title: Molecular and biochemical characterization of methionine aminopeptidase of Babesia bovis as a potent drug target.
abstract: Aminopeptidases are increasingly being investigated as therapeutic targets in various diseases. In this study, we cloned, expressed, and biochemically characterized a member of the methionine aminopeptidase (MAP) family from Babesia bovis (B. bovis) to develop a potential molecular drug target. Recombinant B. bovis MAP (rBvMAP) was expressed in Escherichia coli (E. coli) as a glutathione S-transferase (GST)-fusion protein, and we found that it was antigenic. An antiserum against the rBvMAP protein was generated in mice, and then a native B. bovis MAP was identified in B. bovis by Western blot assay. Further, an immunolocalization assay showed that MAP is present in the cytoplasm of the B. bovis merozoite. Analysis of the biochemical properties of rBvMAP revealed that it was enzymatically active, with optimum activity at pH 7.5. Enhanced enzymatic activity was observed in the presence of divalent manganese cations and was effectively inhibited by a metal chelator, ethylenediaminetetraacetic acid (EDTA). Moreover, the enzymatic activity of BvMAP was inhibited by amastatin and bestatin as inhibitors of MAP (MAPi) in a dose-dependent manner. Importantly, MAPi was also found to significantly inhibit the growth of Babesia parasites both in vitro and in vivo; additionally, they induced high levels of cytokines and immunoglobulin (IgG) titers in the host. Therefore, our results suggest that BvMAP is a molecular target of amastatin and bestatin, and those inhibitors may be drug candidates for the treatment of babesiosis, though more studies 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; Institute of Veterinary Medicine, Zaisan 17042, Ulaanbaatar, Mongolia.
authors.forename: Tserendorj
authors.initials: T
authors.lastname: Munkhjargal
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishizaki
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Azirwan
authors.initials: A
authors.lastname: Guswanto
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Takemae
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Yokoyama
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Electronic address: igarcpmi@obihiro.ac.jp.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology / Q000627:therapeutic use; D016793:Babesia bovis / Q000145:classification / Q000187:drug effects* / Q000201:enzymology*; D001404:Babesiosis / Q000188:drug therapy; D016503:Drug Delivery Systems; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D007074:Immunoglobulin G / Q000097:blood; D020128:Inhibitory Concentration 50; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000494:pharmacology* / Q000627:therapeutic use; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000981:Antiprotozoal Agents; D007074:Immunoglobulin G; D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C016977:amastatin; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex
keywords: Amastatin; Aminopeptidase; Bestatin; Enzyme assay; Parasite