Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3

Abstract

In the malaria parasite Plasmodium falciparum, erythrocytic trophozoites hydrolyse haemoglobin to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block parasite haemoglobin hydrolysis and development, indicating that cysteine proteases are required for these processes. Three papain-family cysteine protease sequences have been identified in the P. falciparum genome, but the specific roles of their gene products and other plasmodial proteases in haemoglobin hydrolysis are uncertain. Falcipain-2 was recently identified as a principal trophozoite cysteine protease and potential drug target. The present study characterizes the related P. falciparum cysteine protease falcipain-3. As is the case with falcipain-2, falcipain-3 is expressed by trophozoites and appears to be located within the food vacuole, the site of haemoglobin hydrolysis. Both proteases require a reducing environment and acidic pH for optimal activity, and both prefer peptide substrates with leucine at the P(2) position. The proteases differ, however, in that falcipain-3 undergoes efficient processing to an active form only at acidic pH, is more active and stable at acidic pH, and has much lower specific activity against typical papain-family peptide substrates, but has greater activity against native haemoglobin. Thus falcipain-3 is a second P. falciparum haemoglobinase that is particularly suited for the hydrolysis of native haemoglobin in the acidic food vacuole. The redundancy of cysteine proteases may offer optimized hydrolysis of both native haemoglobin and globin peptides. Consideration of both proteases will be necessary to evaluate cysteine protease inhibitors as antimalarial drugs.

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

“…Immunoblotting with monospecific antibodies to falcipains confirmed the complete absence of falcipain-1 expression in recombinant parasites, whereas expression of falcipain-2 and falcipain-3 remained very similar to that in WT parasites (Fig. 2C) (5). As final confirmation of the knockout, binding of the generic cysteine protease inhibitor 125 I-labeled DCG04 to a protein previously identified as falcipain-1 (8) was seen in the parental strain, but not recombinant parasites (M. Bogyo, personal communication).…”

“…gDNA was isolated from schizont-stage parasites by using the PureGene DNA isolation kit (Gentra Systems), digested with XmnI or HincII-BsrGI restriction endonucleases, separated on agarose gels, transferred to Hybond-N membranes (Amersham Pharmacia Biosciences), and probed with [␣-32 P]dATP-labeled truncated falcipain-1 (Megaprime DNA labeling system, Amersham Pharmacia Biosciences), as described in ref. 5.…”

Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems

“…Four aspartic proteases called plasmepsins and three cysteine proteases called falcipains are found in the food vacuole (9)(10)(11)(12). All of them are capable of degrading hemoglobin or globin in biochemical assays using recombinant enzyme, although their order of action is unclear (9)(10)(11)(12)(13)(14). Inhibitors of aspartic and cysteine proteases kill parasites in culture and animal models, although their specificity is not strict (13)(14)(15).…”

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).…”