The Putative Glutathione Peroxidase Gene ofPlasmodium falciparum Codes for a Thioredoxin Peroxidase

Abstract

A putative glutathione peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It was active with H2O2 and organic hydroperoxides but, unlike phospholipid hydroperoxide glutathione peroxidase, not with phosphatidylcholine hydroperoxide. With glutathione peroxidases it shares the ping-pong mechanism with infiniteV max and K m when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoperoxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum(Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K mof 10 μm and a k 1′ of 0.55 s−1. The apparent k 1′ for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 × 104m−1 s−1, 3.3 × 103m−1s−1, and 2.5 × 103m−1 s−1, respectively. k 2′ for reduction by autologous thioredoxin is 5.4 × 104m-1s−1 (21.2m−1 s−1for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense. A putative glutathione peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It was active with H2O2 and organic hydroperoxides but, unlike phospholipid hydroperoxide glutathione peroxidase, not with phosphatidylcholine hydroperoxide. With glutathione peroxidases it shares the ping-pong mechanism with infiniteV max and K m when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoperoxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum(Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K mof 10 μm and a k 1′ of 0.55 s−1. The apparent k 1′ for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 × 104m−1 s−1, 3.3 × 103m−1s−1, and 2.5 × 103m−1 s−1, respectively. k 2′ for reduction by autologous thioredoxin is 5.4 × 104m-1s−1 (21.2m−1 s−1for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense. glutathione peroxidase plasma GPx cytosolic GPx phospholipidhydroperoxide GPx thioredoxin peroxidase ofP. falciparum thioredoxin Trx of P. falciparum matrix-assisted laser desorption and ionization time of flight mass spectrometry t-butyl hydroperoxide Plasmodium falciparum, the causative agent of the most severe form of malaria, reportedly displays glutathione peroxidase (GPx)1 activity (1Fairfield A.S. Abosch A. Ranz A. Eaton J.W. Mol. Biochem. Parasitol. 1988; 30: 77-82Crossref PubMed Scopus (59) Google Scholar). Also, a putative GPx gene was isolated from P. falciparum (2Gamain B. Langsley G. Fourmaux M.N. Touzel J.P. Camus D. Dive D. Slomianny C. Mol. Biochem. Parasitol. 1996; 78: 237-248Crossref PubMed Scopus (40) Google Scholar). Complementing enzymes that may constitute a glutathione-dependent antioxidant defense system have also been characterized in Plasmodia species. They can rely on their own GSH biosynthesis (3Ayi K. Cappadoro M. Branca M. Turrini F. Arese P. FEBS Lett. 1998; 424: 257-261Crossref PubMed Scopus (41) Google Scholar), and the pertinent key enzyme, γ-glutamylcysteine synthetase, was identified (4Luersen K. Walter R.D. Müller S. Mol. Biochem. Parasitol. 1999; 98: 131-142Crossref PubMed Scopus (41) Google Scholar). The parasites regenerate GSH from GSSG more efficiently than their host cells (3Ayi K. Cappadoro M. Branca M. Turrini F. Arese P. FEBS Lett. 1998; 424: 257-261Crossref PubMed Scopus (41) Google Scholar), and a plasmodial glutathione reductase was also characterized (5Farber P.M. Becker K. Müller S. Schirmer R.H. Franklin R.M. Eur. J. Biochem. 1996; 239: 655-661Crossref PubMed Scopus (54) Google Scholar).Plasmodia species require an efficient antioxidant defense system, since they have to survive in a pro-oxidant habitat, the red blood cell. Moreover, they have to overcome the oxidant attack of phagocytes during the critical period between dissemination from, and reinvasion of the host cell (6Malhotra K. Salmon D. Le Bras J. Vilde J.L. Infect. Immun. 1988; 56: 3305-3309Crossref PubMed Google Scholar, 7Jensen J.B. Vande Waa J.A. J. Immunol. Methods. 1988; 112: 201-205Crossref PubMed Scopus (80) Google Scholar). Within the infected erythrocytes,Plasmodia species appear to enhance oxidative stress, as indicated by the generation of methemoglobin (8Atamna H. Ginsburg H. Mol. Biochem. Parasitol. 1993; 61: 231-241Crossref PubMed Scopus (180) Google Scholar) and hydroxyalkenals (9Buffinton G.D. Hunt N.H. Cowden W.B. Clark I.A. Biochem. J. 1988; 249: 63-68Crossref PubMed Scopus (42) Google Scholar, 10Schwarzer E. Müller O. Arese P. Siems W.G. Grune T. FEBS Lett. 1996; 388: 119-122Crossref PubMed Scopus (71) Google Scholar). On the other hand, they are known to be sensitive to oxidant killing, as evident from the peroxide nature and pro-oxidant potential of many antimalarial drugs and impaired survival in host cells with disturbed hydroperoxide metabolism (for review see Ref. 11Flohé L. Hecht H.J. Steinert P. Free Radic. Biol. Med. 1999; 27: 966-984Crossref PubMed Scopus (184) Google Scholar). Plasmodial enzymes involved in the antioxidant defense of the parasite have therefore attracted attention as potential targets for the development of novel antimalarials. In this context the putative plasmodial GPx gene appeared particularly intriguing. Encoding a sulfur homologue of the mammalian selenoproteins, the gene product should be substantially less efficient than the host cell enzymes. (12Rocher C. Lalanne J. Chaudiere J. Eur. J. Biochem. 1992; 205: 955-960Crossref PubMed Scopus (136) Google Scholar, 13Maiorino M. Aumann K-D. Brigelius-Flohé R. Doria D. van den Heuvel J. McCarthy J. Roveri A. Ursini F. Flohé L. Biol. Chem. Hoppe-Seyler. 1995; 376: 651-660Crossref PubMed Scopus (222) Google Scholar). The comparatively high sensitivity of Plasmodia species to hydroperoxides could therefore result from the necessity to rely on sulfur-catalyzed hydroperoxide reduction, whereas the host cells make use of the more efficient selenium catalysis. Beyond, the amino acid sequence deduced from the plasmodial GPx gene resembles phospholipid hydroperoxide peroxidase (PHGPx, GPx-4), which displays a degenerate substrate specificity (14Ursini F. Maiorino M. Brigelius-Flohé R. Aumann K.D. Roveri A. Schomburg D. Flohé L. Methods Enzymol. 1995; 252: 38-53Crossref PubMed Scopus (671) Google Scholar). Correspondingly, it could not be taken for granted that the plasmodial GPx homologue is indeed a glutathione peroxidase. The plasmodial GPx gene was therefore heterologously expressed inEscherichia coli, and the protein was purified in sufficient quantities to allow an in-depth functional analysis. As expected, it proved to be a peroxidase acting on a broad spectrum of hydroperoxides with low efficiency. Also, the reaction rates with GSH were surprisingly low. Instead, thioredoxins reduced the enzyme efficiently enough to reclassify this member of the glutathione peroxidase family as a thioredoxin peroxidase and to substitute the acronym PfTPx for the originally introduced glutathione peroxidase of P. falciparum (PfGPx) (2Gamain B. Langsley G. Fourmaux M.N. Touzel J.P. Camus D. Dive D. Slomianny C. Mol. Biochem. Parasitol. 1996; 78: 237-248Crossref PubMed Scopus (40) Google Scholar). The full-length cDNA encoding the PfTPx was amplified by reverse transcriptase-polymerase chain reaction using asynchronous blood stage RNA and cloned into the pET5a vector (Calbiochem-Novabiochem) between the EcoRI and NdeI sites and transformed intoE. coli BL21 (DE3) pLysS. The clone was grown in carbenicillin (1 mg/ml)-supplemented LB media at 37 °C and 180 rpm to an A 600 of 0.5 and induced with 0.4 mmisopropyl-1-thio-β-d-galactopyranoside. The culture was grown for an additional 5 h and controlled for expression by SDS-polyacrylamide gel electrophoresis. For routine preparation, cells were harvested 3 h after induction, resuspended in 50 mm Tris, 1 mm dithiothreitol, pH 8.5 (buffer A), disrupted with a French press at the 900 p.s.i., and centrifuged at 18,000 rpm for 10 min. The supernatant was loaded on a Macro-Prep® High Q Support fitted on a Macro-Prep® High S Support column with a flow rate of 2 ml/min (Bio-Rad). The column was washed with 10 bed volumes of buffer A. The Macro Prep® High S Support column alone was eluted with a NaCl gradient (0–2 m) in the same buffer. Fractions with GPx activity were concentrated by ultrafiltration (Omega Cell, cut-off 10 kDa, Pall Gelman Sciences, An Arbor, MI), loaded onto a Sephacryl S-200 (Amersham Pharmacia Biotech) gel filtration column equilibrated with 0.1 m Tris, 0.1 m NaCl, 5 mm EDTA, pH 7.6, and eluted at a flow rate of 0.5 ml/min. Active fractions were analyzed by SDS-polyacrylamide gel electrophoresis for homogeneity and stored at 4 °C. The molecular mass of denatured PfTPx was estimated by silver-stained (15Butcher L.A. Tomkins J.K. Anal. Biochem. 1985; 148: 348-388Crossref Scopus (58) Google Scholar) SDS-polyacrylamide gel electrophoresis using the Phast SystemTM (Amersham Pharmacia Biotech) with an acrylamide gradient of 8–25% and a 10-kDa ladder as reference. The molecular mass of native PfTPx was estimated by gel filtration on a Sephadex S-100 column equilibrated with a 0.1 m Tris buffer of pH 7.6 containing 5 mm EDTA at a flow rate of 0.5 ml/min. Chymotrypsinogen, bovine serum albumin, blue dextran, and cytochromec were co-chromatographed as reference proteins. Protein concentration was determined according to Bradford (16Bradford M.M. Anal. Biochem. PubMed Scopus Google Scholar) with the from bovine serum as the The molecular was determined by matrix-assisted laser desorption and ionization time of flight mass spectrometry with a mass For this the protein was with washed with the and resuspended in acid in and The protein at a concentration 50 was at were using bovine serum as were for was with an A GPx activity was by the glutathione oxidation at at °C in 0.5 containing in 0.1 m Tris, pH 7.6, 5 3.3 mm and μm t-butyl hydroperoxide L. J. H. of Free and in Scholar). for the hydroperoxide substrate was with hydroperoxide H2O2 and phosphatidylcholine hydroperoxide according to Maiorino M. C. Ursini F. Methods Enzymol. PubMed Scopus Google Scholar). The concentration of hydroperoxides was determined by the GPx reaction to to For of and hydroperoxide bovine GPx for phosphatidylcholine hydroperoxide a of M. J.B. Brigelius-Flohé R. F. Roveri A. Steinert P. Ursini F. Flohé L. J. 1998; PubMed Scopus Google Scholar) was peroxidase activity was determined by GSH by thioredoxin and glutathione reductase by thioredoxin In the thioredoxin was to the thioredoxin reductase of the same species. was taken that the thioredoxin reductase E. was from thioredoxin was by A. and thioredoxin as as thioredoxin reductase of P. falciparum were as Schirmer R.H. R. Becker K. J. Biol. Chem. PubMed Scopus Google Scholar, S. P.M. Becker K. Schirmer R.H. Walter R.D. Mol. Biochem. Parasitol. 1996; PubMed Scopus Google Scholar). For the glutathione peroxidase was at GSH from 3.3 to mm and hydroperoxides peroxidase were with thioredoxin of P. falciparum at between and 5 reaction rates were were by the according to J.W. Biochem. PubMed Scopus (42) Google Scholar) and analyzed as by K. Chem. Google Scholar) for E. coli BL21 (DE3) cells transformed with the expression efficiently PfTPx with as indicated by the of a of The heterologously expressed protein 2 h after and appeared to for at more The a product of apparent electrophoretic homogeneity The apparent as the molecular mass of by spectrometry was less than the molecular mass of for the deduced amino acid sequence of full-length PfTPx an amino acid sequence of the deduced PfTPx sequence in Within the determined molecular mass with that for a sequence to of PfTPx with of glutathione (Swiss-Prot accession GPx accession number GPx accession number and (Swiss-Prot accession number acid of PfTPx in other GPx are the and are with an in of GSH by are with an of PfTPx with mammalian glutathione peroxidases that the plasmodial enzyme is a member of the GPx yet is more to than to of the other GPx and with and respectively. the PfTPx sequence as by expression in E. coli, to the of amino acid in R. Aumann H. G. M. D. Maiorino M. Roveri A. R. Ursini F. E. Flohé L. J. Biol. Chem. Google Scholar). Like PfTPx proved to be when to Sephadex gel filtration The of the enzyme was by GPx activity It eluted with an apparent molecular mass of kDa, whereas activity could be in the fractions to the molecular mass of its As is evident from the of O. R. A. Eur. J. Biochem. PubMed Scopus Google Scholar) and B. B. R. J. Mol. Biol. PubMed Scopus Google Scholar), in GPx are by to the at and of the PfTPx sequence L. Aumann K-D. Brigelius-Flohé R. Schomburg D. Ursini F. K. Active and Scholar). They are in PfTPx and which their was more active with less active with H2O2 and and unlike not at phosphatidylcholine hydroperoxide not In of PfTPx appeared than bovine as a reference GPx as routine by orders of magnitude The mechanism for hydroperoxide reduction by PfTPx was by of the J.W. Biochem. PubMed Scopus (42) Google Scholar) at GSH which were time by and a concentration of which time and to of the reaction the of at of 2 were and the at pertinent time as for the of by GSH in The are as K. Chem. Google Scholar), in which the are by enzyme to the of as As in for of as is for the GSH the apparent max for of a at the The same was by PfTPx with H2O2 and hydroperoxide as as GSH was as substrate It can be by a for substrate ping-pong 1 the limiting max m are of enzyme can be to the of is slower than the reaction the not at as is presumed in 2 3 4 In this the is as the rate k for the reaction of reduced enzyme with and on the nature of the peroxide k is as k k and may be as k since the reaction in 2 should be the k the of the reduced enzyme by GSH according to 3 the of k more 5 The of PfTPx is to that of the glutathione peroxidases L. G. E. Chem. PubMed Scopus Google Scholar, H. Müller Flohé L. Chem. PubMed Scopus Google Scholar, F. Maiorino M. C. 1985; PubMed Scopus Google Scholar, P. Biochem. 1993; PubMed Scopus (71) Google Scholar). the and of PfTPx from those of the Instead, they are to those of a sulfur homologue of by for is μm and is μm the with a of and of μm M. Aumann K-D. Brigelius-Flohé R. Doria D. van den Heuvel J. McCarthy J. Roveri A. Ursini F. Flohé L. Biol. Chem. Hoppe-Seyler. 1995; 376: 651-660Crossref PubMed Scopus (222) Google Scholar), resembles for the reduction of by The apparent 3 for concentration of are the GSH The the at that the which that the m of PfTPx are for the of and apparent rate of PfTPx for the reduction of hydroperoxides by × × × × × × and of PfTPx are from of The is for for the reaction of PfTPx not whereas 1′ on the nature of the hydroperoxide. phosphatidylcholine for are taken from Maiorino M. Aumann K-D. Brigelius-Flohé R. Doria D. van den Heuvel J. McCarthy J. Roveri A. Ursini F. Flohé L. Biol. Chem. 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The rates with thioredoxin of E. coli and in the were to those with 10 mm and autologous plasmodial thioredoxin at a concentration of 5 μm a faster of PfTPx for of Trx coli Trx as substrate was replaced by Trx of E. coli and P. falciparum and glutathione reductase as enzyme was replaced by the autologous thioredoxin With of the the rate was at as high as that with GSH at the in a GSH as substrate was replaced by Trx of E. coli and P. falciparum and glutathione reductase as enzyme was replaced by the autologous thioredoxin With of the the rate was at as high as that with GSH at the on the of PfTPx were analyzed with thioredoxin of P. falciparum and 5 that also with a ping-pong is The a when with the with GSH as substrate. The the at with m Also, the for is than for GSH and the allow the The substrate not the reaction of the reduced enzyme with the hydroperoxide are not for the and the as for a ping-pong therefore can result from a of rate in the of the of a between the enzyme and faster with k than its The limiting K m for the is by It the K and can be taken as a of for The rate is k The high k and with a low K m as a substrate of of the reduction of by are on of as in at 5 μm The the at which a with as the of of PfTPx with GSH and as m × × × the were from of the peroxide substrate. and k 1′ are those for in a For the were from of the peroxide substrate. and k 1′ are those for The glutathione peroxidase family of is the (2Gamain B. Langsley G. Fourmaux M.N. Touzel J.P. Camus D. Dive D. Slomianny C. Mol. Biochem. Parasitol. 1996; 78: 237-248Crossref PubMed Scopus (40) Google Scholar, F. Maiorino M. Brigelius-Flohé R. Aumann K.D. Roveri A. Schomburg D. Flohé L. 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Ursini F. Flohé L. Biol. Chem. Hoppe-Seyler. 1995; 376: 651-660Crossref PubMed Scopus (222) Google Scholar). also in that could be glutathione peroxidases putative thioredoxin peroxidases have been identified accession number and are In mammals, a member of this family been to be a GPx C. J. Biol. Chem. PubMed Scopus Google Scholar), and in amino enzymes are M. D. M. C. T. J. T. A. A. B. 1999; PubMed Scopus Google Scholar). many to glutathione-dependent peroxide metabolism to antioxidant defense in Plasmodium to be a low peroxidase with a for the thioredoxin, is not the to this for in enzyme and activity and for the analysis.