ADP-ribosyl transferase activity in Trypanosoma brucei

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Molecular and Biochemical Parasitology, 14 (1985) 251-259 Elsevier


MBP 00515



1Cell & Molecular Biology Laboratory, University of Sussex, Brighton BNI 9QG, England, and 2Section for Medical Enzymology, Laboratory of Biochemistry, University of Amsterdam, Jan Swammerdam Institute, P.O. Box 60.000, 1005 GA Amsterdam, The Netherlands (Received 5 September 1984; accepted 8 October 1984)

Nuclear adenosine diphosphoribosyi transferase (ADPRT) catalyses the covalent modification ofchromatin proteins by (ADP-ribose)n. This activity, which is entirely dependent on DNA containing strand breaks, is required for efficient DNA excision repair possibly because it regulates DNA ligation. ADPRT activity is also required for cytodifferentiation in a number of different cell types. We report here the presence of ADPRT activity in the blood-stream form of Trypanosoma brucei and its activation by DNA strand breaks formed by exposure to, either exogenously supplied deoxyribonuclease I, or treatment with the methylating agent, dimethylsulphate. 3-Aminobenzamide, but not its chemical analogue 3-aminobenzoic acid, is a competitive inhibitor of ADPRT activity in T. brucei. Intact trypanosomes are readily permeable to this competitive inhibitor of ADPRT activity. Key words: ADP-ribosyl transferase; Adenosine diphosphoribosyl transferase; Trypanosoma brucei; DNA damage


The nuclear ADPRT (EC catalyses the formation of mono-, oligo-, and poly(ADP-ribose)-modified chromatin proteins; the specific substrate for this activity is NAD ÷. This activity, which is entirely dependent on the presence of DNA containing strand breaks [1,2], is required for efficient DNA excision repair [3]. In intact cells the induction of DNA damage by radiation or chemical agents stimulates ADPRT activity and causes a drop in the cellular NAD ÷ content [4]. Inhibition of

Present address: International Institute of Cellular and Molecular Pathology, Avenue Hippocrate 75, 1200 Bruxelles, Belgium. ** Present address: The Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Abbreviations: ADPRT, adenosine diphosphoribosyl transferase; PSG buffer, 60 laM sodium phosphate buffer (p H 8.0) containing 45 mM NaCI and 1% (w/v) glucose; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid; TCA, trichloroacetic acid *

0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

252 (ADP-ribose) n biosynthesis, by a number of competitive inhibitors [5], blocks this drop in the NAD ÷ content, retards the excision repair of DNA and causes a synergistic enhancement of the cytotoxicity of DNA damaging agents [4,6]. The molecular mechanism of ADPRT involvement in DNA repair is still not totally clear. However, it has been shown that the requirement for this activity is neither the incision nor excision events, but rather in the re-ligation step [7,8], probably because it regulates DNA ligase II activity [9]. More recently, it has been demonstrated that ADP-ribosylation may also regulate the activity of DNA topoisomerase I [10,11]. ADPRT activity is also an obligatory requirement for the cytodifferentiation, but not for the proliferation, of primary chick myoblasts [ 12,13]; the expression of foetal functions in cultured rat hepatocytes [14] and for the mitogen stimulation of human peripheral blood lymphocytes [15]. ADPRT activity has also been studied in two protozoan parasites. Inhibition of ADPRT activity blocks the differentiation, but not proliferation, of Trypanosoma cruzi amastigotes to epimastigotes and trypomastigotes [ 16]. The presence of ADPRT activity has also been directly demonstrated in Plasmodium yoelii [ 17]. The presence and characteristics of this enzyme in trypanosomes have not yet been studied directly. In addition, it is not clear whether the protozoan ADPRT activity can be stimulated by DNA strand-breaks and whether the inhibitors of ADPRT activity in higher eukaryotes can also block the activity of this enzyme in trypanosomes. Here we report the detection of ADPRT activity in T. brucei, its activation by DNA damage and its inhibition by 3-aminobenzamide and we show that intact trypanosomes are readily permeable to this drug. MATERIALS AND METHODS

Chemicals. 3-Aminobenzamide was a gift from Dr. M. Tavassoli, synthesised from 3-nitrobenzamide by catalytic hydrogenation [5] as previously described [7]. Benzamide and benzoic acid were obtained from Sigma, Dorset, England. 3-Methoxybenzamide, 3-methoxybenzoic acid and 3-aminobenzoic acid were from Aldrich, Dorset, England. Nicotinamide [U-~4C]adenine-dinucleotide was purchased from Amersham, England. 3-[3H]aminobenzamide, uniformly labelled by an exchange reaction, was from a batch kindly prepared by Dr. R. Wade, Ciba-Geigy, U.K.

Trypanosome isolation. Blood stream T. brucei, stock 427, were grown in white 'Swiss random' female mice to a density of > 109/ml of blood. Trypanosomes were then isolated from blood by DEAE-cellulose chromatography [18]. The isolated trypanosomes were washed twice in 60 mM sodium phosphate buffer (pH 8.0) containing 45 mM NaCI and 1% (w/v) glucose (PSG buffer) and collected by centrifugation at 2 000 )< g for 10 min. Freshly isolated trypanosomes were kept on ice and used within 1 h.

Trypanosome permeabilisation.

Parasites were made permeable to NAD ÷ by an

253 osmotic shock procedure previously described [13] and modified slightly for the permeabilisation of trypanosomes. Washed trypanosomes were resuspended at a density of approximately 2 X 108/ml in a hypotonic solution consisting of 9 mM HEPES (pH 7.8), 5 mM dithiothreitol, 4.5% (w/v) Dextran (Mol. wt. 60 000-90 000), 4.5 mM MgCI2 and 1% (w/v) glucose. Trypanosomes were kept on ice, until more than 90% became permeable; then the solution was made isotonic by the addition of 1/5 vol. of a hypertonic solution. Permeabilisation was routinely achieved after around 15 min. Permeabilisation was monitored by adding 1/5 vol. of a solution of 0.2% (w/v) Trypan Blue in the hypertonic buffer to a small sample of trypanosomes in the hypotonic buffer and estimating the percentage of Trypan Blue positive cells microscopically. The hypertonic solution contained 200 mM HEPES (pH 7.8), 750 mM KCI, 5 mM dithiothreitol, 4.5% (w/v) Dextran, 7.0 mM EGTA, 4.5 mM MgCI2 and 1% (w/v) glucose.

ADPRT assay. The basal or physiological activity: In order to estimate the basal ADPRT activity 200 pl aliquots of permeabilised trypanosomes in the hypotonic buffer were transferred to a water bath at 25°C and the assay was started by the addition of 40 ~tl of the hypertonic buffer containing 6.0 mM isonicotinic acid hydrazide (to prevent NAD ÷ degradation by the glycohydrolase), and 600 ~tM [~'C]NAD ÷ (12.5 Ci mol-~). The reaction was terminated by the addition of 500 ~tl of 20% (w/v) trichloroacetic acid (TCA), containing 1% (w/v) nicotinamide and 2% (w/v) tetrasodium pyrophosphate. The acid-insoluble radioactivity was then estimated by liquid scintillation counting. The DNA content was determined by the method of Kissane and Robins [19]. The total or potential activity: For the estimation of the total ADPRT activity, 0.5% (v/v) Triton X-100 was included in the hypotonic buffer and the assay mixture contained 10 units ml-~ DNase I. In these assays the specific activity of [~4C]NAD÷was reduced to 1.25 Ci mol-~. Identification of (ADP-ribose). To identify the radioactive product of the ADPRT assay, the acid-insoluble material obtained from the total ADPRT assay was dissolved in 0.1 M NaOH and neutralised by the addition of 1/10 volume 1.0 M HCI and 1/10 volume 0.5 M Tris-HC1, pH 7.6, containing 200 mM MgCI2. The solubilised (ADP-ribose)n was then incubated at 37°C in the presence of 100 units ml-~ of one of each of the following enzymes: RNase A (Sigma type IIA), pancreatic DNase I (Sigma type III), micrococcal nuclease (Sigma grade VI), snake venom phosphodiesterase (Boehringer Mannheim) and proteinase K (Merck). After 30 min the reaction was terminated and the remaining acid-insoluble radioactivity was determined by liquid scintillation counting. Permeability of intact trypanosomes to 3-aminobenzamide. Trypanosomes at a density of approximately 2 )< 107/ml in PSG buffer were divided into two fractions; one

254 fraction was permeabilised, while the other was maintained in the P S G buffer on ice. Radioactive 3-[3H]aminobenzamide (from a stock solution o f 100 m M in PSG, specific activity 50 Ci mol-~), was added to each fraction to a final concentration o f 1 mM. At the specified times 200 I~1 aliquots were removed, 1 ml fresh P S G was added and centrifuged at approximately 10000 × g for 15 s. T r y p a n o s o m e s were washed three times using this procedure (total washing time < 2 min). The t r y p a n o s o m e s were then dried a n d the retained radioactivity was measured by liquid scintillation counting. The difference in the radioactivity between the permeabilised and intact t r y p a n o somes was taken as the measure o f 3-aminobenzamide inside the intact trypanosomes. 3 - A m i n o b e n z a m i d e permeability was measured at both 0°C and 37°C. RESULTS

A D P R T activity.

T r y p a n o s o m e s are impermeable to N A D ÷, the substrate for A D P R T . In cells permeabilised to N A D + by a mild h y p o t o n i c shock two levels o f A D P R T activity can be measured: (i) the basal or physiological activity, in the absence o f experimentally-induced D N A strand breaks; and (ii) the potential activity displayed by the presence o f experimentally-induced D N A strand-breaks, f o r m e d by the action o f exogenously supplied D N a s e I. In T. brucei permeabilised to N A D + there is a low but measurable basal A D P R T activity (Fig. 1, I ) . I n d u c t i o n o f D N A strand breaks by 10 units m1-1 D N a s e I increases this activity approximately 10 fold (Fig. 1, A), d e m o n s t r a t i n g that the potential A D P R T activity is much higher than the activity actually present in t r y p a n o s o m e s not subjected to D N A d a m a g i n g agents. This is also evident in t r y p a n o s o m e s which are treated with 100 p.M dimethylsulphate for 15 min


! oot o




Time (min)

Fig. 1. ADPRT activity in T. brucei permeabilised to NAD÷by hypotonic shock. Trypanosomes, freshly isolated from infected mice were made permeable to NAD÷. The basal (I) and total (A) ADPRT activity were measured in the presence of 100 ~M NAD+(specificactivity 12.5 Ci mol-~for basal, and 1.25 Ci mol-~ for total ADPRT assay). For the estimation of the total ADPRT activity the permeahilisation solution also contained 0.5% (v/v) Triton X-100, and 10 units DNase I per ml were included in the assay mixture. The basal ADPRT activity after DNA damage (e) was measured in trypanosomes pre-treated for 15 min with 100 IzM dimethylsulphate prior to permeabilisation.

255 prior to permeabilisation (Fig. 1, o). In these damaged cells the A D P R T activity (measured in the absence of DNase I) is increased 4 fold over the basal, physiological, A D P R T activity. Because the basal A D P R T activity is very low, its measurement required the use of substrate with high specific activity. In subsequent studies A D P R T activity has been measured in the presence of 10 units/ml exogenously supplied DNase I, using [~4C]NAD of specific activity 1.25 Ci mol -l. In order to confirm that the acid-insoluble radioactive material synthesised during the A D P R T assay was in fact (ADP-ribose)n, this material was incubated with a number of hydrolytic enzymes. RNase A, DNase I, micrococcal nuclease and proteinase K failed to degrade this material (Table I). By contrast, 100 units ml -~ snake venom phosphodiesterase degraded 96% of the radioactive acid-insoluble product of the A D P R T assay. This demonstrates that the incorporated radioactive material was (ADP-ribose)n. Further confirmation based on the analysis of the products of snake venom phosphodiesterase digestion could not be carried out due to the relatively low levels of ([l'C]ADP-ribose)n synthesised.

Inhibition of ADPRTactivity.

A number ofbenzamide analogues have been shown to inhibit A D P R T activity in various eukaryotic cells. One such compound, 3-aminobenzamide, completely inhibited the A D P R T activity of T. brucei when present at 100 ~M (equimolar concentration to the substrate) (Fig. 2, l). The same concentration of the acid analogue of this compound, 3-aminobenzoic acid, did not block the synthesis of (ADP-ribose) n (Fig. 2,/,). Addition of 3-aminobenzamide after either 5 or 15 min of incubation with [14C]NAD +, was followed by a reduction in the level of (ADP:ribose) n. Therefore, a degrading enzyme, possibly poly(ADP-ribose) glycohydrolase is active in the permeabilised trypanosomes (Fig. 2, • and 0). This reduction in the level of (ADP-ribose) n was also seen in the absence of the inhibitor after 30 min of incubation. The net reduction in the level of the in vitro synthesised (ADP-ribose) n

TABLE I Sensitivity of ADPRT assay product to enzymehydrolysis(see Methods for experimental details) Enzyme used

labelled product remaining (pmol)

Control RNase A DNase I Micrococcal nuclease Proteinase K Snake venom phosphodiesterase

754 741 735 739 689 34

% product degraded 0 2

3 2 9 96

256 A ,<

I: •~c 150 I

< 20



T i m e (min)

Fig. 2. Inhibition of ADPRT activity. The total ADPRT activity was measured either in the absence (A), or presence of either 100 ~M 3-aminobenzamide (a), or 100 gM 3-aminobenzoic acid (t0 added to permeabilised trypanosomes before the addition of [I'C]NAD ÷. Alternatively 100 p.M 3-aminobenzamide was added either 5 min (0), or 15 min (O) after the start of the assay. Cross bars represent standard deviations of the mean of at least 3 separate measurements.

takes place either because the substrate N A D ÷ is degraded or because the poly(ADPribose) glycohydrolase is more stable than ADPRT under these conditions. The Michaelis constant (Km) for the total, potential ADPRT activity in permeabilised trypanosomes is estimated from a double-reciprocal plot to be 110 :l: 17 I~M (n = 4) (Fig. 3, A); the corresponding maximum velocity (Vmax) is 39 + 5 pmol ADP-ribose 0.5



i°, A



o.~4 +

' o~


o.h 1

1/S(NAD concent ratlon(~lM)~"

Fig. 3. Double-reciprocal plot of ADPRT activity; effect of 3-aminobenzamide. The ADPRT activity was measured after 10 min of incubation under conditions for the estimation of total ADPRT activity either in the presence of 5 I~M (0) or 10 ~tM (m) 3-aminobenzamide; 10 I~M 3-aminobenzoic acid (4), or in the absence of any inhibitor (A).


min-z ~tg-] DNA (n = 4). 3-Aminobenzamide is a competitive inhibitor; the estimated K i, measured at 5 ?M and 10 laM is 4.3 5:0.5 ~tM (n = 6) (Fig. 3,t and 1). The estimated K i values for two other competitive inhibitors, benzamide and 3-methoxybenzamide were 2.6 5:0.4 IxM (n = 6) and 2.9 5:0.5 laM (n = 6), respectively. The acid analogues of these compounds, 3-aminobenzoic acid (Fig. 3, zx), benzoic acid and 3-methoxybenzoic acid, do not inhibit the ADPRT activity. Permeability of trypanosomes to 3-aminobenzamide. When freshly isolated, intact, trypanosomes were incubated at 37°C in the presence of 1 mM 3-[3H]aminobenzamide, this compound entered the cells and equilibrium was reached in approximately 15 min (Fig. 4, -). Incubation at 0°C reduced the rate of permeation by about two fold (Fig. 4, m). The rapid equilibration at 0°C suggests that 3-aminobenzamide gets into trypanosomes by passive diffusion rather than by active transport. DISCUSSION

In eukaryotic cells nuclear ADPRT activity is required for efficient DNA excision repair. A number of studies have also demonstrated that cellular differentiation in primary chick myoblasts [ 12,13], expression of foetal functions in cultured rat hepatocytes [14] and the mitogen stimulation of human resting peripheral blood lymphocytes [15] can be reversibly blocked by inhibitors of ADPRT activity (see [20] for review). ADPRT inhibitors can also reversibly block the morphological differentiation in both T. cruzi [16], and T. brucei (J.D. Barry, personal communication). These latter observations have provided indirect evidence for the presence of ADPRT activity in trypanosomes and its involvement in their cytodifferentiation. Studies

A300 E Q.




.~ 100 "13 m

o 0







Time (rain)

Fig. 4. Permeability of T. brucei to 3-aminobenzamide. Freshly isolated, intact trypanosomes were incubated at either 37°C (A), or 0°C (l) in the presence of 1 mM 3-[3H]aminobenzamide (specific activity 50 Ci mol-~). Each point represents the difference between the radioactivity retained by intact trypanosomes after three washes compared to permeabilised trypanosomes. Cross bars represent standard deviations of the mean of at least 3 separate measurements.

258 reported here directly demonstrate the presence of A D P R T activity in T brucei. The K m and Vmax of total, potential A D P R T activity in permeabilised trypanosomes is 110 + 17 I~M (n = 4) NAD + and 39 + 5 pmol ADP-rihose rain -I ~tg-1 DNA (n = 4), respectively. These values are comparable to those reported for A D P R T activity in higher eukaryotic cells (see [21] for a review). A D P R T activity in T. brucei is blocked by benzamide and its analogues, 3-methoxybenzamide, or 3-aminohenzamide (see Figs. 1, 2 and 3). The estimated K i values for benzamide, 3-methoxybenzamide and 3-aminobenzamide are 2.6 4- 0.4 I~M (n = 6), 2.9 + 0.5 ~tM (n = 6) and 4.3 4- 0.5 I~M (n = 6), respectively. The reported K i values for 3-methoxybenzamide and 3-aminobenzamide in isolated pig thymus nuclei are 1.5 4- 0.3 I~M and 1.8 -I- 0.2 I~M, respectively [5]. As in higher eukaryotic cells, the A D P R T activity in trypanosomes is not inhibited by the acid analogues of these compounds. Intact trypanosomes are readily permeable to 3-aminobenzamide which seems to enter the cells by passive diffusion, as is expected of a small uncharged hydrophobic molecule. The stimulation of A D P R T activity by DNA damaging agents (Fig. 1) suggests that the physiological role of this enzyme in trypanosomes is similar to that in higher eukaryotes, an inference also strongly suggested by its involvement in the morphological differentiation of trypanosomes. African trypanosomes, like T. brucei, depend on antigenic variation to evade the immune response of the vertebrate host. Antigenic variation is caused by repeated switches in the composition of the surface coat and most switches require the duplicative transposition of a coat protein gene [22-24]. Trypanosomes might therefore be more vulnerable to inhibitors of DNA repair than their vertebrate hosts. We are currently testing whether 3-aminobenzamide inhibits antigenic variation of T. brucei in infected rats. ACKNOWLEDGEMENTS This work was supported by an EMBO fellowship to F.F., and financial assistance from the U N D P / W o r l d B a n k / W H O Special Program for Research and Training in Tropical Diseases (T16/181/T7/34). REFERENCES I 2 3 4

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8 9 10

11 12

13 14

15 16 17 18 19 20 21 22 23


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