Abstract
Aminopeptidases are emerging as exciting novel drug targets and vaccine candidates in parasitic infections. In this study, we describe for the first time an aminopeptidase from three highly pathogenic Leishmania species. Intronless genes encoding a leucyl aminopeptidase (lap) were cloned from Leishmania amazonensis, Leishmania donovani, and Leishmania major, which encoded 60-kDa proteins that displayed homology to leucyl aminopeptidases from Gram-negative bacteria, plants, and mammals. The lap genes were present as a single copy in each genome, and lap mRNA was detected by reverse transcription-PCR in all life-cycle stages of L. amazonensis. Lap assembled into catalytically competent 360-kDa hexamers and demonstrated potent amidolytic activity against synthetic aminopeptidase substrates containing leucine, methionine, and cysteine residues, representing the most restricted substrate specificity of any leucyl aminopeptidase described to date. Optimal activity was observed against L-leucyl-7-amido-4-methylcoumarin (k(cat)/K(m) approximately 63 s(-1) x mm(-1)) with a pH optimum of 8.5. Leishmania Lap activity was inhibited by metal ion chelators and enhanced by divalent manganese, cobalt, and nickel cations, although only zinc was detected in the purified Lap by inductively coupled plasma atomic emission spectroscopy, indicating that zinc is the natural Lap cofactor. Activity was potently inhibited by bestatin and apstatin in a slow binding competitive fashion, with K(i)* values of 3 and 44 nm, respectively. Actinonin was a tight binding competitive inhibitor (K(i) approximately 1 nm), whereas arphamenine A (K(i) approximately 70 microm) and L-leucinol (K(i) approximately 100 microm) were non-tight binding competitive inhibitors. Lap was not secreted by Leishmania in vitro and was localized to the parasite cytosol.
Highlights
Protozoan parasites of the genus Leishmania cause visceral, cutaneous, and mucosal diseases in humans, collectively referred to as leishmaniasis
The lap genes were present as a single copy in each genome, and lap mRNA was detected by reverse transcription-PCR in all life-cycle stages of L. amazonensis
Cloning, Sequencing, and Analysis of Leishmania lap Genes—The lap genes isolated from Leishmania consisted of an ORF of 1698 base pairs, encoding polypeptides of 565 amino acids with predicted molecular masses of 60 129.00, 60 041.30, and 60 274.10 Da for L. amazonensis, L. donovani, and L. major, respectively, with predicted pI values of 9.1–9.2
Summary
Protozoan parasites of the genus Leishmania cause visceral, cutaneous, and mucosal diseases in humans, collectively referred to as leishmaniasis. There is a pressing need for the identification of novel leishmanial virulence factors, drug targets, and vaccines to improve our understanding, prevention, and treatment of leishmaniasis. The peptidases of parasitic protozoans (for review, see Ref. 2) are becoming increasingly important as virulence factors, drug targets, and vaccine candidates in parasitic infections. Aminopeptidases, which catalyze the removal of N-terminal amino acid residues from peptides and proteins [6], are emerging as novel and exciting anti-parasite targets. Synthetic broad-spectrum aminopeptidase inhibitors [8] and 1,2-aminoalcohol inhibitors of Lap [9] have shown promise as drugs against malaria, and the aminopeptidase inhibitor arphamenine A has activity against Trypanosoma brucei, a kinetoplastid protozoan that is a close relative of Leishmania [10]
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