ADP-Ribosyltransferase Activity in Trypanosoma· brucei

F ARZIN F ARZANEH1 , SYDNEY SHALL 2 , PAUL MICHELS3 ,

and PIET BORST4

Introduction

The nuclear enzyme ADPRT (Ee: 2.4.2.30), which is entirely dependent on the pre­sence of DNA-containing strand breaks for its activity [1], is required for efficient DNA excision repair [2]. ADPRT activity is also an obligatory requirement for the expression of the differentiated phenotype in a number of eukaryotic cells (see [3]). Williams [4] has shown that inhibition of ADPRT activity blocks the differentiation, but not proliferation, of Trypanosoma cruzi amastigotes to epimastigotes and trypo­mastigotes. Although the presence of ADPRT activity in Plasmodium yoelii has been demonstrated [6], the presence of this enzyme in trypanosomes has not been directly investigated. 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 repor the detection of ADPRT activity in T. brucei, its activation by DNA damage and its inhibition by benzamide and its analogues. We also show that intact trypa­nosomes are readily permeable to the ADPRT inhibitor 3-aminobenzamide.

Presence of ADPRT Activity in Trypanosoma bruce;

In freshly isolated blood-stream trypanosomes, which are permeabilised to NAD by a mild hypotonic shock, there is a low but detectable level of basal ADPRT activity (Fig. 1 -). Induction of DNA strand breaks by exogenous DNase I increases this

Harris Birthright Research Center for Fetal Medicine, Department of Obstetrics and Gynaecology, Kings College School of Medicine, Denmark Hill, London SE5 8RX, Great Britain

2 Cell and Molecular Biology Laboratory, University of Sussex, Brighton BNl 9QG, Great Britain 3 Section for Medical Enzymology, University of Amsterdam, Jan Swamerdam Institute,

1005 GA Amsterdam, The Netherlands. Present address: International Institute of Cellular and Molecular Pathology, Avenue Hippocrat 75,1200 Bruxelles, Belgium

4 Section for Medical Enzymology, University of Amsterdam, Jan Swamerdam Institute, 1005 GA Amsterdam, The Netherlands. Present address: The Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

ADP-Ribosylation of Proteins (ed. by F.R. Althaus, H. Hilz, and S. Shall) © Springer-Verlag Berlin Heidelberg 1985

368 F. Farzaneh et al.

~ Z C 300 Cl

~ rJ) Ol

"'5 200 E Q.

Ol rJ)

100 0 .c '':

I a. C «

Time (min) Fig. 1. ADPRT activity in permeabilised T. brucei. Trypanosomes were permeabilised to NAD by a mild hypotonic shock. ADPRT activity was measured either in the absence of exogenous nucle­ases (-), or in the presence of 10 U mr' of DNase I (.) for the estimation of basal and potential activities, respectively. The basal ADPRT activity in trypanosomes pre-treated with 100 11M dimethylsulphate, prior to permeabilisation (e)

activity approximately tenfold (Fig. 1 .). The basal ADPRT activity can also be stimu­lated fourfold by exposure of the trypanosomes to 100 pM DMS for 15 min, prior to permeabilisation (Fig. 1 e).

In order to confirm that the acid-insoluble radioactive material synthesised during the ADPRT assay is in fact (ADP-ribose)n' this material was digested with a number of hydrolytic enzymes. RNase A, DNase I, Micrococcal nuclease and proteinase K failed to degrade this material. However, snake venome phosphodiesterase degraded 96% of the acid-insoluble radioactive material (Table 1). This strongly suggests that the incor­porated radioactive material was (ADP-ribose)n'

Inhibition of ADPRT Activity

The ADPRT activity in permeabilised T. brucei is completely inhibited by 100 pM 3-aminobenzamide (equimolar concentration to the substrate) (Fig. 2 D). At this concentration, 3-aminobenzoic acid did not block the synthesis of (ADP-ribose)n

Table 1. Sensitivity of ADPRT assay product to enzyme hydrolysis

Enzyme used pmol [' 4 C1-ADP-ribose %(ADP-ribose)n remaining degraded

Control 754 0 RNase A 741 2 DNase I 735 3 Micrococcal nuclease 739 2 Proteinase K 689 9 Snake venom phosphodiesterase 34 96

'I

ADP-Ribosyltransferase Activity in Trypanosoma brucei

~ Z o en 450 ~ rJ) C1)

-0 E 300 Q. ~

C1) rJ)

.& 150

.;: I

Q. o <l:

40

Time (min)

369

Fig. 2_ Inhibition of ADPRT activity. The potential ADPRT activity was measured either in the presence (->, or absence of either 100 !lM 3-aminobenzamide (~), or 100!lM 3-aminobenzoic acid (6). Alternatively 100 !lM 3-aminobenzamide was added either 5 min (+), or 15 min (0) after the start of the assay

(Fig. 26). The addition of 3-aminobenzamide after either 5 or 15 min of incubation with [14 C]-NAD, was followed by a reduction in the level of already synthesized (ADP-ribose)n (Fig. 2 + and 0). Therefore, a degrading enzyme, possibly poly(ADP­ribose) glycohydrolase, is present and active in the permeabilised trypanosomes.

~

<l: Z o Ol :::i... "­c: ·E "­Q) IJ)

o .0 .... I

Q. C <l:

-0 E Q.

0.4

0_3

0_2

> ........ .-

a 0 ~04 ~08

+ _1 1/s (NAD concentration (}-1M»

Fig. 3. Double-reciprocal plot of ADPRT activity. The total ADPRT activity was measued in the absence (A) or presence of 5 !lM (+) or 1O!lM (-> 3-aminobenzamide, or 10 !lM 3-aminobenzoic acid (6)

370

E 300 Q. U

"C Q) c: ·2 200 Q) .... >--:~ u co 100 o '5 co 0::

o,--~_--,-_~_-,-_~_-,-_~---,

o 10 20 30

Time (min)

F. Farzaneh et al.

Fig. 4. Permeability of T. brucei to 3-aminobenzamide. Intact or per­meabilised trypanosomes were incu­bated at either 4° C (-> or 37° C (A) in a physiological saline containing 1 mM [3 H)-3-aminobenzamide. After three very rapid washes, the retained radioactivity was measured. Results are expressed as the difference be­tween intact and permeabilised try­panosomes which served as control

The Km and V max of the potential ADPRT activity (measured in the presence of 10 U ml-1 of exogenous DNase I), estimated from a double-reciprocal plot, are 110 ± 17 pM NAD and 39 ± 5 pmol ADP-ribose min-1 Mg-1 DNA, respectively. (Fig. 3 A).

3-Aminobenzamide is a competitive inhibitor of ADPRT activity in T. brucei. The Ki , measured at 5 pM and 10 pM, is 4.3 ± 0.5 pM (Fig. 3 • and .). The estimated Ki values for two other competitive inhibitors, benzamide and 3-methoxybenzamide, are 2.6 ± 0.4 pM and 2.9 ± 0.5 pM, respectively (results not shown). The acid analogues of these compounds, 3-aminobenzoic acid (Fig. 3 L:.), benzoic acid and 3-methoxy­benzoic acid, do not inhibit the ADPRT activity.

Freshly isolated intact trypanosomes are permeable to 3-aminobenzamide. When incubated in a physiological saline in the presence of [3 H]-3-aminobenzamide, at either 4°C or 37°C, this compound readily permeates the trypanosomes (Fig. 4. and A). The faster rate of permeation at 37°C may be due to the increased mobility of the trypanosomes and better mixing of the incubation mixture at this temperature.

Discussion

Studies reported here demonstrate the presence of ADPRT activity in T. brucei and its inhibition by benzamide and its amide, but not acid, analogues. The stimulation of ADPRT activity by DNA damage suggests that the role of this enzyme in trypanosomes may be similar to that in higher eukaryotic cells. It has already been shown that inhibi­tion of ADPRT activity blocks the differentiation, but not proliferation, in T. cruzi [5] and T. brucei (J.D. Barry, pers. comm.). We have previously demonstrated the formation of DNA strand breaks and the obligatory involvement of ADPRT activity in the cytodifferentiation, but not proliferation, of avian myoblasts in culture [3]. Based on this and other similar observations (see [4]) we have suggested that DNA strand break formation and rejoining, modulated by ADPR T activity, may be involved in the

ADP-Ribosyltransferase Activity in Trypanosoma brucei 371

developmental regulation of cellular differentiation. We have postulated that this may be due to the possible involvement of gene amplification, mobility in the genome and/ or regional chromatin relaxation mediated by the action of topoisomerases in this process.

African trypanosomes, like T brucei, depend on antigenic variation to evade the immune response of their vertebrate hosts. Antigenic variation is caused by repeated switches in the composition of the surface coat (variable surface glycoproteins, VSGs) and most switches require the duplicative transposition of a VSG gene [7]. We are currently investigating the possible involvement of ADPRT activity in VSG gene switching in T brucei.

References

1. Benjamin RG, Gill DM (1980) Poly(ADP-ribose) synthesis in vitro programed by damaged DNA. J Bioi Chern 255:10943-10508

2. Durkacz WD, Omidiji 0, Gray DA, Shall S (1980) (ADP-ribose)n participates in DNA excision repair. Nature (London) 283:593-596

3. Farzaneh F, Zalin R, Brill D, Shall S (1982) DNA strand breaks and ADP-ribosyl transferase activation during cell differentiation. Nature (London) 300: 362-366

4. Williams GT, Johnstone AP (1983) ADP-ribosyl transferase, rearrangement of DNA, and cell differentiation. Biosci Rep 3:815-830

5. Williams GT (1983) Trypanosoma cruzi: Inhibition by ADP-ribosyl transferase antagonists of intracellular and extracellular differentiation. Exp Parasitol 56:409-415

6. Okolie EE, Onyezili NI (1983) ADP-ribosyl transferase in Plasmodium (malaria Parasite). Biochem J 209:687-693

7. Borst P, Cross GAM (1982) The molecular basis for trypanosome antigenic variation. Cell 29: 291-303