Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development

Abstract

Plasmodium falciparum-derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobinhydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrinactin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo. IntroductionPlasmodium falciparum causes the most severe form of human malaria and is becoming increasingly resistant to available antimalarial drugs. New chemotherapy-based approaches to fight the disease are therefore urgently needed. Parasite proteases that are involved in P falciparum development appear to be good targets. Mounting evidence suggests that cysteine proteases are involved in host cell rupture and release of merozoites. In the presence of such inhibitors, merozoites mature normally but are unable to escape from host erythrocytes. [1][2][3][4] The cluster of merozoites inside a red blood cell (RBC) is enclosed within 2 membranes: an inner parasitophorous vacuole membrane (PVM) and an outer RBC membrane. The rupture of these 2 membranes apparently releases the merozoites for another round of RBC invasion. In a recent study by Salmon et al,5 the authors propose a 2-step process for parasite release from the host erythrocyte: an initial exit of merozoites enclosed within the PVM followed by a rapid escape from the PVM by a proteolysisdependent mechanism. This study suggests that the RBC membrane is lost independently of the PVM. In another report, Winograd et al 6 used videomicroscopy to study the release of merozoites and concluded that an aperture is made through the PVM and RBC membranes to allow merozoites to exit in an orderly fashion. Merozoites were released together with the residual body containing hemozoin, leaving behind the red cell membrane and some...

Structural and Functional Characterization of Falcipain-2, a Hemoglobinase from the Malarial Parasite Plasmodium falciparum

“…The S2 pocket is the major determinant of specificity for most cysteine proteases (66), and its predominantly hydrophobic nature in falcipain-2 agrees well with the observation that all characterized falcipain-2 cleavage motifs of its specific intracellular protein targets, including ankyrin (NVSAR2FWLSD) (67), band 4.1 (SQEEIK2KHHASI) (17), and the falcipain-2 precursor (KYLLD2QMNYF) (18), contain a hydrophobic residue at the P2 position (marked in bold in the preceding sequences). Experiments with fluorogenic substrates have also shown that falcipain-2 has a strong preference for substrates with a hydrophobic residue, particularly Leu, at the P2 position (18).…”

“…During the late trophozoite and schizont stages, falcipain-2 is also involved in the degradation of erythrocyte-membrane skeletal proteins including ankyrin and the band 4.1 protein (17). This activity displays a pH optimum in the range of 7.0 -7.5 and is thought to contribute to destabilization of the erythrocyte membrane, leading to host cell rupture and release of the mature merozoites.…”

Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d

“…Identifying the proteases expressed in both the asexual and the sexual stages and their in vivo accessibilities to inhibitors is an important component of drug development. Previous work has implicated falcipains 2A/B and 3 in asexual hemoglobin digestion and, possibly, RBC emergence, and recombinant falcipains 2A and 3 have been used in a number of drug screening assays (6,7,11,29,31,33,34). The data from this study demonstrating that gametocytes express falcipain 3 and not falcipain 2A/B, coupled with the possibility that falcipain 3 could be essential for asexual growth, suggest that falcipain 3 is a prime drug target.…”

Plasmodium falciparum falcilysin: an unprocessed food vacuole enzyme

“…It also suggests that the enzyme may serve another purpose in mature schizonts, as the majority of hemoglobin degradation is already complete at schizogony. Both plasmepsin II and falcipain-2 have been shown to cleave host membrane proteins such as band 4.1 and spectrin [16,17]. The significance of these proteolytic events [27] in serum-free RPMI media supplemented with 0.5% Albumax (Gibco BRL, Grand Island, NY), was generated from parasites (3% parasitemia and 2% hematocrit) harvested by saponin lysis at multiple time points during the intraerythrocytic life cycle.…”