Molecular cloning and characterization of ouabain-insensitive Na+-ATPase in the parasitic protist, Trypanosoma cruzi
Molecular cloning and characterization of ouabain-insensitive Na+-ATPase in the parasitic protist, Trypanosoma cruzi
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DOI：
10.1016/j.bbamem.2006.04.025
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发表时间：
2006-06-01
影响因子：
3.4
通讯作者：
Aoki, Takashi
中科院分区：
文献类型：
--
作者：
Iizumi, Kyoichi;Mikami, Yuko;Aoki, Takashi
维持细胞内低钠浓度对几乎所有生物体都至关重要。Na+外流通常由动物中的P型ATP酶（Na+/K+-ATP酶）和真菌和植物中的Na+-ATP酶（称为ENA）控制。寄生于哺乳动物细胞的克氏锥虫必须经历对宿主细胞外高Na+浓度和宿主细胞内低Na+浓度的剧烈适应。然而，尚未确定克氏锥虫Na+外排泵。本文报道了克氏锥虫Na ~+-ATPase基因的克隆和鉴定，该基因与真菌和植物的ENA相似，称为TcENA。TcENA是一种在寄生虫整个生命周期中表达的质膜蛋白。TcENA在昆虫期外鞭毛体和血流锥鞭毛体中的转录水平高于细胞内无鞭毛体，这可能反映了宿主细胞外高Na+浓度的影响。对在哺乳动物细胞中异源表达的TcENA的生化分析首次证实TcENA的ATP酶活性受Na+和K+的双重刺激，对Na+/K+-ATP酶的特异性抑制剂哇巴因不敏感。此外，过量生产TcENA的上鞭毛体表现出对高Na+胁迫的耐受性增加。我们的研究结果表明，TcENA作为一个钠泵，并提供见解的离子稳态的调节寄生原生生物。(c)2006 Elsevier B.V保留所有权利。
Maintaining low intracellular sodium concentrations is vital for almost all organisms. Na+ efflux is generally governed by P-type ATPases, Na+/K+-ATPase in animals and Na+-ATPase, called ENA, in fungi and plants. Trypanosoma cruzi, which parasitizes mammalian cells, must undergo drastic adaptations to high Na+ concentrations outside and low Na+ concentrations inside host cells. However, T cruzi Na+ efflux pumps have not been identified. We report here the cloning and characterization of the gene encoding Na+-ATPase in T cruzi, which resembled fungal and plant ENAs, termed TcENA. TcENA was a plasma membrane protein expressed throughout the parasite life cycle. The transcription level of TcENA was higher in insect stage epimastigotes and blood stream trypomastigotes than in intracellular amastigotes, probably reflecting the high Na+ concentration outside the host cells. Biochemical analysis of TcENA expressed heterologously in mammalian cells demonstrated, for the fist time, that the ATPase activity of TcENA is stimulated by both Na+ and K and is insensitive to ouabain, a specific inhibitor of Na+/K+-ATPases. Furthermore, epimastigotes overproducing TcENA showed increased tolerance to high Na+ stress. Our findings suggest that TcENA acts as a sodium pump and provide insights into the regulation of ion homeostasis in the parasitic protist. (c) 2006 Elsevier B.V All rights reserved.