title: Cloning, expression and functional characterization of heme detoxification protein (HDP) from the rodent malaria parasite Plasmodium vinckei.
abstract: 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.
authors.affiliation: 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.
authors.forename: Awakash
authors.initials: A
authors.lastname: Soni
authors.affiliation: Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226031, India.
authors.forename: Manish
authors.initials: M
authors.lastname: Goyal
authors.affiliation: Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226031, India.
authors.forename: Kirtika
authors.initials: K
authors.lastname: Prakash
authors.affiliation: 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.
authors.forename: Jyoti
authors.initials: J
authors.lastname: Bhardwaj
authors.affiliation: 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.
authors.forename: Arif Jamal
authors.initials: AJ
authors.lastname: Siddiqui
authors.affiliation: 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. Electronic address: sk_puri@cdri.res.in.
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Puri
mesh_terms: D000595:Amino Acid Sequence; D000962:Antimalarials / Q000494:pharmacology; D003001:Cloning, Molecular; D015870:Gene Expression; D006418:Heme / Q000378:metabolism; D006420:Hemeproteins / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010961:Plasmodium / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000962:Antimalarials; D006420:Hemeproteins; D015800:Protozoan Proteins; C020753:hemozoin; D006418:Heme; D006639:Histidine
keywords: Haemoglobin; Heme and Malaria; Heme detoxification protein; Hemozoin; Plasmodium vinckei