School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
PLoS One. 2011;6(9):e24820. doi: 10.1371/journal.pone.0024820. Epub 2011 Sep 28.
Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani.
杜氏利什曼原虫是一种原生动物寄生虫，存在于哺乳动物宿主的巨噬细胞中。水通道蛋白家族的蛋白质是寄生虫-宿主界面的重要组成部分。寄生虫-宿主界面可能是化疗的潜在靶点。对 L. major 和 L. infantum 基因组的分析表明，存在 5 种水通道蛋白 (AQPs)，分别注释为 AQP9 (230aa)、AQP 假定蛋白 (294aa)、AQP 样蛋白 (279aa)、AQP1 (314aa) 和 AQP 样蛋白 (596aa)。我们在此报告了来自 L. donovani 的 AQPs 的结构建模、定位和功能特征。LdAQP1、LdAQP9、LdAQP2860 和 LdAQP2870 具有典型的 NPA-NPA 基序，而 LdAQP 假定蛋白具有非典型的 NPM-NPA 基序。在除 AQP1 之外的所有 AQP 的羧基末端的第二个 NPA 盒中，发现了缬氨酸/丙氨酸残基，而不是精氨酸。在这方面，这四个 AQP 类似于植物中的液泡内在蛋白，它们定位于细胞内细胞器。表达 GFP 标记的 AQP 的 L. donovani 的共焦显微镜显示 LdAQP9 和 LdAQP2870 的细胞内定位。实时 PCR 检测表明除 LdAQP2860 外，所有水通道蛋白均有表达，但其水平无法检测到。AQP 的三维同源建模表明，LdAQP1 的结构与 aquaglyceroporin 的拓扑相似性大于与大肠杆菌的 aquaporin 的拓扑相似性。LdAQP1 的孔在形状和大小上与其他孔非常不同。LdAQP2860 的腔在几何形状上非常不规则且不明确。为了进行功能表征，将四个 AQP 蛋白在酵母中异源表达。在缺乏关键 aquaglyceroporin 的 fps1Δ 酵母细胞中，仅 LdAQP1 表现出对渗透压敏感的表型，表明甘油转运活性。然而，在甘油产生缺失的酵母 gpd1Δ 菌株中表达 LdAQP1 和 LdAQP 假定蛋白赋予了对渗透压敏感的表型，表明水转运活性或 aquaporin 功能。我们的分析首次表明存在亚细胞水通道蛋白，并提供了 Leishmania donovani 中水通道蛋白的结构和功能特征。
