Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development.
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.
Stack
Colin M
CM
Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Level 6, Building 4, Corner of Thomas and Harris Street, Ultimo, Sydney, New South Wales 2007, Australia.
Lowther
Jonathan
J
Cunningham
Eithne
E
Donnelly
Sheila
S
Gardiner
Donald L
DL
Trenholme
Katharine R
KR
Skinner-Adams
Tina S
TS
Teuscher
Franka
F
Grembecka
Jolanta
J
Mucha
Artur
A
Kafarski
Pawel
P
Lua
Linda
L
Bell
Angus
A
Dalton
John P
JP
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007477:Ions; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000502:physiology*; D052940:Merozoites / Q000737:chemistry; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D053058:Trophozoites / Q000737:chemistry
D000596:Amino Acids; D000962:Antimalarials; D007477:Ions; D011994:Recombinant Proteins; D007931:Leucyl Aminopeptidase; D007930:Leucine