Four aspartic proteases occur in the Plasmodium falciparum food vacuole.

Abstract

Hemoglobin (Hb) is a major food source for the malaria parasite during the intraerythrocytic blood stage. Hb is ingested into a specialized acidic compartment, the food vacuole (FV), where it is broken down into short peptides and the released haem is incorporated into hemozoin. Amino acids are then liberated from these peptides outside the FV and used by the parasite. This Hb proteolysis process is catalyzed by a series of enzymes, including plasmepsins. Plasmepsins are aspartic protease enzymes, two of which, plasmepsin (PM) I and PM II, have been well characterized. These enzymes are capable of cleaving native Hb. A cysteine protease enzyme, falcipain, and a metalloprotease, falcilysin, are involved in further degrading the protein to short peptides. Given the central role of these processes in the blood stage, these enzymes represent an important potential drug target for novel antimalarials. Indeed, several protease inhibitors exhibit activity both in vitro and in vivo. In the current climate of resurgent malaria and serious problems of drug resistance, a deeper understanding of these processes is vital.The sequence of the Plasmodium falciparum genome has revealed ten aspartic protease genes, including nine plasmepsins (I, II, IV–X) and histo-aspartic protease (HAP). Two key questions then arise. Which of these genes are expressed in the blood stage? Which play a role in the FV and what is their enzymatic activity? These questions have been addressed by Banerjee et al. [1xFour plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine. Banerjee, R et al. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 990–995Crossref | PubMed | Scopus (276)See all References[1]. They have shown that seven of these genes are expressed during the blood stage (PM I, II, IV, V, IX, X and HAP), but only four of these proteins are localized in the FV (PM I, II, IV and HAP). Interestingly, these enzymes are closely related. Their genes occur as a cluster on chromosome 14, exhibit 50–70% amino acid sequence homology and are initially expressed as 51-kDa proenzymes and are cleaved into 37-kDa active enzymes. Two active-site motifs are conserved in these plasmepsins, but HAP exhibits two conservative amino acid substitutions and one aspartate is replaced by histidine. PM IV and HAP are expressed somewhat later in the life cycle than PM I and II.PM IV, although similar to PM I and II in many respects, exhibits significantly reduced activity against native Hb, although it shows a marked synergistic effect with PM II. The authors also addressed whether HAP is an active enzyme given that itlacks one active-site aspartate moiety. Although recombinant HAP is inactive, the native protein is shown to have strong proteolytic activity against globin (although not against native Hb). Similar to PM IV, HAP also exhibits a synergistic effect with PM II against native Hb.The study has thus shown that four aspartic proteases probably act in concert in breaking down Hb in the FV. The emerging picture suggests that PM I and II probably initiate hemoglobin proteolysis, releasing globin that is then proteolyzed further by PM IV and HAP, and subsequently by falcipain and falcilysin. What is also significant is the unique active site and unusual inhibitor profile of HAP, which marks this enzyme as a particularly interesting candidate for novel antimalarial drugs.