title: Molecular Characterization of Babesia bovis M17 Leucine Aminopeptidase and Inhibition of Babesia Growth by Bestatin.
abstract: The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 μM, respectively, while the Ki was 2210 ± 358 μM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 μM] than B. gibsoni [IC50 = 460.8 ± 114.45 μM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Gabriel Oluga
authors.initials: GO
authors.lastname: Aboge
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Shinuo
authors.initials: S
authors.lastname: Cao
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Mohamad Alaa
authors.initials: MA
authors.lastname: Terkawi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Masatani
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Younkyoung
authors.initials: Y
authors.lastname: Goo
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: AbouLaila
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Nishikawa
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Xuenan
authors.initials: X
authors.lastname: Xuan
mesh_terms: D000818:Animals; D016793:Babesia bovis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D004285:Dogs; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D011480:Protease Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D016054:DNA, Protozoan; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex