Ankyrin Peptide Blocks Falcipain-2-mediated Malaria Parasite Release from Red Blood Cells

Abstract

Falcipain-2 (FP-2) is a dual-function protease that cleaves hemoglobin at the early trophozoite stage and erythrocyte membrane ankyrin and protein 4.1 at the late stages of parasite development. FP-2-mediated cleavage of ankyrin and protein 4.1 is postulated to cause membrane instability facilitating parasite release in vivo. To test this hypothesis, here we have determined the precise peptide sequence at the hydrolysis site of ankyrin to develop specific inhibitor(s) of FP-2. Mass spectrometric analysis of the hydrolysis products showed that FP-2-mediated cleavage of ankyrin occurred immediately after arginine 1,210. A 10-mer peptide (ankyrin peptide, AnkP) containing the cleavage site completely inhibited the FP-2 enzyme activity in vitro and abolished all of the known functions of FP-2. To determine the effect of this peptide on the growth and development of P. falciparum, the peptide was delivered into intact parasite-infected red blood cells (RBCs) via the Antennapedia homeoprotein internalization domain. Growth and maturation of trophozoites and schizonts was markedly inhibited in the presence of the fused AnkP peptide. <10% of new ring-stage parasites were detected compared with the control sample. Together, our results identify a specific peptide derived from the spectrin-binding domain of ankyrin that blocks late-stage malaria parasite development in RBCs. Confocal microscopy with FP-2-specific antibodies demonstrated the proximity of the enzyme in apposition with the RBC membrane, further corroborating the proposed function of FP-2 in the cleavage of RBC skeletal proteins.Plasmodium falciparum causes the most severe form of human malaria and is responsible for nearly all malaria-induced mortality. Clinical manifestations of malaria are caused by the intraerythrocytic life cycle of P. falciparum. The parasite undergoes distinct morphologic changes during its 48-h life cycle inside human red blood cells (RBCs) 1 (1). After invasion into RBCs, the parasites mature from ring stage to trophozoites and then to schizonts. Mature segmented schizonts finally rupture the host erythrocytes releasing merozoites, which rapidly invade other erythrocytes to reinitiate the cycle. The mechanism of merozoite release from host RBCs is largely unknown. The cluster of merozoites inside a red blood cell is enclosed within two membranes: an inner parasitophorous vacuole membrane (PVM) and an outer RBC membrane. The rupture of these two membranes precedes the release of merozoites for subsequent round of RBC invasion. Several studies have shown that the merozoite release is susceptible to protease inhibitors. In the presence of such inhibitors, merozoites mature normally but are unable to escape from RBCs (2-4). Furthermore, a number of plasmodial proteases have been isolated and are shown to have activities against known RBC membrane skeletal proteins (5), although a functional role of these proteases in the rupture of the RBC membrane during merozoite escape has not been established.The issue of malaria parasite release fro...

Plasmodium falciparum cysteine protease falcipain-1 is not essential in erythrocytic stage malaria parasites

“…In addition, because of the inhibition of development or independent effects, cysteine protease inhibitors block the release of merozoites from schizontinfected erythrocytes at the completion of the erythrocytic cycle (18,(20)(21)(22). In in vitro studies, falcipain-2 and falcipain-3 hydrolyzed hemoglobin (4, 5) and falcipain-2 also hydrolyzed the erythrocyte cytoskeletal proteins ankyrin and band 4.1 (25,26), but until recently, specific roles of cysteine proteases in the parasite life cycle have been uncertain.…”

“…Cysteine protease-mediated activation of plasmepsin aspartic proteases also may contribute indirectly to hemoglobin hydrolysis, although the proteases responsible for this processing are unknown (16,22). If cysteine proteases independently mediate erythrocyte rupture, potential candidates include falcipain-2, which cleaves the erythrocyte cytoskeletal proteins ankyrin and band 4.1 in vitro (25,26); falcipain-3, which is maximally expressed late in the life cycle (5); serine repeat antigen family proteins, which share homology with cysteine proteases and are expressed in mature schizonts (28); and other putative cysteine proteases (32). Falcipain-1, which is expressed throughout the erythrocytic cycle and has been localized to merozoites (8), also may play a role in erythrocyte rupture and invasion, but our data indicate that it is not required for these processes.…”

Molecular and functional aspects of parasite invasion

“…This study revealed that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane, both steps involving the action of distinct proteases [11]. A contribution of falcipain-2 in this release from red blood cells was postulated [12], but the disruption of the gene encoding falcipain 2 failed to confirm this prediction. However, these experiments substantiated a role of this protease in hemoglobin degradation [13].…”

Molecular Epitopes of the Ankyrin−Spectrin Interaction

“…The human erythrocyte spectrin tetramer consists of two α and two β chains, arranged as shown. Each spectrin repeat of the tetramer is depicted as an ellipse; repeats of interest in studying spectrin-ankyrin binding (13,14,15, and 16) are shaded and numbered. Divided ellipses represent the dimerization sites formed from the N-terminus of α-spectrin and the Cterminus of β-spectrin.…”

“…1). Much is known about the associations of this ubiquitous protein's multiple isoforms (8,9), the static and kinetic properties of the ankyrin-Band 3 interaction (10), the consequences of ankyrin mutations for cardiac (11,12) and other functions (12,13). Characterization of its molecular structure, however, has been confined largely to its Band 3 binding domain of so-called ankyrin repeats.…”