Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum
Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum
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DOI：
10.1074/jbc.275.14.10683
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发表时间：
2000-04-07
影响因子：
4.8
通讯作者：
Ullman, B
中科院分区：
文献类型：
--
作者：
Carter, NS;Ben Mamoun, C;Ullman, B
恶性疟原虫是人类疟疾最致命形式的病原体，不能从头合成嘌呤，因此，从宿主获得嘌呤是不可或缺的营养需求。这种嘌呤补救过程是通过将预先形成的嘌呤转运到寄生虫体内而启动的。我们已经确定了一个编码恶性疟原虫核苷转运蛋白PfNT 1的基因，并分析了其在红细胞内寄生虫发育过程中的功能和表达。PfNT 1预测了具有11个跨膜结构域的422个氨基酸的多肽，其与平衡型核苷转运蛋白家族的其他成员同源。Southern分析和BLAST搜索的基因组研究所（TIGR）疟疾数据库表明，PfNT 1是一个单拷贝基因位于14号染色体上，北方分析的RNA从红细胞内阶段的寄生虫表明，PfNT 1的表达在整个无性生活周期，但在早期滋养体阶段显着升高。PfNT 1在非洲爪蟾卵母细胞中的功能性表达显著增加了它们摄取天然存在的D-腺苷（K-m = 13.2 μ M）和D-肌苷（K-m = 253 μ M）的能力。值得注意的是，PfNT 1与哺乳动物核苷转运蛋白不同，也具有转运立体异构体L-腺苷（K-m > 500 μ M）的能力。一组嘌呤和嘧啶核苷和碱基及其类似物的抑制研究表明，PfNT 1对嘌呤和嘧啶核苷具有广泛的底物特异性。这些数据提供了令人信服的证据，PfNT 1编码一个功能性嘌呤/嘧啶核苷转运蛋白，其表达在恶性疟原虫生命周期的无性阶段受到强烈的发育调控。此外，不寻常的能力，运输L-腺苷和重要贡献的嘌呤运输寄生虫的生存，使PfNT 1治疗评价的一个有吸引力的目标。
Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter hom P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14, Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K-m = 13.2 mu M) and D-inosine (K-m = 253 mu M) Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K-m > 500 mu M). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation.