Kaiser Annette, Langer Barbara, Przyborski Jude, Kersting David, Krüger Mirko
Medical Research Centre, Institute for Pharmacogenetics, University Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany.
Institute for Pharmacology, Hufelandstrasse 55, 45147 Essen, Germany.
PLoS One. 2015 Nov 5;10(11):e0140994. doi: 10.1371/journal.pone.0140994. eCollection 2015.
During its development the malaria parasite P. falciparum has to adapt to various different environmental contexts. Key cellular mechanisms involving G-protein coupled signal transduction chains are assumed to act at these interfaces. Heterotrimeric G-proteins are absent in Plasmodium. We here describe the first cloning and expression of a putative, non-canonical Ras-like G protein (acronym PfG) from Plasmodium. PfG reveals an open reading frame of 2736 bp encoding a protein of 912 amino acids with a theoretical pI of 8.68 and a molecular weight of 108.57 kDa. Transcript levels and expression are significantly increased in the erythrocytic phase in particular during schizont and gametocyte formation. Most notably, PfG has GTP binding capacity and GTPase activity due to an EngA2 domain present in small Ras-like GTPases in a variety of Bacillus species and Mycobacteria. By contrast, plasmodial PfG is divergent from any human alpha-subunit. PfG was expressed in E. coli as a histidine-tagged fusion protein and was stable only for 3.5 hours. Purification was only possible under native conditions by Nickel-chelate chromatography and subsequent separation by Blue Native PAGE. Binding of a fluorescent GTP analogue BODIPY® FL guanosine 5'O-(thiotriphosphate) was determined by fluorescence emission. Mastoparan stimulated GTP binding in the presence of Mg2+. GTPase activity was determined colorimetrically. Activity expressed as absolute fluorescence was 50% higher for the human paralogue than the activity of the parasitic enzyme. The PfG protein is expressed in the erythrocytic stages and binds GTP after immunoprecipitation. Immunofluorescence using specific antiserum suggests that PfG localizes to the parasite cytosol. The current data suggest that the putitative, Ras-like G-protein might be involved in a non-canonical signaling pathway in Plasmodium. Research on the function of PfG with respect to pathogenesis and antimalarial chemotherapy is currently under way.
在其发育过程中，恶性疟原虫必须适应各种不同的环境。涉及G蛋白偶联信号转导链的关键细胞机制被认为在这些界面起作用。疟原虫中不存在异源三聚体G蛋白。我们在此描述了从疟原虫中首次克隆和表达一种假定的非经典类Ras G蛋白（简称PfG）。PfG显示出一个2736 bp的开放阅读框，编码一个912个氨基酸的蛋白质，理论pI为8.68，分子量为108.57 kDa。转录水平和表达在红细胞期显著增加，特别是在裂殖体和配子体形成期间。最值得注意的是，由于多种芽孢杆菌属和分枝杆菌中小类Ras GTP酶中存在EngA2结构域，PfG具有GTP结合能力和GTP酶活性。相比之下，疟原虫的PfG与任何人类α亚基都不同。PfG在大肠杆菌中作为组氨酸标签融合蛋白表达，仅稳定3.5小时。仅在天然条件下通过镍螯合色谱法并随后通过蓝色原胶PAGE分离才能进行纯化。通过荧光发射测定荧光GTP类似物BODIPY® FL鸟苷5'O-(硫代三磷酸)的结合。在Mg2+存在下，马斯托帕兰刺激GTP结合。通过比色法测定GTP酶活性。以绝对荧光表示的活性，人类同源物比寄生虫酶的活性高50%。PfG蛋白在红细胞期表达，免疫沉淀后结合GTP。使用特异性抗血清进行的免疫荧光表明PfG定位于寄生虫胞质溶胶。目前的数据表明，假定的类Ras G蛋白可能参与疟原虫中的非经典信号通路。目前正在对PfG在发病机制和抗疟化疗方面的功能进行研究。
