[01] Nahla O. M. Ali dr_nahla2004@yahoo.com, Department of Parasitology, Faculty of Veterinary Medicine, University of Khartoum, Khartoum, Sudan;Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.

The hypoxanthine-guaninephosphoribosyltransferase (HGPRT) enzyme of Plasmodium berghei, plays a key role in the salvage of preformed purine nucleotides from parasite-infected erythrocytes. Since P. berghei cannot synthesize purines de novo, development of inhibitors specific for the parasite HGPRT should be an effective method of chemotherapy. This gene has been annotated as encoding PRTase activity and proposed as essential for survival of the P. berghei. The aim of this work is to identify the HGPRT from P. berghei using bioinformatic tools in searching the FullMal database for EST sequences for a cDNA clone with full length HGPRT, design specific oligonucleotides primers and then isolate PbHGPRT from the cDNA clone. The PCR analysis produced an amplicon of 696 bp which is in agreement with the predicted size from the nucleotides sequence deduced from the HGPRT cDNA open reading frame. The significance of the above results is discussed in the light of existing literature.

Plasmodium berghei, HGPRT, Phosphoribosyltransferase, Bioinformatics, EST, cDNA

[01] Ali NOM, Ibrahim ME, Grant KM, Mottram JC. (2010). Molecular cloning, characterization and overexpression of a novel cyclin from Leishmania Mexicana mexicana. Pakistan Journal of Biological Sciences 13(16): 775-784. [02] Ali NOM, Croof HI, Abdalla HS. (2011). Molecular Diagnosis of Trypanosoma evansi Infection in Dromedary Camels from Eastern and Western regions of the Sudan. Emirates Journal of Food and Agriculture 23(4): 320-329. [03] Ali NOM, Ibrahim ME, Grant KM, Mottram JC. (2012a). Leishmania mexicana: Expression; characterization & activity assessment of E. coli-expressed recombinant CRK3. European Review for Medical and Pharmacological Sciences. 16(10): 1338-1345. [04] Ali NOM, Ibrahim ME, Abdalla HS, Mukhtar MM, El-Hassan AM, Mottram JC. (2012b). Isolation and characterization of a cdc2-related protein kinase 3 (CRK3) from a Sudanese strain of Leishmania donovani. International Research Journal of Pharmacy and Pharmacology 2(9): 215-224. [05] Ali NOM, Grant KM, Mottram JC. (2013). Expression, Purification, and Biochemical Characterization of a Recombinant Protein Kinase CRK1 of Leishmania mexicana. Scholars Journal of Applied Medical Sciences 1(2): 35-40. [06] Ali NOM. Secretome Analysis of Plasmodium falciparum intraerythrocytic asexual stage using SDS-PAGE electrophoresis. International Journal of Science and Research. 2014; 3(6): 351-355. [07] Altschul SF, Gish W, Miller W, Myers EW ,Lipman DJ. Basic local alignment search tool. J. Mol. Biol. 1990; 215: 403-10. [08] Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS, Heiges M, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Miller JA, Nayak V, Pennington C, Pinney DF, Roos DS, Ross C, Stoeckert CJJr, Treatman C, Wang H. PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Res. 2009 Jan; 37 (Databaseissue): D539-43. [09] Bahl A, Brunk B, Coppel RL, Crabtree J, Diskin SJ, Fraunholz MJ, Grant GR, Gupta D, Huestis RL, Kissinger JC, Labo P, Li L, McWeeney SK, Milgram AJ, Roos DS, Schug J, Stoeckert CJJr. PlasmoDB: the Plasmodium genome resource. An integrated database providing tools for accessing, analyzing and mapping expression and sequence data (both finished and unfinished). Nucleic Acids Res. 2002 Jan1; 30(1): 87-90. [10] Baszczyňski O, Hocková D, Janeba Z, Holý A, Jansa P, Dračínský M, Keough DT, Guddat LW. The effect of novel [3-fluoro-(2-phosphonoethoxy) propyl] purines on the inhibition of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases. Eur J. Med. Chem. 2013 Sep; 67: 81-9. [11] Büngener W and Nielsen G. Nukleinsäurens toff wechsel bei experimenteller Malaria. 2. Einbau von Adenosin und Hypoxanthin in die Nukleinsäuren von Malaria parasite (Plasmodium berghei und Plasmodium vinckei) Z Tropen med Parasitol. 1968 Jun; 19(2): 185–197. [12] Craig SP 3rd and Eakin AE. Purine phosphoribosyltransferases. J. Biol. Chem. 2000 Jul7; 275(27): 20231-4. [13] Franke-Fayard B, Trueman H, Ramesar J, Mendoza J, vanderKeur M, et al. A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle. Molecular and Biochemical Parasitology. 2004; 137: 23–33. [14] Frevert U, Engelmann S, Zougbede S, Stange J, Ng B, et al. Intravital observation of Plasmodium berghei sporozoite infection of the liver. PLoS Biol. 2005; 3: e192. [15] Gomes FC, Ali NO, Brown E, Walker RG, Grant KM, Mottram JC. (2010). Leishmania Mexicana cyclin-dependent kinase complex CRK3:CYCA is active in the absence of T-loop phosphorylation. Molecular and Biochemical Parasitology. 171(2):89-96. [16] Gutteridge WE and Trigg PI. Incorporation of radioactive precursors into DNA and RNA of Plasmodium knowlesi in vitro. J. Protozool.1970 Feb; 17(1): 89–96. [17] Gutteridge WE and Coombs GH. (1977). Biochemistry of Parasitic Protozoa, University Park Press, Baltimore. [18] Hillyer JF, Barreau C,Vernick KD. Efficiency of salivary gland invasion by malaria sporozoites is controlled by rapid sporozoite destruction in the mosquito haemocoel. Int. J. Parasitol. 2007; 37: 673–681. [19] Jin Y, Kebaier C,Vanderberg J. Direct microscopic quantification of dynamics of Plasmodium berghei sporozoite transmission from mosquitoes to mice. Infect. Immun. 2007; 75: 5532–5539. [20] Kelley WN and Wyngaarden JB. (1983). In Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL and Brown MS. (eds), The Metabolic Basis of Inherited Disease, 5th edn, pp. 1115-1143. McGraw-Hill NewYork. [21] Keough DT, Hocková D, Holý A, Naesens LM, Skinner-Adams TS, Jersey Jd, Guddat LW. Inhibition of hypoxanthine-guaninephosphoribosyltransferase by acyclic nucleoside phosphonates: a new class of antimalarial therapeutics. J. Med. Chem. 2009 Jul 23; 52(14): 4391-9. [22] Keough DT, Ng AL, Winzor DJ, Emmerson BT,deJersey J. Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthinephosphoribosyltransferase and comparison with the human enzyme. Mol. Biochem. Parasitol. 1999; 98: 29–41. [23] King JG and Hillyer JF. Infection-Induced Interaction between the Mosquito Circulatory and Immune Systems. PLoSPathog. 2012; 8: e1003058. [24] Königk E. Salvage syntheses and their relationship to nucleic acid metabolism. Bull World Health Organ. 1977; 55(2-3): 249–252. [25] Krečmerová M, Dračínský M, Hocková D,Holý A, Keough DT, Guddat LW. Synthesis of purine N9-[2-hydroxy-3-O-(phosphonomethoxy) propyl] derivatives and their side-chain modified analogs as potential antimalarial agents. Bioorg. Med. Chem. 2012 Feb1; 20(3)1222-30. [26] Krenitsky TA, Papaioannou R, Elion GB. Human hypoxanthine phosphoribosyltransferase. I. Purification, properties, and specificity. J. Biol. Chem. 1969; 244: 1263–1270. [27] Lee CC, Craig SPIII, Eakin AE. A single amino acid substitution in the human and a bacterial hypoxanthine phosphoribosyltransferase modulates specificity for the binding of guanine. Biochemistry. 1998; 37: 3491–3498. [28] Lukow I, Schmidt G, Walter RD, Königk E. Adenosin monophosphat-Salvage-Synthese bei Plasmodium chabaudi. Z Tropen med Parasitol. 1973 Dec; 24(4): 500–504. [29] Musick WD. Structural features of the phosphoribosyltransferases and their relationship to the human deficiency disorders of purine and pyrimidine metabolism. CRC Crit. Rev. Biochem. 1981; 11(1): 1-34. [30] Olliaro PL and Yuthavong Y. An overview of chemotherapeutic targets for antimalarial drug discovery. Pharmacol. Ther. 1999; 81: 91–110. [31] Queen SA, VanderJagt DL, Reyes P. Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum. Mol. Biochem. Parasitol. 1988 Aug; 30(2): 123-33. [32] Queen SA, Vander Jagt DL, Reyes P. Characterization of adenine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum. Biochem. Biophys. Acta. 1989 Jul6; 996(3): 160-5. [33] Reyes P, Rathod PK, Sanchez DJ, Mrema JE, Rieckmann KH, Heidrich HG. Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum. Mol. Biochem. Parasitol. 1982 May; 5(5): 275–290. [34] Schimandle CM, Mole LA, Sherman IW. Purification of hypoxanthine-guaninephosphoribosyltransferase of Plasmodium lophurae. Mol. Biochem. Parasitol. 1987 Feb; 23(1): 39–45. [35] Sculley DG, Dawson PA, Emmerson BT, Gordon RB. A review of the molecular basis of hypoxanthine-guaninephosphoribosyltransferase (HPRT) deficiency. Hum. Genet. 1992 Nov; 90(3): 195-207. [36] Shahabuddin M and Scaife J. The gene for hypoxanthine phosphoribosyltransferase of Plasmodium falciparum complements a bacterial HPT mutation. Mol. Biochem. Parasitol. 1990 Jun; 41(2): 281-8. [37] Sherman IW. Biochemistry of Plasmodium (malarial parasites). Microbiol. Rev. 1979 Dec; 43(4): 453-95. [38] Subbayya IN and Balaram H. A point mutation at the subunit interface of hypoxanthine-guanine-xanthine phosphoribosyltransferase impairs activity: role of oligomerization in catalysis. FEBS Lett. 2002 Jun 19; 521(1-3): 72-6. [39] Sujay Subbayya IN and Balaram H. Evidence for multiple active states of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase. Biochem. Biophys. Res. Commun. 2000; 279: 433–437. [40] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22: 4673-80. [41] Ullman B and Carter D. Hypoxanthine-guaninephosphoribosyltransferase as a therapeutic target in protozoal infections. Infect. Agents. Dis. 1995 Mar; 4(1): 29-40. [42] Vasanthakumar G, Davis RLJr, Sullivan MA, Donahue JP. Cloning and expression in Escherichia coli of a hypoxanthine-guaninephosphoribosyltransferase-encoding cDNA from Plasmodium falciparum. Gene. 1990 Jul 2; 91(1): 63-9. [43] Vincke IH. (1954). Natural history of Plasmodium berghei. Indian J. Malariol. 8: 245–256. [44] Vincke IH and Lips M. (1948). Un nouveau Plasmodium d’un rongeur sauvage du Congo: Plasmodium berghei. Ann. Soc. Belge. Med. Trop. 28: 97–104. [45] Walter RD and Königk E. Hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase from Plasmodium chabaudi, purification and properties. Tropen. Med. Parasitol. 1974 Jun; 25(2): 227–235. [46] Watanabe J, Wakaguri H, Sasaki M, Suzuki Y, Sugano S. Comparasite: a database for comparative study of transcriptomes of parasites defined by full-length cDNAs. Nucleic Acids Research, 2007, Vol.35, Database issue D431–D438. [47] Webster HK and Whaun JM. Purine metabolism during continuous erythrocyte culture of human malaria parasites (P. falciparum). Prog. Clin. Biol. Res. 1981; 55: 557–573. [48] Webster HK, Whaun JM. Antimalarial properties of bredinin. Prediction based on identification of differences in human host-parasite purine metabolism. J. Clin. Invest. 1982 Aug; 70(2): 461-9. [49] Webster HK, Whaun JM, Walker MD, Bean TL. Synthesis of adenosine nucleotides from hypoxanthine by human malaria parasites (Plasmodium falciparum) in continuous erythrocyte culture: inhibition by hadacidin but not alanosine. Biochem. Pharmacol. 1984 May1; 33(9): 1555-7. [50] Wu Y, Fairfield AS, Oduola A, Cypess RH. The Malaria Research and Reference Reagent Resource (MR4) Center—creating African opportunities. Afr. J. Med. Med. Sci. 2001; 30 Suppl: 52-4. [51] Yamada KA and Sherman IW. Purine metabolism by the avian malarial parasite Plasmodium lophurae. Mol. Biochem. Parasitol. 1981 Aug; 3(4): 253–264.