Distribution and Biochemical Properties of an M1-family Aminopeptidase in Plasmodium falciparum Indicate a Role in Vacuolar Hemoglobin Catabolism

Abstract

Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the food vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68-and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the food vacuole lumen. Determination of steadystate kinetic parameters for both aminoacyl-␤-naphthylamide and unmodified dipeptide substrates over the pH range 5.0 -8.5 reveals that k cat is relatively insensitive to pH, whereas K m increases at pH values below 6.5. At the pH of the food vacuole lumen (5.0 -5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the food vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.Human malaria is responsible for around one million deaths annually (1). Five species of the genus Plasmodium cause malaria in humans as they replicate within host erythrocytes. The cytoadherent properties of intraerythrocytic Plasmodium falciparum coupled with its ability to invade mature erythrocytes make it the most virulent of the species that infect humans. During its erythrocytic replication cycle, P. falciparum endocytoses and catabolizes over two-thirds of soluble erythrocyte proteins (2, 3), the majority of which is hemoglobin. Hemoglobin catabolism provides amino acids for protein synthesis, general metabolism, and isoleucine import (4, 5) and may also prevent premature hemolysis by reducing the colloid osmolarity of the erythrocyte (6). Blocking hemoglobin catabolism with protease inhibitors prevents parasite replication; therefore, enzymes that catalyze this process are attractive targets for the development of novel anti-malarial drugs (7).Hemoglobin is extensively catabolized by the parasite within an acidic organelle called the food vacuole or digestive vacuole. In the vacuole, numerous types of endo-and exopeptidases act in a complementary and concerted manner to catalyze the hydrolysis of the ␣-and ␤-globin chains of hemoglobin. Aspartic proteases (plasmepsin I, II, IV, and histo-aspartic protease) and cysteine endoproteases (falcipain-2, -2Ј, and -3) initiate cleavage of the globin chains and generate polypeptide fragments (8, 9). The metallopeptidase fa...

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“…Details of the expression in Escherichia coli and purification of recombinant PfA-M1 (residues 192 to 1,085) will be described separately (53). Pf-LAP lacking the N-terminal Asn-rich region (residues 79-605) was expressed with a C-terminal hexahistidine tag in E. coli and purified as previously described (43).The estimated molecular mass of the purified species from size exclusion chromatography (343 kDa) was in good agreement with the predicted mass for the hexameric enzyme (357 kDa).…”

Anti-malaria drug development targeting the M1 alanyl and M17 leucyl aminopeptidases

“…55 By contrast, studies by Dalal and Klemba 30 using P. falciparum parasites stably expressing yellow fluorescent protein-tagged PfM1AAP localized this enzyme to the DV and nucleus only. These cellular locations were further supported by immunoelectron microscopy 33 , and therefore Klemba and colleagues proposed that the PfM1AAP functions within the DV, releasing aminoacids from peptides generated by other endopeptidases (falcipain, plasmepsins and dipeptidase), and may have a separate role within the nucleus. In support of this theory, Ragheb et al 33 showed that while PfM1AAP exhibited less activity in the pH range 5.0 -5.5 than at a neutral pH environment (consistent with previously published data 42 ), it is still nevertheless stable and functional at acidic pH.…”

“…These cellular locations were further supported by immunoelectron microscopy 33 , and therefore Klemba and colleagues proposed that the PfM1AAP functions within the DV, releasing aminoacids from peptides generated by other endopeptidases (falcipain, plasmepsins and dipeptidase), and may have a separate role within the nucleus. In support of this theory, Ragheb et al 33 showed that while PfM1AAP exhibited less activity in the pH range 5.0 -5.5 than at a neutral pH environment (consistent with previously published data 42 ), it is still nevertheless stable and functional at acidic pH. The turnover rate (k cat ) of the enzyme did not alter dramatically at pH 5.5, and although the K m did increase with decreasing pH the authors suggested that this could be compensated for by an increased substrate concentration within the lumen of the DV.…”

“…Earlier studies by Gavigan et al 29 using electron microscopy did not show any effect on the DV when parasites where incubated in vitro with bestatin. Ragheb et al 33 attributed differences between their immunocytochemical localization observations (DV and nucleus) and that of Allary et al 53 (cytoplasm) to the use of different fixation methods. In an attempt to equate the collective immunocytochemical and biochemical data we have suggested that PfM1AAP could be associated with the DV by insertion within the DV membrane with the catalytic domain orientated towards the cytoplasm; in this location the enzyme may readily cleave peptides emerging from the DV at its optimum pH for activity, 31 and inhibition of its function could conceivably perturb the DV function and structure.…”

“…[21][22][23][24] Peptides produced in the DV may be further processed to dipeptides by a dipeptidyl aminopeptidase I, an orthologue of mammalian cathepsin C. 25,26 Small peptides and dipeptides may also be reduced to free aminoacids within the DV and/or transported to the parasite cytosol for processing by aminopeptidases. 20,[27][28][29][30][31][32][33] Aminopeptidases are essential in releasing aminoacids from haemoglobin-derived peptides and are thus central to the growth and development of malaria parasites in the erythrocyte. 31,34 The P. falciparum genome encodes five aminopeptidases that are potential candidate enzymes for releasing free aminoacids from haemoglobin-derived peptides; 34 these include three highly specific enzymes, a post-prolyl aminopeptidase (PfAPP) that is located in the DV and an aspartic aminopeptidase (M18AAP) and a prolyl aminopeptidase (S33PAP) located in the cytoplasm.…”