Abstract
Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs.
Highlights
Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are essential for protein synthesis and function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum
We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and represents a target for novel antimalarial drugs
One essential pathway that has been a particular target for antimalarial drug discovery is the catabolism of erythrocyte hemoglobin; between 65 and 75% of the host cell hemoglobin is degraded in a process that results in the liberation of free amino acids (4 – 6)
Summary
Parasite Culture—The asexual intra-erythrocytic stages of P. falciparum parasites were cultured in RPMI 1640 medium containing 10% human serum [24]. Expression of Recombinant Malarial M17 Leucyl Aminopeptidase in Insect Cells—Successful functional expression of the M17 leucyl aminopeptidase (PfLAP) was achieved using a truncated form of the enzyme (Fig. 1A) This truncated form was prepared from the synthesized codon-biased leucyl aminopeptidase gene and was amplified by PCR using 5Ј primers containing the additional bases CACC to allow directional cloning of the gene into the Gateway entry vector pENTR/D-TOPO (Invitrogen). Ki values were determined from Dixon plots of 1/vs versus inhibitor concentration when [S] ϽϽ Km. The metal ion dependence of PfLAP was determined by assaying activity after preincubation of the enzyme (10 min, 37 °C) in 50 mM Tris-Cl, pH 8.0, containing a given metal chloride, before addition of H-Leu-NHMec substrate (10 M). To determine the strength of the groupings, bootstrap values for nodes were calculated by analyzing 1,000 bootstrap replicate data sets
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