The immunologically protective P-4 antigen of Leishmania amastigotes -: A developmentally regulated single strand-specific nuclease associated with the endoplasmic reticulum
The immunologically protective P-4 antigen of Leishmania amastigotes -: A developmentally regulated single strand-specific nuclease associated with the endoplasmic reticulum
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DOI：
10.1074/jbc.m002149200
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发表时间：
2000-12-01
影响因子：
4.8
通讯作者：
McMahon-Pratt, D
中科院分区：
文献类型：
--
作者：
Kar, S;Soong, L;McMahon-Pratt, D
纯化的膜相关利什曼原虫无鞭毛体蛋白 P-4 已被证明可诱导针对感染的保护性免疫，并优先在美国皮肤利什曼病患者的外周血单核细胞中引发 T 辅助细胞 1 样反应。由于该分子对未来的疫苗研究可能很重要，因此对编码 P-4 膜蛋白的 L. pifanoi 基因进行了克隆和测序。 Southern 印迹分析表明，L. pifanoi 中存在 6 个串联排列的 P-4 基因拷贝； P-4 基因的同源物存在于所检查的利什曼原虫属的所有其他物种中。 DNA 衍生的蛋白质序列数据表明与柑橘青霉 (Penicillium citrinum) 的 Pi 锌依赖性核酸酶 (20.8%) 和杜氏利什曼原虫 (Leishmania donovani) 3' 核苷酸酶的 C 末端结构域 (33.7%) 具有同一性。与这些序列分析一致，纯化的 L. pifanoi P-4 蛋白质具有单链核酸酶（DNA 和 RNA）和磷酸单酯酶活性，优先选择 UMP > TMP > AMP >> CMP，使用抗结合蛋白抗体的双标记免疫荧光显微镜分析表明，P-4 蛋白位于无鞭毛体的内质网中。 Northern印迹分析表明该基因在细胞内无鞭毛体阶段（哺乳动物宿主）选择性表达，但在寄生虫的前鞭毛体阶段（昆虫）不表达。基于其亚细胞定位和单链特异性核酸酶活性，讨论了无鞭毛体中 P-4 核酸酶在 RNA 稳定性（基因表达）或 DNA 修复中的可能作用。
The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to induce protective immunity against infection and to elicit preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. As this molecule is potentially important for future vaccine studies, the L. pifanoi gene encoding the P-4 membrane protein was cloned and sequenced. Southern blot analyses indicate the presence of six tandemly arrayed copies of the P-4 gene in L. pifanoi; homologues of the P-4 gene are found in all other species of the genus Leishmania examined. DNA-derived protein sequence data indicated an identity to the Pi zinc-dependent nuclease of Penicillium citrinum (20.8%) and the C-terminal domain of the 3' nucleotidase of Leishmania donovani (33.7%), Consistent with these sequence analyses, purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoesterase activity, with a preference for UMP > TMP > AMP >> CMP, Double-labeling immunofluorescence microscopic analyses employing anti-binding protein antibodies revealed that the P-4 protein is localized in the endoplasmic reticulum of the amastigote. Northern blot analyses indicated that the gene is selectively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. Based upon its subcellular localization and single-stranded specific nuclease activity, possible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed.