Molecular and biochemical characterization of methionine aminopeptidase of Babesia bovis as a potent drug target.
Molecular and biochemical characterization of methionine aminopeptidase of Babesia bovis as a potent drug target.
复制标题
牛巴贝虫甲硫氨酸氨肽酶作为有效药物靶标的分子和生化特征。
DOI：
10.1016/j.vetpar.2016.02.024
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
I.
中科院分区：
文献类型：
--
作者：
Munkhjargal;T.;Ishizaki;T.;Guswanto;A.;Takemae;H.;Yokoyama;N.;and Igarashi;I.
氨基肽酶越来越多地被研究作为各种疾病的治疗靶点。在这项研究中，我们克隆、表达了牛巴贝斯虫（B. bovis）的甲硫氨酸氨肽酶（MAP）家族成员，并对其进行生化表征，以开发潜在的分子药物靶点。重组B. bovisMAP (rBvMAP) 在大肠杆菌 (E. coli) 中作为谷胱甘肽 S-转移酶 (GST) 融合蛋白表达，我们发现它具有抗原性。在小鼠体内产生了针对 rBvMAP 蛋白的抗血清，然后产生了天然 B. bovisMAP 在 B 中被鉴定。牛维斯蛋白质印迹测定。此外，免疫定位测定显示 MAP 存在于 theB 的细胞质中。牛裂殖子。 rBvMAP 的生化特性分析表明它具有酶活性，在 pH 7.5 时具有最佳活性。在二价锰阳离子存在下观察到酶活性增强，并被金属螯合剂乙二胺四乙酸 (EDTA) 有效抑制。此外，BvMAP 的酶活性被作为 MAP (MAPi) 抑制剂的amastatin 和 bestatin 以剂量依赖性方式抑制。重要的是，MAPi 还被发现在体外和体内均能显着抑制巴贝虫寄生虫的生长；此外，它们还能在宿主体内诱导高水平的细胞因子和免疫球蛋白（IgG）滴度。因此，我们的结果表明BvMAP是amastatin和bestatin的分子靶点，这些抑制剂可能是治疗巴贝斯虫病的候选药物，尽管需要更多的研究来证实这一点。
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 fromBabesia bovis(B. bovis) to develop a potential molecular drug target. RecombinantB. bovisMAP (rBvMAP) was expressed inEscherichia 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 nativeB. bovisMAP was identified inB. bovisby Western blot assay. Further, an immunolocalization assay showed that MAP is present in the cytoplasm of theB. bovismerozoite. 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 ofBabesiaparasites bothin vitroandin 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.