Montalvetti Andrea, Rohloff Peter, Docampo Roberto
Laboratory of Molecular Parasitology, Department of Pathobiology and Center for Zoonoses Research, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA.
J Biol Chem. 2004 Sep 10;279(37):38673-82. doi: 10.1074/jbc.M406304200. Epub 2004 Jul 12.
We cloned an aquaporin gene from Trypanosoma cruzi (TcAQP) that encodes a protein of 231 amino acids, which is highly hydrophobic. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA) which have been shown, in other aquaporins, to be involved in the formation of an aqueous channel spanning the bilayer. TcAQP was sensitive to endo H treatment, suggesting that the protein is N-glycosylated. Oocytes of Xenopus laevis expressing TcAQP swelled under hyposmotic conditions indicating water permeability, which was abolished after preincubating oocytes with very low concentrations of the AQP inhibitors HgCl(2) and AgNO(3). glycerol transport was detected. No Immunofluorescence microscopy of T. cruzi expressing GFP-TcAQP showed co-localization of TcAQP with the vacuolar proton pyrophosphatase (V-H(+)-PPase), a marker of acidocalcisomes. This localization was confirmed by Western blotting and immunofluorescence staining using polyclonal antibodies against a C-terminal peptide of TcAQP. In addition, there was a strong anterior labeling in a vacuole, close to the flagellar pocket, that was distinct from the acidocalcisomes and that was identified by immunogold electron microscopy as the contractile vacuole complex. Taking together, the presence of an aquaporin in acidocalcisomes and the contractile vacuole complex of T. cruzi, provides support for the role of these organelles in osmotic adaptations of these parasites.
我们从克氏锥虫中克隆了一个水通道蛋白基因（TcAQP），该基因编码一个由231个氨基酸组成的高度疏水的蛋白质。该蛋白质有六个假定的跨膜结构域以及两个标志性基序天冬酰胺-脯氨酸-丙氨酸（NPA），在其他水通道蛋白中，这些基序参与形成跨越双层膜的水通道。TcAQP对内切糖苷酶H处理敏感，表明该蛋白质是N-糖基化的。表达TcAQP的非洲爪蟾卵母细胞在低渗条件下肿胀，表明具有水通透性，在用极低浓度的水通道蛋白抑制剂HgCl₂和AgNO₃预孵育卵母细胞后，这种通透性消失。检测到甘油转运。对表达绿色荧光蛋白-TcAQP的克氏锥虫进行免疫荧光显微镜观察，结果显示TcAQP与液泡质子焦磷酸酶（V-H⁺-PPase）没有共定位，V-H⁺-PPase是酸性钙小体的标志物。使用针对TcAQP C末端肽的多克隆抗体进行蛋白质印迹和免疫荧光染色，证实了这种定位。此外，在靠近鞭毛袋的一个液泡中有强烈的前部标记，该液泡与酸性钙小体不同，通过免疫金电子显微镜鉴定为收缩泡复合体。综上所述，克氏锥虫酸性钙小体和收缩泡复合体中存在水通道蛋白，为这些细胞器在这些寄生虫渗透适应中的作用提供了支持。
