A 95 kDa protein of Plasmodium vivax and P. cynomolgi visualized by three-dimensional tomography in the caveola-vesicle complexes (Schuffner's dots) of infected erythrocytes is a member of the PHIST family
A 95 kDa protein of Plasmodium vivax and P. cynomolgi visualized by three-dimensional tomography in the caveola-vesicle complexes (Schuffner's dots) of infected erythrocytes is a member of the PHIST family
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DOI：
10.1111/j.1365-2958.2012.08060.x
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发表时间：
2012-06-01
影响因子：
3.6
通讯作者：
Galinski, Mary R.
中科院分区：
文献类型：
--
作者：
Akinyi, Sheila;Hanssen, Eric;Galinski, Mary R.
间日疟原虫和食蟹猴疟原虫在受感染的红细胞膜表面内产生许多小囊泡复合物（CVC）结构。这些与在恶性疟原虫感染的红细胞表面表达的电子致密球突起形成对比。在这里，我们调查的三维（3-D）结构的CVC和身份的主要表达的95 kDa CVC蛋白。通过来自食蟹猴疟原虫提取物的单克隆抗体对免疫沉淀物进行液相色谱串联质谱分析，将该蛋白鉴定为疟原虫螺旋散布亚端粒（PHIST）超家族的成员，计算质量为81 kDa。我们命名为PvPHIST/CVC-8195和PcyPHIST/CVC-8195，分析了它们的结构特征，包括PEXEL基序，重复序列和C-末端PHIST结构域，并显示PHIST/CVC-8195在滋养体中最高表达。我们使用电子断层扫描（ET）生成CVC的3-D图像，并使用免疫-ET显示PHIST/CVC-8195定位于CVC管状延伸的细胞质侧。在体内尝试靶向基因破坏。pcyphist/cvc-8195基因未被破坏，但通过乙胺嘧啶选择回收了含有带有tgdhfr选择盒的附加体的寄生虫。这表明PHIST/CVC-8195对于这些疟疾寄生虫的存活是必不可少的。
Plasmodium vivax and P. cynomolgi produce numerous caveolavesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob protrusions expressed at the surface of Plasmodium falciparum-infected erythrocytes. Here we investigate the three-dimensional (3-D) structure of the CVCs and the identity of a predominantly expressed 95 kDa CVC protein. Liquid chromatography tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P. cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC-8195 and PcyPHIST/CVC-8195, analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-8195 is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography (ET), and used immuno-ET to show PHIST/CVC-8195 localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-8195 gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-8195 is essential for survival of these malaria parasites.