 to trypsin-treated genetically mutant human erythrocytes, missing various erythrocyte membrane proteins. AMA1 failed to bind trypsin-treated Kx null (McLeod) erythrocytes, which lack the Kx protein. Furthermore, treatmentofhumanerythrocyteswithtrypsin,followedby␣-chymotrypsin, cleaved Kx and destroyed the binding of AMA1 to human erythrocytes. Lastly, the rate of invasion of Kx null erythrocytes by P. falciparum was significantly lower than Kx-expressing erythrocytes. Taken together, our data suggest that AMA1 plays an important, but not exclusive, role in invasion of human erythrocytes through a process that involves exposure or modification of the erythrocyte surface protein, Kx, by a trypsin-like enzyme. T he complex multistep process of invasion of erythrocytes byPlasmodium falciparum begins with the binding of any surface of the merozoite, followed by reorientation to put the merozoite's apical end in close apposition to the erythrocyte surface. The apical end of merozoites contains the organelles of invasion, including the micronemes that contain receptors such as members of the Duffy binding protein family of erythrocytebinding ligands (1). A junction forms between P. knowlesi merozoites and the erythrocyte before the merozoite is brought into the erythrocyte. The junction only forms and invasion only occurs between P. knowlesi merozoites and Duffy blood group positive human erythrocytes. Duffy null cells fail to form a junction and are not invaded by P. knowlesi merozoites (2). However, trypsin-or neuraminidase-treated human Duffy null erythrocytes could form a junction and were invaded by P. knowlesi merozoites (3) despite the fact that untreated and trypsin-treated human Duffy null cells fail to bind any of the P. knowlesi Duffy binding proteins expressed on COS-7 cells (2). This result indicates that molecules other than the parasite Duffy binding protein family are exposed by enzyme treatment to form the junction between Duffy negative human erythrocytes and P. knowlesi merozoites. One of the possible junction-forming molecules is apical merozoite antigen 1 (AMA1), which is found in the mic...

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

“…Antibodies against AMA1 inhibit invasion ( Thomas et al, 1984;Deans et al, 1988;Hehl et al, 2000;Kocken et al, 2000;Mitchell et al, 2004), as do phage-displayed peptides derived from (Urquiza et al, 2000) or with affinity for Plasmodium AMA1 (Li et al, 2002;Keizer et al, 2003). Together with trans-species complementation experiments (Triglia et al, 2000) and heterologous expression experiments (Fraser et al, 2001;Kato et al, 2005), these data suggest that AMA1 plays a role in host cell invasion, perhaps as an adhesin. However, it has not previously been possible to disrupt AMA1 for phenotypic analysis, in any apicomplexan parasite, presumably because AMA1 is an essential gene (Mital and Ward, unpublished results;Hehl et al, 2000;Triglia et al, 2000).…”

“…Many microneme proteins are thought to function as host cell adhesins (Tomley and Soldati, 2001), and AMA1 has been postulated to play such a role (Fraser et al, 2001;Kato et al, 2005;Pizarro et al, 2005). The ability of AMA1/AMA1-myc and ⌬ama1/AMA1-myc parasites to bind to host cells was therefore compared using a laser scanning cytometer-based attachment assay.…”

Babesia divergens Apical Membrane Antigen 1 and Its Interaction with the Human Red Blood Cell

“…Furthermore, they found that the contiguous domain I/II was required for efficient binding. In contrast, Kato and others (19) report that the RBC protein Kx is the receptor for PfAMA1 and that it is domain III of PfAMA1 that mediates this binding. We have obtained similar binding profiles for native BdAMA1 to wildtype and Kx null cells (data not shown), which demonstrates that Kx is not the erythrocyte receptor for BdAMA1.…”

“…Attempts to explore its erythrocyte-binding capabilities have provided contradictory results, with the identification of either domains I and II of Plasmodium yoelii AMA1 (10) or domain III of PfAMA1 (19) being critical for this function. We took advantage of the high yields of native BdAMA1 available due to the extremely high parasitemia obtained in vitro and showed conclusively that the processed fragments of BdAMA1 (48, 44, and 37 kDa) (Fig.…”

Preclinical profiling of the immunogenicity of a two-component subunit malaria vaccine candidate based on virosome technology

“…[17][18][19]23,24 Loop 1 of domain III binds to the erythrocyte membrane protein Kx, and invasion of Kx null erythrocytes is reduced, indicating a significant role of AMA-1 and Kx in parasite invasion of erythrocytes. 25 We have previously shown the development of synthetic peptides based on these two proteins for inclusion into a multivalent subunit malaria vaccine. [26][27][28][29][30] Peptide UK-39 is based on the NANP repeats of CSP 30 and peptide AMA49-C1 on the erythrocyte-binding loop 1 of domain III from AMA-1.…”