Soni Awakash, Goyal Manish, Prakash Kirtika, Bhardwaj Jyoti, Siddiqui Arif Jamal, Puri Sunil K
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226031, India; Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India.
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226031, India.
Gene. 2015 Jul 15;566(1):109-19. doi: 10.1016/j.gene.2015.04.037. Epub 2015 Apr 16.
Malaria parasite resides within the host red blood cells, where it degrades vast amount of haemoglobin. During haemoglobin degradation, toxic free heme is liberated which subsequently gets converted into hemozoin. This process is facilitated by action of various proteins viz. heme detoxification protein (HDP), and histidine rich proteins II and III (HRP II & III). Out of these, HDP is the most potent in hemozoin formation and plays indispensible role for parasite survival. Despite this, the detailed study of HDP from rodent and simian parasite has not been performed till date. Here, we have cloned and sequenced hdp gene from different malaria parasites Plasmodium vinckei, Plasmodium yoelii, Plasmodium knowlesi, and Plasmodium cynomolgi. Furthermore, HDP from P. vinckei (PvHDP) was over-expressed and purified for detailed characterization. The PvHDP is cytosolic, expressed throughout the intra erythrocytic stages and its expression is higher in late trophozoite and schizont stages of parasite. The PvHDP interacts with free heme (KD=89 nM) and efficiently converts heme into hemozoin in a time and concentration dependent manner. Moreover, PvHDP showed activity in acidic pH and over a broad range of temperature. Histidine modification of PvHDP using DEPC showed reduction in heme binding and hemozoin formation, thus emphasizing the importance of histidine residues in heme binding and subsequent hemozoin production. Furthermore, applicability of PvHDP to screen anti-plasmodial agents (targeting heme to hemozoin conversion) was also determined using chloroquine, and mefloquine as reference antimalarials. Results showed that these drugs inhibit heme polymerization effectively in a concentration dependent manner. In conclusion, our study identified and biochemically characterized HDP from rodent malaria parasite P. vinckei and this will help to develop a high throughput assay to evaluate new antimalarials targeting hemozoin pathway.
疟原虫寄生于宿主红细胞内，在其中降解大量血红蛋白。在血红蛋白降解过程中，有毒的游离血红素被释放出来，随后转化为疟色素。这一过程由多种蛋白质的作用促成，即血红素解毒蛋白（HDP）、富含组氨酸的蛋白质II和III（HRP II和III）。其中，HDP在疟色素形成中最为有效，对疟原虫的存活起着不可或缺的作用。尽管如此，迄今为止尚未对来自啮齿动物和猿类疟原虫的HDP进行详细研究。在此，我们从不同的疟原虫物种——文氏疟原虫、约氏疟原虫、诺氏疟原虫和食蟹猴疟原虫中克隆并测序了hdp基因。此外，对来自文氏疟原虫的HDP（PvHDP）进行了过表达和纯化，以进行详细表征。PvHDP存在于细胞质中，在整个红细胞内期均有表达，在疟原虫的晚期滋养体和裂殖体阶段表达较高。PvHDP与游离血红素相互作用（解离常数KD = 89 nM），并以时间和浓度依赖的方式有效地将血红素转化为疟色素。此外，PvHDP在酸性pH值和较宽的温度范围内均表现出活性。使用焦碳酸二乙酯（DEPC）对PvHDP进行组氨酸修饰后，血红素结合和疟色素形成减少，从而强调了组氨酸残基在血红素结合及随后的疟色素产生中的重要性。此外，还以氯喹和甲氟喹作为参考抗疟药，确定了PvHDP在筛选抗疟原虫药物（靶向血红素向疟色素转化）方面的适用性。结果表明，这些药物以浓度依赖的方式有效地抑制了血红素聚合。总之，我们的研究鉴定并对来自啮齿动物疟原虫文氏疟原虫的HDP进行了生化表征，这将有助于开发一种高通量检测方法，以评估针对疟色素途径的新型抗疟药。
