Plasmodium falciparum Falcilysin

Abstract

The malaria parasite Plasmodium falciparum degrades host cell hemoglobin within an acidic food vacuole. The metalloprotease falcilysin has previously been identified as an important component of this catabolic process. Using random peptide substrate analysis, we confirm that recombinant falcilysin is highly active at acidic pH, consistent with its role in hemoglobin degradation. Unexpectedly, the enzyme is also robustly active at neutral pH, but with a substantially different substrate specificity. Imaging studies confirm the location of falcilysin in the food vacuole and reveal association with vesicular structures elsewhere within the parasite. These data suggest that falcilysin may have an expanded role beyond globin catabolism and may function as two different proteases in its two locations.Plasmodium falciparum is the causative agent of the deadliest form of human malaria. Each year 500 million infections and up to 2 million deaths are attributed to Plasmodium parasites (1). This disease is a major public health issue in many areas of the world where the increasing prevalence of drugresistant strains underscores the need to identify new drug targets. Examination of the parasite's unique metabolic pathways, such as hemoglobin degradation, provides numerous attractive candidates for chemotherapeutic development.Plasmodium infects and develops within human erythrocytes where hemoglobin is degraded in a semi-ordered cascade of proteases to provide energy and nutrients to the growing parasite (2). Hemoglobin degradation occurs in the unique acidic environment of the food vacuole. A family of aspartic proteases, the plasmepsins, mediates the initial cleavage within a conserved hinge region of the hemoglobin alpha chains (3, 4). The denatured globin is further degraded by plasmepsins in concert with a family of cysteine proteases, falcipain-2 and -3 (2, 4 -7). The resulting globin peptides serve as substrates for the zinc metalloprotease falcilysin (FLN).1 FLN prefers to degrade small globin peptides up to 20 amino acids in length at a pH optimum of 5.2 (8), consistent with the acidic environment of the food vacuole (9, 10). The temporal expression pattern of FLN is similar to that of other globinases yet differs by the absence of proteolytic processing during biosynthesis (11).FLN is a member of the M16 family of the clan ME zinc metalloproteases. This family is characterized by an inverted active site motif of HXXEH (12), where the two histidines and a glutamic acid residue located 75-84 residues C-terminal to this motif are involved in coordinating the catalytic zinc ion. The M16 family is divided into subfamilies A and B. Subfamily B includes the mitochondrial processing peptidase (MPP) and the chloroplast stromal processing peptidase; both enzymes are involved in removing N-terminal targeting peptides (13-15). Falcilysin belongs to subfamily A whose members are oligoendopeptidases involved in processing biologically important peptides such as yeast ␣ factor (16, 17), insulin (18), and transforming growth fact...

Distal Substrate Interactions Enhance Plasmepsin Activity

“…This peptide bond is inaccessible in native Hb, and proteolysis at this position is thought to lead to denaturation of Hb and exposure of additional cleavage sites to PMs and other downstream proteases such as falcipain-2 (21,22), falcilysin (23,24), and dipeptidyl-aminopeptidase I (25). Our study supports the idea that PMs are unique among aspartic proteases in their ability to cleave native Hb well.…”

Damage to the Blood-Brain Barrier during Experimental Cerebral Malaria Results from Synergistic Effects of CD8+T Cells with Different Specificities

“…Clearly, this neuropathology is mediated by more than one epitope. In this study, we describe the identification of a new H-2K b epitope, which we dubbed Pb2, IITDFENL from bergheilysin, a protein that plays essential roles in hemoglobin degradation and protein trafficking in blood stage parasites (37)(38)(39). In P. yoelii parasites, the orthologue is also known to be expressed in the liver stage (40).…”

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

“…Although gelatinolytic activity is relatively crude and nonquantitative, the strongest regions of hydrolysis were observed at two distinct pH values with peaks at pH 2.0, no activity at pH 3.0, and a second peak of activity at pH 5.0 -7.0. Falcilysin was recently shown to be expressed in both the food vacuole and within vesicular structures elsewhere in P. falciparum (45). Moreover, the enzyme was shown to have dual specificity, displaying globinolytic activity at acidic pH and a distinctly different substrate preference at neutral pH, prompting the authors to suggest that falcilysin functions as two different proteases in different anatomic loca-tions within the parasite (45).…”

“…Falcilysin was recently shown to be expressed in both the food vacuole and within vesicular structures elsewhere in P. falciparum (45). Moreover, the enzyme was shown to have dual specificity, displaying globinolytic activity at acidic pH and a distinctly different substrate preference at neutral pH, prompting the authors to suggest that falcilysin functions as two different proteases in different anatomic loca-tions within the parasite (45). Although dual peaks in gelatinolytic activity were seen for MEP-1, the protease was only detected in the intestine of adult worms, suggesting a single function in Hb digestion.…”