Characterization of Native and Recombinant Falcipain-2, a Principal Trophozoite Cysteine Protease and Essential Hemoglobinase ofPlasmodium falciparum

Abstract

Trophozoites of the malaria parasite Plasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mM glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum food vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.

Electrostatic properties in the catalytic site of papain: A possible regulatory mechanism for the reactivity of the ion pair

“…However, to put these hypothesis more theoretically grounded is necessary to evaluate in each enzyme the overall electrostatic contribution of the negatively and positively charged residues. A comparative study involving proteinases belonging to the papain family, including falcipain isoforms from the protozoa Plasmodium falciparum 6 and the isoforms cruzipain and cruzipain2 from the protozoa Trypanosoma cruzi, 80 are under progress and will be presented elsewhere.…”

Novel cinnamic acid/4-aminoquinoline conjugates bearing non-proteinogenic amino acids: Towards the development of potential dual action antimalarials

“…Recombinant FP2 and FP3 were expressed as previously described [49,50] and assays of inhibition of the enzymes were performed as previously reported [9]. In brief, equal amounts (1 nM) of recombinant FP2 and FP3 were incubated with different concentrations of test compounds (added from 10 mM stocks in DMSO) in 100 mM sodium acetate, pH 5.5, 10 mM dithiothreitol for 30 min at room temperature before addition of the substrate benzoxycarbonyl-Leu-Arg-7-amino-4-methyl-coumarin (final concentration 25 mM).…”

A Multi-enzyme Cascade of Hemoglobin Proteolysis in the Intestine of Blood-feeding Hookworms

“…After extensive in vitro and in silico screening of A. caninum cDNA libraries and expressed sequence tags, respectively, we did not identify an orthologue of Na-APR-2 from A. caninum. 2 Cysteine proteases of P. falciparum digest both native Hb and reduced globin (5,6,36,37). Although the reducing environment of the digestive vacuole is an unresolved issue, erythrocytes contain 1.2 mM GSH and 0.006 mM cysteine (32).…”