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 steady-state kinetic parameters for both aminoacyl-β-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.
Highlights
Grant AI077638. □S The on-line version of this article contains supplemental Table S1 and Figs
The idea that PfA-M1 plays a catalytic role in the vacuole has been questioned on the basis of an apparent pH profile for PfA-M1 catalysis that precludes significant activity at pH values below 6 [18], such as that found in the P. falciparum food vacuole (26 –29)
Parasite Culture and Isolation—P. falciparum 3D7 parasites were cultured in human Oϩ erythrocytes (Interstate Blood Bank) in RPMI 1640 medium supplemented with 27 mM sodium bicarbonate, 11 mM glucose, 0.37 mM hypoxanthine, 10 g/ml gentamicin, and 5 g/liter Albumax I (Invitrogen)
Summary
Parasite Culture and Isolation—P. falciparum 3D7 parasites were cultured in human Oϩ erythrocytes (Interstate Blood Bank) in RPMI 1640 medium supplemented with 27 mM sodium bicarbonate, 11 mM glucose, 0.37 mM hypoxanthine, 10 g/ml gentamicin, and 5 g/liter Albumax I (Invitrogen). Purification of Native PfA-M1—PfA-M1 was purified from a modified 3D7 parasite line that expressed PfLAP with a C-terminal HA tag [24]. The flow-through, which contained PfA-M1 activity, was adjusted to pH 7.5 by adding Tris base to 15 mM and injected onto a Mono Q 5/50 GL column equilibrated with 20 mM TrisHCl, pH 7.5. PfA-M1 eluted at around 330 mM NaCl. Fractions with PfA-M1 activity were concentrated in an Ultra-4 centrifugal device (Amicon) and injected onto a Superdex 200 10/30 gel filtration column (GE Healthcare) equilibrated in 50 mM Tris-HCl, pH 7.5, 200 mM NaCl. Fractions were subjected to immunoblotting with antiPfA-M1 serum to assess the distributions of the PfA-M1 species. Protein concentrations were calculated by quantitative immunoblotting as described under “Immunoblotting and immunolocalization” using a standard curve generated with recombinant PfA-M1.
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