Cloning and Characterization of a Leucyl Aminopeptidase from Three Pathogenic Leishmania Species

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 Ϸ 63 s ؊1 ⅐mM ؊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 Ϸ 1 nM), whereas arphamenine A (K i Ϸ 70 M) and Lleucinol (K i Ϸ 100 M) were non-tight binding competitive inhibitors. Lap was not secreted by Leishmania in vitro and was localized to the parasite cytosol.Protozoan parasites of the genus Leishmania cause visceral, cutaneous, and mucosal diseases in humans, collectively referred to as leishmaniasis. Leishmaniasis is prevalent in 88 countries, with 12 million people currently infected, a further 350 million at risk, and 2 million new cases reported per year.No vaccine exists, and therapies are inadequate (1). 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. However, only three peptidases from Leishmania, lysosomal cysteine peptidases (3), a cell-surface metallopeptidase (4), and a cytosolic serine oligopeptidase (5) have received attention.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. Vaccination of sheep with Fasciola leucyl aminopeptidase (Lap) 1 elicited high anti-Lap titers that conferred protection against fascioliasis and fascioliasis-related liver damage (7). Synthetic broad-spec...

A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism

“…Mn 2ϩ , Co 2ϩ , and Mg 2ϩ enhanced enzyme activity, whereas Zn 2ϩ and Ca 2ϩ supported very little CGase activity (Table 3). This result, which suggests that Mn 2ϩ , Co 2ϩ , or Mg 2ϩ is the preferred metal for activation of CGase, is similar to the result seen with bovine lens LAP, which catalyzes Cys-Gly hydrolysis in the presence of Mn 2ϩ but not in the presence of Zn 2ϩ (35). CGase Activity and the cga Gene Are Present in Multiple T. denticola Strains-To demonstrate that the CGase activity from T. denticola was not strain-specific, enzyme assays were done on extracts from five other T. denticola strains as well as from T. vincentii.…”

“…The optimal pH for several other M17 family members has been determined to be somewhat basic (pH 7.9 -8.5) with Leu-X as substrate (35,46,50). However, in the one case where it was examined, bovine lens aminopeptidase (46), the optimal pH for Cys-Gly hydrolysis with the same enzyme was more acidic (pH 6.8).…”

“…Leucine aminopeptidase activity was measured using L-Leup-NA as substrate (35). The reaction was done in 50 mM TrisHCl (pH 8.0) with 0.2 mM MnCl 2 and 2 mM L-Leu-p-NA, unless noted otherwise.…”

Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk,Patella caerulea

“…Our purification of the enzyme from Patella is an extremely fast procedure. The yield (approximately 5 mg of pure enzyme per liter of the mollusk extract) is well comparable with enzyme production by DNA recombinant techniques without the need of further manipulation, such as the removal of polyhistidine tag, to obtain the native enzyme form 35,36 . Thus, given the abundance and availability of the starting material, purification could make large amounts of protein available for structural studies.…”

Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic ArchaeonPyrococcus furiosus

“…It will be interesting to see what the structural analyses reveal about this site. Such an exchange of divalent cations has been observed in certain leucine aminopeptidases (LAPs) (18,23,31 (31). Hence, such an exchange of metals may be occurring in the P. furiosus KAP.…”