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 I/II/III, domains I/II 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, treatment of human erythrocytes with trypsin, followed by alpha-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.

Fig. 1.

Schematic representation of P. falciparum AMA1 and expression vectors for domains I/II/III, domains I/II, and domain III of AMA1. (A) AMA1 consists of a signal sequence, a prosequence, domains I, II, and III, a predicted transmembrane (TM), and a cytoplasmic domain (Cyt). (B) Domains I/II/III, domains I/II, and domain III of AMA1 were amplified and cloned into the T8 vector that contains a signal sequence, a multicloning site, and a glycosylphosphatidylinositol (GPI) anchor. CHO cells that are transfected with this plasmid, pT8-AMA1 domains I/II/III (Top), domains I/II (Top Middle) or domain III (Bottom Middle), express proteins for domains I/II/III, domains I/II or domain III of P. falciparum AMA1 on their surface.

Fig. 2.

AMA1 domain III binds erythrocytes that are trypsin treated. Domains I/II/III, domains I/II, domain III, EBA-175, domain II of Duffy antigen binding protein from P. vivax, domain II of JESEBL from the Dd2 clone of P. falciparum, and DBL2 were expressed in CHO-K1 cells. Normal and enzyme-treated erythrocytes from two individuals were either untreated or treated with trypsin, neuraminidase, α-chymotrypsin, trypsin followed by neuraminidase, or trypsin followed by α-chymotrypsin before addition to transfected cells. Transfected cells with five or more attached erythrocytes were counted as rosettes, and the number of rosettes per 2.0 × 10⁵ CHO cells was calculated after standardization for the number of CHO cells. The transfection efficiency of all constructs into CHO cells was 80–90%. The numbers that are above the bar of Duffy protein represent the number of rosettes for the average of two experiments. The height of the bar are the average of two experiments (white and black circles). D, domain.

Fig. 3.

AMA1 domain III fails to bind trypsin-treated Kx_null erythrocytes. Two erythrocytes from normal individuals (normal 1 and 2), Duffy_null blood group (Duffy null 1 and 2), Kell_null blood group (Kell null 1 and 2), McLeod blood group, which lacks Kx protein and has reduced Kell protein, (McLeod 1, 2, and 3), PP_iP^k negative blood group were treated with trypsin before addition to transfected CHO-K1 cells. McLeod 1 is from a patient of CGD, and McLeod 2 and 3 are from non-CGD donors with point mutation in the XK gene. The data of four independent experiments are shown. Kell null 2 RBC was also Duffy null. D, domain.

Fig. 4.

The Kx protein is proteolytically modified. Immunoblot of ghosts from normal erythrocytes, Kx_null (McLeod) erythrocytes from a patient who had CGD, and the following treatment of normal erythrocytes: trypsin (T), T followed by α-chymotrypsin, T followed by neuraminidase, and neuraminidase followed by T. The erythrocyte binding data are from Figs. 2 and 3 (+, binding; –, no binding). Molecular masses of the standards (kDa) are shown on the left.