Trypanothione dependent peroxide metabolism in Crithidia fasciculata and Trypanosoma brucei

Molecular and Biochemical Parasitology, 24 (1987) 39--45

39

Elsevier MBP 00800

Trypanothione dependent peroxide metabolism in Crithidia fasciculata and Trypanosoma brucei Graeme B. Henderson, Alan H. Fairlamb and Anthony Cerami Laboratory of Medical Biochemistry, The Rockefeller University, New York, NY, U.S.A.

(Received 20 October 1986; accepted 8 January 1987)

A trypanothione-dependent peroxidase activity has been identified in the insect trypanOsomatid Crithidia fasciculata and in the mammalian trypanosome Trypanosoma brucei. Using organic hydroperoxides as oxidant, specificperoxidase activities in these organisms are 5.0 and 1.0 nmol min x (108 cells) ~ respectively. The T. brucei peroxidase had an activity of 0.4 nmol min t (108 cells) ~using hydrogen peroxide as oxidant. The enzyme is specific for the NI,Na-bis(glutathionyl)spermidineconjugate (dihydrotrypanothione); Nl-mono-glutathionylspermidine is not a substrate. Experiments to demonstrate that this parasite peroxidase may contain selenium were inconclusive. However, bloodstream T. brucei can incorporate radiolabelled selenite into proteins. Key words: Crithidia fasciculata; Trypanosoma brucei; Trypanothione; Peroxide metabolism

Introduction The ability of t r y p a n o s o m e s and leishmanias to withstand oxidant d a m a g e by reactive oxygen species is essential for the survival of these organisms in their m a m m a l i a n host [1]. Toxic oxygen metabolites such as O~, H202, and HO" may be produced internally as a product of normal aerobic metabolism of the parasite or externally as part of the host defences in response to invasion. In m a m m a l i a n cells, low intracellular concentrations of superoxide and hydrogen peroxide are maintained by the sequential action of superoxide dismutase, glutathione peroxidase and glutathione reductase [2]; in this way m a m m a l i a n cells minimize the formation of extremely reacCorrespondence address: Dr. G.B. Henderson, Laboratory of Medical Biochemistry',The Rockefeller University, 1230 York Ave., New York, NY 10021, U.S.A. Abbreviations: GR, glutathione disulfide reductase; TR, try-

panothione reductase; GSH, glutathione; trypanothione disulfide (T(S)2), N 1,NS-bis(L-3,-glutamyl-L-hemicystinylglycyl)spermidine; dihydro-trypanothione (T(SH)2), N1,NS-bis(L3,-glutamyl-L-cysteinylglycyl)spermidine;N~-mono-glutathionyl-spermidine, N~-L-~-glutamyl-L-cysteinylglycylspermidine.

tive free radicals such as the hydroxyl radical ( H O ' ) , for which there is no enzymatic defence. Although trypanosomatids possess superoxide dismutase activity [1,3,4], which effectively prevents accumulation of the superoxide anion radical, they have long been thought to be deficient in the enzymatic mechanisms for the removal of the product, H202, as glutathione peroxidase and catalase are either completely lacking or present in only trace amounts [4]. Although glutathione reductase activity has been reported in T. cruzi [4], it was subsequently shown that glutathione disulfide reduction in trypanosomatids is dependent upon a thiol-containing co-factor [5]. Since all trypanosomatids so far studied possess the unique co-factor, N ~,NS-bis(gluta thionyl)spermidine (trypanothione) (Fig. 1) and an N A D P H - d e p e n d e n t flavoprotein disulfide reductase (trypanothione reductase) which maintains this metabolite in the dithiol form within the cell [5,6], we have investigated whether the function of glutathione peroxidase in other cells was replaced in the parasite cell by an analogous trypanothione peroxidase. In this p a p e r we report that such a t r y p a n o t h i o n e - d e p e n d e n t peroxidase activity can be detected in the m a m m a l i a n para-

0166-6851/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

40

A HOOC

B

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0

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/ 0 HOOC

S

\ H

HOOC

(CH2}3 NH l (ICH2)4 NH

0

O

H

HS/

0 HOOC

/SH \ H

(ICH213 NH l (IC.2)4 NH

0

Fig. 1. (A) Structure of trypanothione disulfide (T(S)2). (B) Dihydro-trypanothione (T(SH)2).

site T. brucei and in the insect trypanosomatid Crithidia fasciculata. Materials and Methods

Reagents. All chemicals and enzyme reagents were purchased from Sigma Chemical Co., St. Louis, MO or from Boehringer Mannheim Biochemicals, Indianapolis, IN, and were of the highest grade commercially available. Naz75SeO3 (3.4 mCi mg-1) was purchased from Amersham, Arlington Heights, IL. Yeast glutathione disulfide reductase (GR) was from Sigma and C. fasciculata trypanothione disulfide reductase was purified as previously described [7]. Trypanothione disulfide (T(S)2), dihydro-trypanothione (T(SH)2) and NLmono-glutathionylspermidine were chemically synthesised as described previously [8]. Organisms. C. fasciculata was cultured as described previously [3]. Cells were harvested by centrifugation at late log phase and washed with Hanks' balanced salt solution before use. T. brucei ( E A T R O 427) was obtained from 400 g Sprague - Dawley rats 3 days after intraperitoneal inoculation with approximately 4 x 107 trypanosomes. The parasites were purified from infected blood by DEAE-cellulose chromatography [9] and washed with 60 mM phosphate buffer, pH 8.0 containing 45 mM NaC1 and 50 mM glucose. Enzyme extracts. The organisms were completely disrupted by sonication (4 x 20 s, with 1 min cooling periods, 4°C) in 50 mM potassium phos-

phate pH 7.0 containing 1 mM EDTA. Lysis was monitored microscopically. The lysis products were centrifuged (100000 x g for 1 h) and the resulting supernatants were dialysed against the same buffer (4 x 50 vol) before analysis. Glutathione peroxidase activity was measured by coupling it to N A D P H oxidation which was measured spectrophotometrically at 340 nm and 27°C. The assay mixture contained 50 mM potassium phosphate pH 7.0, 1 mM E D T A , 0.25 mM N A D P H , 0.1 mM glutathione (GSH) and 5 U ml ~ yeast glutathione reductase. Trypanothione peroxidase was measured in the same way but in this case the assay mixture contained 0.3 U ml 1 pure C. fasciculata trypanothione reductase (TR) and 0.05 mM T(SH)2. After measuring background N A D P H oxidation in the absence or presence of extracts, initial rates were determined on addition of 0.05 mM hydrogen peroxide, t-butyl hydroperoxide or cumene hydroperoxide.

Selenium labelling experiments. Five rats were injected subcutaneously with 50 p,Ci Naz75SeO3 three days prior to infection with T. brucei and again with 50 ~xCi intraperitoneally at inoculation. After three days normal high parasitemia was observed and the trypanosomes were isolated and purified as described above. The isolated parasites were washed with phosphate buffered saline by centrifugation until the supernatant wash contained no residual radioactivity (4 times in all). 75Se was detected on a TM analytic scintillation spectrometer. This instrument provided an efficiency of approximately 5%. Results

When a dialysed extract of C. fasciculata was incubated with glutathione reductase, N A D P H and glutathione (GSH) and ButOOH added, no increase in the rate of N A D P H oxidation was observed, indicating the absence of glutathione peroxidase activity (Fig. 2). If ButOOH was added to a mixture of TR and T(SH)2, no change in the rate of N A D P H oxidation was observed showing that TR itself does not possess peroxidase activity (Fig. 2). Other experiments showed that the background rate of N A D P H oxidation is due to

41 Extract

+GR+GSH v

~ ~ '

ButOOH

-~ T(SH)2 + TR

~ _ ~ r

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ButOOH

,. Extract

'l Extract

Ext ract

+TR

~~T(SH)2

0~1 Abs340 .

Fig. 2. Trypanothione dependent peroxidase activity in C.

fasciculata. Figures are spectrophotometric tracings of A 340 nm against time. Assay conditions as described in Materials and Methods. Additions are: extract, C. fasciculata dialysed enzyme extract (0.8 mg protein); GR, yeast glutathione reductase (5 U ml 1); GSH, glutathione (0.1 mM); ButOOH, t-butyl hydroperoxide (0.02 mM); T(SH)2, dihydro-trypanothione (0.02 mM); TR, trypanothione reductase (0.3 U ml-1).

T(SH)2 auto-oxidation. Subsequent addition of C. fasciculata extract led to a marked increase in N A D P H oxidation. To eliminate the possibility of T(SH)2 oxidation occurring due to disulfide exchange with protein disulfides, the extract was preincubated with TR and T(SH)2 prior to the addition of B u t O O H (Fig. 2); net peroxidase activity could only be observed after the addition of ButOOH. A similar result is obtained if the order of addition of substrates is reversed (Fig. 2) showing that this peroxidase activity is entirely dependent on the presence of dihydro-trypanothione. Enzymatic activity could be abolished by heating the extract at 90°C for 3 min. N~-Mono glutathionylspermidine, a co-metabolite of trypanothione in trypanosomes would not serve in place of T(SH)2, indicating that the enzyme is specific for the bis-glutathionylspermidine conjugate. Experiments with a freshly prepared ex-

tract of bloodstream T. brucei demonstrated a similar trypanothione-dependent peroxidase activity (not shown). Using ButOOH as substrate, the amount of intracellular trypanothione peroxidase activity was measured in C. fasciculata and bloodstream T. brucei. Activity is proportional to protein added only at higher concentrations of either extract (Fig. 3). With cumene hydroperoxide similar rates were obtained (not shown). The non-proportionality at lower protein concentrations could be due to dissociation of the enzyme into inactive (or less active) subunits; a similar phenomenon has been reported with glutathione peroxidase [10]. From the linear portion of these graphs, specific activities of trypanothione peroxidase were determined to be 5.2 and 1.0 nmol rain -1 (10 s cells) -1 for C. fasciculata and T. brucei, respectively (assuming all activity in the cells is released into the supernatant). The rate of non-enzymatic reaction of T(SH)2 with H202 is much higher than that with the organic hydroperoxides, cumene hydroperoxide or ButOOH. The rate of T(SH)2 oxidation to T(S)2 by H202 in the absence of enzyme extracts is proportional to the concentration of peroxide (Fig. 4). On addition of 7". brucei extract an increased rate of T(SH)2 oxidation was observed, such that the net enzyme dependent T(SH)2 oxidation could be calculated. Again the enzyme activity is nonlinear at lower protein concentration as found when ButOOH was used as oxidant (Fig. 4, insert). From this experiment it was calculated that T. brucei is able to metabolise hydrogen peroxide at a rate of 0.40 nmol min i (108 cells)-l. It was not possible to demonstrate a similar activity with H202 as substrate in C. fasciculata due to the presence of a catalase-like activity in these organisms [11] and the addition of the catalase inhibitor aminotriazole did not improve the analysis. In order to assess the significance of non-enzymatic removal of H202 by T(SH)2 or GSH, rate expressions for these reactions were experimentally determined by coupling the formation of disulfide forms to N A D P H oxidation using C. fasciculata TR or yeast GR. These reactions were found to obey the following rate equations at 27°C in the concentration range 0.01 to 0.2 mM:

42

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l

i

i

i

i

i

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10

20

30

40

50

60

70

80

Dialysad Extract

(~ul)

Fig. 3. Peroxidase activity as a function of cell extract. (B) C.

fasciculata. (i) T. brucei. 1 ml dialysed extract = 10 TM cells.

T(SH)2 + H 2 0 2 , T(S)2 + 2 H 2 0 Rate = 1.33 [ T ( S H ) 2 ] 0"631 [ H 2 0 2 ] 0"981 M -1 min 1 2 GSH + H2O 2 ) GSSG + 2 H 2 0 Rate = 1.175 [ G S H ] 0'713 [ H 2 0 2 ] °'953 M -1 min -l. From these rate expressions it is clear that the rate of H202 consumption by T(SH)2 is only marginally greater than that of G S H (given equivalent thiol concentrations). This result is consistent with the redox potential of trypanothione which has been determined to be only slightly more negative than that of glutathione (E' 0 = -0.242 V and -0.230 V for T(SH)2 and GSH respectively) [12]. The relative contribution of these non-enzymatic versus enzymatic reaction rates will be discussed below. In mammalian cells, glutathione-dependent peroxidases can be classified into two types: a selenium-containing enzyme, which can utilise H202 or alkyl hydroperoxides; and a non-seleniumcontaining class of enzymes ( G S H sulfotransferases), which display an apparent peroxidase ac-

i 0.05

1 0.10 H202]

i 0.15

i 0.20

mM

Fig. 4. Oxidation of dihydro-trypanothione as a function of H202 concentration and T. brucei extract. Non-enzymaticoxidation (i); plus T. brucei extract (i) 0.01 ml, (A) 0.03 ml and (O) 0.06 ml. Rate due to T. brucei dialysed extract is shown in insert. tivity only with alkyl hydroperoxides [13]. Thus selenium (in the form of a seleno-cysteine residue) is thought to be essential for H 2 0 2 peroxidase activity [15]. We therefore investigated whether selenite (an inorganic precursor of seleno-cysteine in glutathione peroxidase [14]) could be incorporated into bloodstream T. b r u c e i proteins. Trypanosomes were isolated from [75Se]selenite-labelled rats and after extensive washing were found to contain a total of 438 000 dpm, approximately equivalent to 1.4 nmol of selenium. After lysis, centrifugation and extensive dialysis, 54 and 10.2% of the initial radioactivity was found to be associated with pellet and supernatant respectively. Thus, a total of 64% of the [75Se]selenite taken up by the trypanosomes had been converted into a non-dialysable form, presumably by incorporation into protein of molecular weight > 10000. After concentration, the radioactive supernatant was fractionated by gel filtration. As shown in Fig. 5, the ma-

43

jority of the radioisotope applied was recovered in two high molecular weight peaks; 9% eluted with the void volume of the column (Fig. 5, fractions 24-30) and 53% in a peak (Fig. 5, fractions 31-39) with a relative molecular weight slightly greater than that of T. brucei trypanothione reductase (M r 110000). Both labelled peaks were pooled and concentrated, but no trypanothionedependent peroxidase activity could be detected. In a subsequent experiment, large amounts of unlabelled extract were chromatographed but only a small percentage ( < 1%) of the applied enzyme activity could be recovered in the labelled protein region (Fig. 5, fractions 31-39). No peroxidase activity was recovered elsewhere. The reason for the apparent poor stability of the enzyme is not known. Discussion

Mammalian cells maintain low levels of H 2 0 2 to protect sensitive cellular components such as membrane lipids, protein thiol residues and nu-

1.5

cleic acids from oxidative damage [15]. The removal of U202 is achieved by means of the concerted action of glutathione peroxidase and glutathione reductase, thus N A D P H serves as the electron donor with G S H acting as a redox carrier (Fig. 6A). Our findings [5], as well as others [16], indicate that both enzymes in this cycle are absent in dialyzed extracts of trypanosomatids. Instead, in trypanosomes the removal of H202 is achieved in an analogous fashion by the cyclical action of trypanothione and the enzymes trypanothione peroxidase and trypanothione reductase (Fig. 6B). As for mammalian systems, N A D P H serves as electron donor. Contrary to previous reports, a recent study has shown that bloodstream T. brucei does not have a deficient capacity to metabolise H 2 0 2 [17], and most importantly, these authors clearly demonstrated the involvement of trypanothione in this process. However, these workers concluded that a trypanothione peroxidase was absent in this organism, in direct contradiction to our results. The reason for this is not clear. The same authors also find that T. brucei (whole cells) is able to metabolise H 2 0 2 a t a rate of 2.6 nmol min ~ (108 cells) 1 at 37°C [17], i.e., approximately 1.3 nmol min -1 (108 cells) -1 at 27°C (assuming a 2-fold increase in reaction rate per 10°C). In our strain of T. brucei we find activities of ROOH

300

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20

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Fig. 5. Ultragel ACA 44 filtration of [75 Se]selenite-labelled T. brucei extract. ( - - - ) Protein concentration, by absorbance at 280 nm. (---) 75Se radioactivity per 8 ml fraction.

B~ trypanosomatids

Fig. 6. Removal of peroxides in mammalian cells (A) and trypanosomatids (B). GPx and TPx are glutathione and trypanothione peroxidase respectively.

44

0.4 nmol min -I (10 s cells) i at 27°C for the trypanothione peroxidase when using H 2 0 2 as oxidant and 20 nmol min -I (108 cells) -1 for trypanothione reductase (using T ( S ) 2 as substrate). Thus, from our present data, such a cycle would account for about one third of the rate of H20 2 consumption reported in the whole cells [17]. This calculation does not take into account the fact that the in vivo concentration of T(SH)2 (0.16 mM) is higher than in the assay conditions. Owing to the instability of the enzyme activity, we have not carried out a kinetic analysis. However, if the trypanothione peroxidase has kinetics similar to glutathione peroxidase, then the actual rate in vivo would be proportional to the T(SH)2 concentration [18]; in which case the trypanothione peroxidase activity measured for T. brucei would account for almost all of the H20 2 consumption observed in whole cells [17]. In vivo, the major source of H202 generation in bloodstream T. brucei is believed to arise from the pool of reduced ubiquinone in mitochondria [16]. An upper limit for H202 production by mitochondrial preparations of T. brucei has been determined to be in the range 0.2-0.7 nmol min -1 (108 cells) 1 [16]. Assuming a low K m for the trypanothione peroxidase, the specific activity calculated here would be sufficient to consume intracellularly produced H202. This would be in complete agreement with the recent finding [17] that H202 is below the limit of detection (< 0.5 IxM) in this organism and in contrast to a previous report [19]. It is also important to note that non-enzymatic reduction of H202 by either GSH or T(SH)2 (as shown by the experimentally determined rate expression for these processes) would be quantitatively insignificant processes in vivo given the intracellular concentrations of these thiols in bloodstream T. brucei (0.44 and 0.16 mM for GSH and T(SH)2 respectively; A.H.F. and G.B.H., unpublished experiments). It is probable that this trypanothione-dependent peroxidase activity represents the sole mechanism for the removal of H202 in T. brucei and similar trypanothione dependent peroxidase activities might be expected in other species of trypanosomes and leishmanias which possess the flavoenzyme trypanothione reductase. Purification of the activities will be necessary to further char-

acterise these enzymes, in particular to determine if a selenium containing amino acid is present (as in the H20: metabolising glutathione peroxidase). The selenite labeling experiment reported here would indicate that bloodstream T. brucei can incorporate selenite (or possibly a metabolic product of selenite synthesised by the rat host) into protein. In conclusion, a specific function for trypanothione in trypanosomes has now been demonstrated. This finding greatly enhances the importance of the 'trypanothione system' as a target for chemotherapy. Inhibition of trypanothione biosynthesis, the trypanothione peroxidase or of trypanothione reductase (the latter enzyme being essential for returning and maintaining trypanothione in the reduced (dihydro) form) would be expected to have a significant effect on the ability of these organisms to respond to oxidative stress exerted extracellularly by the mammalian host or intracellularly by trypanocidal drugs which can increase steady state peroxide and superoxide concentrations.

Acknowledgements The authors thank Ms. Helen Shim for technical assistance and Dr. Harvey Cohen (Rochester University) for helpful discussion. This work was supported by a grant from the Rockefeller Foundation (RF 85078, #127), grants from the National Institutes of Allergy and Infectious Diseases (AI 21429 and AI 19428) and by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases.

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45 4 Boveris, A., Sies, H., Martino, E.E., Docampo, R., Turrens, J.F. and Stoppani, A.O.M. (1980) Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi. Biochem. J. 188,643-648. 5 Fairlamb, A.H. and Cerami, A. (1985) Identification of a novel, thiol-containing co-factor essential for glutathione reductase enzyme activity in trypanosomatids. Mol. Biochem. Parasitol. 14, 187-198. 6 Fairlamb, A.H., Blackburn, P., Ulrich, P., Chait, B.T. and Cerami, A. (1985) Trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. Science 227, 1485-1487. 7 Shames. S.L., Fairlamb, A.H., Cerami, A. and Walsh, C.T. (1986) Purification and characterisation of trypanothione reductase from Crithidia fasciculata: a new member of the family of disulfide-containing flavoprotein reducrases. Biochemistry 25, 3519-3526. 8 Henderson, G.B., Ulrich, P., Fairlamb, H.A. and Cerami, A. (1986) Synthesis of the trypanosomatid metabolites trypanothione, and Nl-mono and NS-mono-gluta thionylspermidine. J. Chem. Soc. Chem. Commun. 593-594. 9 Lanham, S.M. (1968) Separation of trypanosomes from the infected blood of rats and mice by anion exchange. Nature 218, 1273-1274. 10 Stults, F.H., Forstrom, J.W., Chiu, D.T. and Tappel, A.L. (1977) Rat liver glutathione peroxidase: purification and study of multiple forms. Arch. Biochem. Biophys. 183, 490-497.

11 Wertlieb, D.M. and Guttman, H.N. (1963) Catalase in insect trypanosomatids. J. Protozool. 10, 109-112. 12 Fairlamb, A.H. and Henderson, G.B. (1987) Metabolism of trypanothione and glutathionylspermidine in trypanosomes. In: Host-Parasite Molecular Recognition and Interaction in Protozoal Infections (Chang, K.P., and Snary, D., eds.) pp. 29-4(/, NATO ASI series. 13 Burk, R.F,, Nishiki, K., Lawrence, R.A. and Chance, B. (1978) Peroxide removal in selenium-dependent and selenium-independent glutathione peroxidases in haemoglobin-free perfused rat liver. J. Biol. Chem. 253, 43--46. 14 Forstrom, W., Zakowski, J.J. and Tappel, A.L. (1978) Identification of the catalytic site of rat liver glutathione peroxidase as seleno-cysteine. Biochemistry 17,263%2644. 15 Halliwell, B. and Gutteridge, J.M.C. (1986) lron and free radical reactions: two aspects of antioxidant protection. Trends Biochem. Sci. 11,372-375. 16 Meshnick, S.R., Blobstein, S.H., Grady, R.W. and Cerami, A. (1978) An approach to the development of new drugs for African trypanosomiasis. J. Exp. Med. 148, 569-579. 17 Penketh, P.G. and Klein, R.A. (1986) Hydrogen peroxide metabolism in Trypanosoma brucei. Mol. Biochem. Parasitol. 20, 111-121. 18 Wendel, A. (1981) Glutathione Peroxidase. Methods Enzymol. 77, 325-333. 19 Meshnick, S.R., Chang, K.P. and Cerami, A. (1977) Heme lysis of the bloodstream forms of Trypanosoma brucei. Biochem. Pharmacol. 27,293%2945.