Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche
Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche
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DOI：
10.1016/j.bbagen.2018.08.015
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发表时间：
2018-12-01
影响因子：
3
通讯作者：
de Koning, Harry P.
中科院分区：
文献类型：
--
作者：
Campagnaro, Gustavo D.;Nascimento, Janaina de Freitas;de Koning, Harry P.
背景：克氏锥虫是南美锥虫病的病原体，它不能合成自己的嘌呤，只能依靠宿主的补救。在其他原生动物中，嘌呤摄取是由平衡核苷转运蛋白（ENTs）介导的。结果：TcrNB 1对次黄嘌呤（Km为93.8 ± 4.7nM）和鸟嘌呤有很高的亲和力，对腺嘌呤有中等亲和力。发现TcrNT 1是一种高亲和力鸟苷/肌苷转运蛋白（肌苷K-m为1.0 +/- 0.03 μ M;鸟苷K-i为0.92 +/- 0.2 μ M）。TcrNT 2编码高亲和力胸苷转运蛋白（Km = 223.5 +/- 7.1 nM），明显偏好2 '-脱氧嘧啶。TcrNB 2，其活性不能在我们的系统中确定，可能是一种低亲和力嘌呤核碱基转运蛋白，鉴于其序列和预测的结构相似性，主要利什曼原虫NT 4。所有4个转运蛋白基因在无鞭毛体形态中均高表达，而在非分裂期表达较低。与氨基嘌呤相比，Cruzi更偏爱氧代嘌呤，并有效地转运2 ′-脱氧嘧啶。霸王cruzi ENTs显示出异常高的底物亲和力，以适应其细胞内定位。cruzi嘌呤和嘧啶转运蛋白，包括第一个编码嘧啶选择性原生动物转运蛋白的基因。
Background: Trypanosoma cruzi, the causative agent of Chagas' disease is unable to synthesise its own purines and relies on salvage from the host. In other protozoa, purine uptake has been shown to be mediated by Equilibrative Nucleoside Transporters (ENTs).Methods: To investigate the functionality of T. cruzi-encoded ENT transporters, its four putative ENT genes (TcrNB1, TcrNB2, TcrNT1 and TcrNT2) were cloned and expressed in genetically adapted Trypanosoma brucei procyclic cells from which the nucleobase transporter locus was deleted.Results: TcrNB1 displayed very high affinity for hypoxanthine (K-m 93.8 +/- 4.7 nM for) and guanine, and moderate affinity for adenine. TcrNT1 was found to be a high-affinity guanosine/inosine transporter (inosine K-m is 1.0 +/- 0.03 mu M; guanosine K-i is 0.92 +/- 0.2 mu M). TcrNT2 encoded a high-affinity thymidine transporter (K-m = 223.5 +/- 7.1 nM) with a clear preference for 2'-deoxypyrimidines. TcrNB2, whose activity could not be determined in our system, could be a low-affinity purine nucleobase transporter, given its sequence and predicted structural similarities to Leishmania major NT4. All 4 transporter genes were highly expressed in the amastigote forms, with much lower expression in the non-dividing stages.Conclusions: The data appear to show that, surprisingly, T. cruzi has a preference for oxopurines over amino-purines and efficiently transports 2'-deoxypyrimidines. The T. cruzi ENTs display exceptionally high substrate affinity as an adaptation to their intracellular localisation.General significance: This study reports the first cloning of T. cruzi purine and pyrimidine transporters, including the first gene encoding a pyrimidine-selective protozoan transporter.