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A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi
FEMS MicrobiologyLetters67 (1990)145-150 Published by Elsevier
145
FEMSLE 03872
A
major cysteine proteinase is developmentally regulated in Trypanosoma cruzi Oscar Campetella, Javier Martinez and J u a n Jos6 Cazzulo lnsrimto de lnvestigacianes BioquJmicas Fundacibn Campomar. Facultad de Ciencias Exactas y Naturales. Universldad de Buenos Aires -- CONICET, Buenos Aires, Repbblica Argentina
ReceivedS August1989 Revisionreceivedand accepted3 October1989 Key words: Tryp a n o so ma cruzi; Cysteine proteinases; Western blots; Polyacrylamide gels
1. SUMMARY
2. INTRODUCTION
Epimastigotes of different stocks of Trypanos o m a cruzi contain similar levels of proteinase
Three cysteine proteinases have been purified to electrophoretic homogeneity from cell free extracts of epimastigotes of different strains of T r y p a n o s o m a cruzi, the causative agent of Chagas" disease [1]. One of them is a lysosomal glycoprorein of molecular weight of about 60 kD& strongly inhibited by L . t r o n s - e p o x y s u c c i n y l - l e u c y l a m i d o (4-guanidino) butane (E-64), leupeptin, tosyl-Llysine chloromethyl ketone (TLCK) and organomercurial reagents, insensitive to phenyl methyl sulfonyl fluoride (PMSF) and activated by 2mercaptoethanol, which presents 65% of identify with papain and cathepsin L in its N-terminus [2-4]. We have recently shown that this enzyme is the major proteolytic activity detectable in cell-free extracts by clectrophoresis in polyacrylamide gels containing fibrinogen or hemoglobin [5]. We present here evidence indicating that epimastigotes of. different stocks and clones of the parasite contain cysteine proteinases, detected both as activity in fibrinogen-containing gels and in Western blots reacted with a polyclonal antiserum. The pro-
activity on azocasein; a m a s t i g o t e s and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.
Correspondence: Dr, Juan Jos~ Cazzulo. lnstituto de Investigaclones Bioquitn~a$ Fundacibn Campomar,A. Machado 151, 1405 BuenosAires. Argentina.
0378-1097/90/$03.50© 1990Federationof EuropeanMicrobiologicalSocieties
146 teinase thus detected is present, in the same range of molecular weight, in epimastigotes of all stocks and clones tested, as well as in the other two main developmental forms o f the parasite, trypomastigote and amastigote.
Buenos Aires, Buenos Aires, Argentina, a n d the clone D m 28e [6], obtained from Dr. A n t o n i o Morello, F a c u l t a d de Medicina, U n i v e r s i d a d de Chile, Santiago, Chile. Epimastigotes were cultured in B H T medium, as previously described [7]. Trypomastigotes and amastigotes o f the R A strain were obtained from infected V e t o cultures [8].
3. M A T E R I A L S A N D M E T H O D S
3.2. Sample preparation 3. I. Parasite stocks and culture
Parasite homogenates used for enzyme activity determination, b o t h in fibrinogen-containing gels and on azocasein in vitro, were m a d e by suspension o f the cells in sample buffer c o n t a i n i n g 0.04 M T r i s - H C l , p H 6.8, 4 M urea, 0 . 1 ~ S D S a n d 2% T r i t o n X-100 [9] a n d freezing at - 2 0 ° C . T h e purified enzyme was obtained as previously de-
T h e parasite stocks used were T u l 0, T u l 2, A W E Corpus Christi, Y, Peru, Sonia, and the clone C A - I / 7 2 , obtained from D r . Elsa L. Segura, Instituto F a t a l a Chaben, Buenos Aires, Argentina; R A , obtained from Dr. Stella M a r l s Gonzfilez C a p p a , F a e u l t a d de Medicina, Universidad de
1A 1
2
3
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1C E
T
4.
5
6
1B 7
9
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P'2'
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T
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2 A E TP
12
34567
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974 660 45.0 -
290-
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1160 974~ 66.0 4 5 0
-
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Fig- I. Protelnase activity in polyac~lamide gels cc~tainin 8 fibrinogen, run in the presence of SDS. When whole parasite homogeaat~s were used, the amount run was 5 × 106 cells per lane. (A), activity in epimastisotcs of Tul 2 (1). clone CA-I/72 (2), Sonia (3), clone Dm28C (4); Peril (5); Tul O (6); Y (7). Corpus Christi (8), AWP (9) and purified enzyme (P, 0.2/lg) p' and 2', same as P and 2. but the gel was washed and incubated in the presence of lfi ~M E-64. (B and C), proteinase activity in am~tszigotes(A), epimastigotes (E) and trypomastigotes (T) of the RA strain. The gels were incubated at 37 °C for 2 h (B), or 18 h (C). Fig. 2. Detection of proteins reactive with a polyclonal rabbit serum against purified eysteine proteinase, in Western blots of total parasite extracts. Letters and numbers are as in Fig. 1, hut the amount of material used was 2× 10s cells for A and I", 1 × 107 cells for epimastigotes of all stocks and clones tested, and l ~g for the purified enzyme.
147 scribed [2]. When used for activity gels, the samples were not boiled [9]. When the parasites were to be used for Western blots, the final washing of the cells was performed in the presence of proteinas¢ inhibitors (0.5 mM TLCK. 2 mM PMSF, 5 /tM lenpeptin, 5 # M E-64 and 5 ,aM peptstatin), the pellets were dissolved in Laemmli's sample buffer [10], and the samples were boiled as usual before loading onto the gels. 3.3. Electrophoresis Minigels ( 9 × 7 cm, 0.5 m m thick), 12.5% acrylamide, were made up as described by Laemmii [10], except that fibrinogan was added to the small pore s~,!ution, at a final concentration of 240 ~tg/ml [9], and ,'an at 70 V for about 2 h. When the bromophenol blue used as marker almost reached the bottom, the run was stopped and the gels (after two washings in distilled water, 15 rain each) were incubated at 3 7 ° C in 0.1 M morpholino ethane sulfonic acid (MES) buffer, pH 3.9, containing 0.5 mM DTI'~ for the times stated in the legend to Fig. 1. The reaction was stopped and the remaining protein stained by incbuation for 45 rain at 5 0 ° C in a solution containing 0.2% Coomassie Brilliant Blue R 250 in methanol/acetic acid/water (50/7/43, v / v / v ) . After destaining in 10% methanol, 7% acetic acid, the activity bands were observed as colorless bands over a blue background. 3.4. Enzyme assay The enzyme activity was determined on azocasein at pH 5 in the presence of 10 mM 2-mcrcaptocthanol, as previously described [2]. Urea and the detergents contained in the sample buffer were diluted 100-fold in the reactin mixtuft, and did not interfere with the enzyme activity. 3.5. Protein determination It was performed by the method of Lowry et ai. [111. 3.6. Preparation of a polyclonal rabbit antiserum Rabbits were immunized by intramuscular injection (hind legs) of 50 ~g of purified enzyme in 1 ml of PBS, emulsified (1 : 1, v / v ) with corn-
plete Freund adjuvant (GIBCO). Three more doses were injected subcutaneously, emulsified with incomplete Freund adjuvant (GIBCO), at 20 dayintervals. The presence of specific antibodies were detected by the method of Ouchterlony (titr¢ 1/160): only one band, with identical migration, was observed when both, the purified enzyme or the cell-free extract, were used as antigens. This was confirmed with Western blots, as described below. 3.Z Western blots The samples were run in 10,% standard (15-12 cm, 1.5 mM thick) polyacrylamide gels in the presence of SDS, elcctrotransferred to nitrocellulose filters, washed for 1 h with Tris-saline buffer, pH 7.6, containing 370 bovine serum albumin, and probed with the antiserum, diluted 1/1OO0 in the same solution. After 1 h incubation at room temperature, and repeated washings, the fihcrs were treated with t2~l-Staphylococcus aureas A protein (105 c.p.m.ml 1) for 1 h at room temperature in the same buffer. After repeated washings the filters were dried and exposed to sensitive X-ray film for autoradiography. 3.8. Chemicals Azocasein, fibrinogen, 2-mercaptoethanol, leupcptin, TLCK, E-64, pepstatin A and PMSF were obtained from Sigma Chemical Co, St. Louis, MO, U.S.A. All other chemicals used were analytica reagents of the highest purity available.
4. RESULTS A N D DISCUSSION Homogenates of T. cruzi epimastigotes of differem clones and stocks, prepared in sample buffer containing urea, SDS and Triton X-t00, presented similar proteinase activity on azocasein, namely (activities, expressed as AA,~o n m ) ' h -1 - m g of protein 1, mean of 3 determinations 4- standard error, in parenthesis): Tul 2 (2.89 ± 0.56); Tul 0 (1.58 + 0.15); Sonia (3.63 ± 0.74); Peril (1.83 40.09); AWP (3.13 4- 0.68); Y (2.56 + 0.23); Corpus Christi (3.04 4- 0.51); D m 28c (2.90 4- 0.44) and CA-1/72 (1.93_+0.19). The specific activities in epimastigotes, anastigotes and trypomastigotes of
148
the RA strain were 2.02 + 0.16, 0.27 4-0.04 and 0.34 _ 0.02, respectively; these differences are statistically significant ( P < 0.001). The azocaseinase activity measured in vitro correlated quite well with the activity observed in fibrioogen-containing polyacrylamide gels, after electrophoresis in the presence of SDS. Fig. I A shows that epimastigotes from all the stocks and clones studied presented proteinase activity, migrating in most cases as a double band, with an electrophoretic mobility similar or identical with that of the purified enzyme; there were small but reproducible differences in enzyme mobility among some of the different stocks and clones, Dm28c showing always the lowest mobility. When epimastigotes, amastigotes and trypomastigotes of the RA strain were compared (Fig. 1B and C), the activity in the former appeared as a main band with the same mobility as the purified enzyme, with traces of a higher molecular weight component. In the case of trypomastigotes, two bands of similar intensity were observed, migrating with the same mobilities. In the case of the amastigotes) there were again two bands, but these had a slightly higher molecular weight. Even with much longer incubation periods (18 h/Fig. 1C) no visible bands could be observed at molecular weight values higher than 66 kDa, or lower than 45 kDa. This is not due to enzyme inactivation by the urea-SDS-Triton sample buffer used, since cell-free extracts in 0.25 M sucrose-5 mM KCI yielded similar results [5]. Fig. 1A also shows that washing and incubation of the gel with solutions containing the cysteine prot¢inase-specific inhibitor E-64, at a concentration of 10 pM, abolished completely the activity. Preincubation with the same concentration of inhibitor inhibited by 95-100~ in the azocaseinase assay in all cases (not shown). Although only two lanes are shown, for the sake of briefness, this result was obtained in all cases. When the proteins present in parasite homogenates were subjected to SDS-PAGE and clectrotransferred to nitrocellulose, and the Western blots were probed with a polyclonai monuspecifie antiserum against the purified proteinase (Fig. 2), the immunoreaetive material in epimastigotes appeared exclusively as a thick band with an electrophoretie mobility identical or very close to that of the purified
enzyme, in most cases with a fainter smear at a higher molecular weight value. This would correspond to the double band observed in the fibrinogen-containing gels (Fig. 1). In the case of the amastigotes, there were two clearly separated bands, of slightly higher molecular weight, in good agreement with the results shown in Fig. 1B and C. The double band observed for the purified enzyme in the Western blots, and very seldom seen in fibrinogen-containing gels (not shown), might be an artifact. Alternatively, it is possible that two proteins very close in molecular weight and with very similar properties, would co-purify in the procedure used; indeed, the elution profile in the Mono Q step indicates mieroheterogeneities [3]. Differential expression of immunological cross-reactive protein variants might be responsible for the different patterns observed in amastigotes and epimastigotes. The results presented in this communication suggest that (a) the major proteinase activities present in the three main developmental forms of T. cruzi, and in epimastigutes of different stocks and clones, belong exclusively to the cysreine proteinase class; (b) the three different experimental approaches followed indicate that epimastigores of different parasite stocks and clones have similar proteinase levels, but that the enzyme is present at lower levels in amastigotes and trypomastigotes. This su~ests that, as recently shown for the 28 kDa cysteine proteinase from T. brueei [12], the expression of the T. cruzi enzyme is developmentally regulated. (c) the pattern of proteinase activities seems to be considerably simpler in T. eruzi, as compared with the African trypanosomes [9]. It is noteworthy that incubation of the gels for up to 18 h, or even 24 h (not shown) failed to detect significant proteinase activity in other regions of the gels. Among the other proteinases purified from T. cruzi [1]1, one has the same molecular weight, and the other, of molecular weight 200 kDa, might become inactive when dissociated. We can not rule out the presence of other proteinases unable to degrade fibrinogen at pH 4 and to cross-react wit hthe antibodies against the purified eysteioc proteinase; (d) the enzyme we have previously purified [2,3] from epimastigotes of the Tul 2 stock, or closely related isocnzymic variants,
149 seems to be the m a i n ¢ysteine proteinase present in different stocks and clones of the parasite.
ACKNOWLEDGEMENTS W e are indebted to Dr. F r a n k AshaU, from the D e p a r t m e n t of M ~ l i c a l Protozoology, London School of Hygiene a n d T r o p i c a l Medicine, for his suggestion o f the use of minigels, and also for his generous gift of a sample of E-64, and to Dr. A.C.C. Frasch for helpful discussions. T h i s w o r k was performed with grants from the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research and T r a i n i n g in T r o p i c a l Diseases, from the Swedisch Agency for Research Cooperation w i t h Developing Countries ( S A R E C ) , and from the Consejo N a c i o n a l de Investigaciones Cientificas y T ~ n i c a s de la Repfiblica A r g e n t i n a ( C O N I C E T ) . JJC is a m e m b e r of the C a r r e r a del Investigador Cienfifico, and O C a Research Fellow, from C O N I C E T , and ,fM holds a student schollarship from the University of Buenos Aires.
REFERENCES [1] Cazzulo, JJ. (1984) Comp. Biochem. Physiol. 79 B+ 309-320. [2] Bomempi. E.. Franke de Cazzulo, B.M.. Ruiz, A.M. and Cazzu[o, J.J. (1984) Comp. Biochem. Physiol. 7"/ B, 599-604. [3l Cazzulo. JJ.. Couso, g., RaimondL A., Wernstndt, C. and H¢llman, U. (1989) MOL Biochem. paras[teL 33, 33-42. 141 BontempL E.. Martine~ J. and Cazzulo, J-l. (1989) MoL Biochem. Paras[teL 33. 43-48. [5] Martinet, J., Silherstein, E. and Cazzulo, JJ. (1989) Anales Asoc. Quim. Argentina 77,113-117. 16] Conteras, V.T., SaBes, J.M., Thomas" N., Morel, C.M. and Goldenber 8. S. (1985) Mol. Biochem. Paras[tel, 16. 315-327. [7] Cazzulo. J.J., Franke de Cazzulo, B.M., Engel. C. and Cannata, J.J.B. (1985) MoL Biochem. Paras[tel. 16, 329-343. [8] Andrews, N.W. and CollL W. (1982) J. ProtozooL 29, 264-269. [9] Lonsdale-Eceles" J.D. and Mpimbaza, G.W.N. (1986) Eur. J. Biocher~ 155, 469-473. [10] Laemmli, U.K. (1970) Nature 227, 680-685. Ill] Lowry, O.H., Rosebrough, NJ., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [12] Pamer. E.G., So, M. and Davis, C.E. (1989) Mol. Biochem. Paras[teL 33, 27-32.
145
FEMSLE 03872
A
major cysteine proteinase is developmentally regulated in Trypanosoma cruzi Oscar Campetella, Javier Martinez and J u a n Jos6 Cazzulo lnsrimto de lnvestigacianes BioquJmicas Fundacibn Campomar. Facultad de Ciencias Exactas y Naturales. Universldad de Buenos Aires -- CONICET, Buenos Aires, Repbblica Argentina
ReceivedS August1989 Revisionreceivedand accepted3 October1989 Key words: Tryp a n o so ma cruzi; Cysteine proteinases; Western blots; Polyacrylamide gels
1. SUMMARY
2. INTRODUCTION
Epimastigotes of different stocks of Trypanos o m a cruzi contain similar levels of proteinase
Three cysteine proteinases have been purified to electrophoretic homogeneity from cell free extracts of epimastigotes of different strains of T r y p a n o s o m a cruzi, the causative agent of Chagas" disease [1]. One of them is a lysosomal glycoprorein of molecular weight of about 60 kD& strongly inhibited by L . t r o n s - e p o x y s u c c i n y l - l e u c y l a m i d o (4-guanidino) butane (E-64), leupeptin, tosyl-Llysine chloromethyl ketone (TLCK) and organomercurial reagents, insensitive to phenyl methyl sulfonyl fluoride (PMSF) and activated by 2mercaptoethanol, which presents 65% of identify with papain and cathepsin L in its N-terminus [2-4]. We have recently shown that this enzyme is the major proteolytic activity detectable in cell-free extracts by clectrophoresis in polyacrylamide gels containing fibrinogen or hemoglobin [5]. We present here evidence indicating that epimastigotes of. different stocks and clones of the parasite contain cysteine proteinases, detected both as activity in fibrinogen-containing gels and in Western blots reacted with a polyclonal antiserum. The pro-
activity on azocasein; a m a s t i g o t e s and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.
Correspondence: Dr, Juan Jos~ Cazzulo. lnstituto de Investigaclones Bioquitn~a$ Fundacibn Campomar,A. Machado 151, 1405 BuenosAires. Argentina.
0378-1097/90/$03.50© 1990Federationof EuropeanMicrobiologicalSocieties
146 teinase thus detected is present, in the same range of molecular weight, in epimastigotes of all stocks and clones tested, as well as in the other two main developmental forms o f the parasite, trypomastigote and amastigote.
Buenos Aires, Buenos Aires, Argentina, a n d the clone D m 28e [6], obtained from Dr. A n t o n i o Morello, F a c u l t a d de Medicina, U n i v e r s i d a d de Chile, Santiago, Chile. Epimastigotes were cultured in B H T medium, as previously described [7]. Trypomastigotes and amastigotes o f the R A strain were obtained from infected V e t o cultures [8].
3. M A T E R I A L S A N D M E T H O D S
3.2. Sample preparation 3. I. Parasite stocks and culture
Parasite homogenates used for enzyme activity determination, b o t h in fibrinogen-containing gels and on azocasein in vitro, were m a d e by suspension o f the cells in sample buffer c o n t a i n i n g 0.04 M T r i s - H C l , p H 6.8, 4 M urea, 0 . 1 ~ S D S a n d 2% T r i t o n X-100 [9] a n d freezing at - 2 0 ° C . T h e purified enzyme was obtained as previously de-
T h e parasite stocks used were T u l 0, T u l 2, A W E Corpus Christi, Y, Peru, Sonia, and the clone C A - I / 7 2 , obtained from D r . Elsa L. Segura, Instituto F a t a l a Chaben, Buenos Aires, Argentina; R A , obtained from Dr. Stella M a r l s Gonzfilez C a p p a , F a e u l t a d de Medicina, Universidad de
1A 1
2
3
A
1C E
T
4.
5
6
1B 7
9
P
P'2'
A
E
T
9"/4 660450290 -
2 A E TP
12
34567
B 9
974 660 45.0 -
290-
,nw
1160 974~ 66.0 4 5 0
-
290
-
Fig- I. Protelnase activity in polyac~lamide gels cc~tainin 8 fibrinogen, run in the presence of SDS. When whole parasite homogeaat~s were used, the amount run was 5 × 106 cells per lane. (A), activity in epimastisotcs of Tul 2 (1). clone CA-I/72 (2), Sonia (3), clone Dm28C (4); Peril (5); Tul O (6); Y (7). Corpus Christi (8), AWP (9) and purified enzyme (P, 0.2/lg) p' and 2', same as P and 2. but the gel was washed and incubated in the presence of lfi ~M E-64. (B and C), proteinase activity in am~tszigotes(A), epimastigotes (E) and trypomastigotes (T) of the RA strain. The gels were incubated at 37 °C for 2 h (B), or 18 h (C). Fig. 2. Detection of proteins reactive with a polyclonal rabbit serum against purified eysteine proteinase, in Western blots of total parasite extracts. Letters and numbers are as in Fig. 1, hut the amount of material used was 2× 10s cells for A and I", 1 × 107 cells for epimastigotes of all stocks and clones tested, and l ~g for the purified enzyme.
147 scribed [2]. When used for activity gels, the samples were not boiled [9]. When the parasites were to be used for Western blots, the final washing of the cells was performed in the presence of proteinas¢ inhibitors (0.5 mM TLCK. 2 mM PMSF, 5 /tM lenpeptin, 5 # M E-64 and 5 ,aM peptstatin), the pellets were dissolved in Laemmli's sample buffer [10], and the samples were boiled as usual before loading onto the gels. 3.3. Electrophoresis Minigels ( 9 × 7 cm, 0.5 m m thick), 12.5% acrylamide, were made up as described by Laemmii [10], except that fibrinogan was added to the small pore s~,!ution, at a final concentration of 240 ~tg/ml [9], and ,'an at 70 V for about 2 h. When the bromophenol blue used as marker almost reached the bottom, the run was stopped and the gels (after two washings in distilled water, 15 rain each) were incubated at 3 7 ° C in 0.1 M morpholino ethane sulfonic acid (MES) buffer, pH 3.9, containing 0.5 mM DTI'~ for the times stated in the legend to Fig. 1. The reaction was stopped and the remaining protein stained by incbuation for 45 rain at 5 0 ° C in a solution containing 0.2% Coomassie Brilliant Blue R 250 in methanol/acetic acid/water (50/7/43, v / v / v ) . After destaining in 10% methanol, 7% acetic acid, the activity bands were observed as colorless bands over a blue background. 3.4. Enzyme assay The enzyme activity was determined on azocasein at pH 5 in the presence of 10 mM 2-mcrcaptocthanol, as previously described [2]. Urea and the detergents contained in the sample buffer were diluted 100-fold in the reactin mixtuft, and did not interfere with the enzyme activity. 3.5. Protein determination It was performed by the method of Lowry et ai. [111. 3.6. Preparation of a polyclonal rabbit antiserum Rabbits were immunized by intramuscular injection (hind legs) of 50 ~g of purified enzyme in 1 ml of PBS, emulsified (1 : 1, v / v ) with corn-
plete Freund adjuvant (GIBCO). Three more doses were injected subcutaneously, emulsified with incomplete Freund adjuvant (GIBCO), at 20 dayintervals. The presence of specific antibodies were detected by the method of Ouchterlony (titr¢ 1/160): only one band, with identical migration, was observed when both, the purified enzyme or the cell-free extract, were used as antigens. This was confirmed with Western blots, as described below. 3.Z Western blots The samples were run in 10,% standard (15-12 cm, 1.5 mM thick) polyacrylamide gels in the presence of SDS, elcctrotransferred to nitrocellulose filters, washed for 1 h with Tris-saline buffer, pH 7.6, containing 370 bovine serum albumin, and probed with the antiserum, diluted 1/1OO0 in the same solution. After 1 h incubation at room temperature, and repeated washings, the fihcrs were treated with t2~l-Staphylococcus aureas A protein (105 c.p.m.ml 1) for 1 h at room temperature in the same buffer. After repeated washings the filters were dried and exposed to sensitive X-ray film for autoradiography. 3.8. Chemicals Azocasein, fibrinogen, 2-mercaptoethanol, leupcptin, TLCK, E-64, pepstatin A and PMSF were obtained from Sigma Chemical Co, St. Louis, MO, U.S.A. All other chemicals used were analytica reagents of the highest purity available.
4. RESULTS A N D DISCUSSION Homogenates of T. cruzi epimastigotes of differem clones and stocks, prepared in sample buffer containing urea, SDS and Triton X-t00, presented similar proteinase activity on azocasein, namely (activities, expressed as AA,~o n m ) ' h -1 - m g of protein 1, mean of 3 determinations 4- standard error, in parenthesis): Tul 2 (2.89 ± 0.56); Tul 0 (1.58 + 0.15); Sonia (3.63 ± 0.74); Peril (1.83 40.09); AWP (3.13 4- 0.68); Y (2.56 + 0.23); Corpus Christi (3.04 4- 0.51); D m 28c (2.90 4- 0.44) and CA-1/72 (1.93_+0.19). The specific activities in epimastigotes, anastigotes and trypomastigotes of
148
the RA strain were 2.02 + 0.16, 0.27 4-0.04 and 0.34 _ 0.02, respectively; these differences are statistically significant ( P < 0.001). The azocaseinase activity measured in vitro correlated quite well with the activity observed in fibrioogen-containing polyacrylamide gels, after electrophoresis in the presence of SDS. Fig. I A shows that epimastigotes from all the stocks and clones studied presented proteinase activity, migrating in most cases as a double band, with an electrophoretic mobility similar or identical with that of the purified enzyme; there were small but reproducible differences in enzyme mobility among some of the different stocks and clones, Dm28c showing always the lowest mobility. When epimastigotes, amastigotes and trypomastigotes of the RA strain were compared (Fig. 1B and C), the activity in the former appeared as a main band with the same mobility as the purified enzyme, with traces of a higher molecular weight component. In the case of trypomastigotes, two bands of similar intensity were observed, migrating with the same mobilities. In the case of the amastigotes) there were again two bands, but these had a slightly higher molecular weight. Even with much longer incubation periods (18 h/Fig. 1C) no visible bands could be observed at molecular weight values higher than 66 kDa, or lower than 45 kDa. This is not due to enzyme inactivation by the urea-SDS-Triton sample buffer used, since cell-free extracts in 0.25 M sucrose-5 mM KCI yielded similar results [5]. Fig. 1A also shows that washing and incubation of the gel with solutions containing the cysteine prot¢inase-specific inhibitor E-64, at a concentration of 10 pM, abolished completely the activity. Preincubation with the same concentration of inhibitor inhibited by 95-100~ in the azocaseinase assay in all cases (not shown). Although only two lanes are shown, for the sake of briefness, this result was obtained in all cases. When the proteins present in parasite homogenates were subjected to SDS-PAGE and clectrotransferred to nitrocellulose, and the Western blots were probed with a polyclonai monuspecifie antiserum against the purified proteinase (Fig. 2), the immunoreaetive material in epimastigotes appeared exclusively as a thick band with an electrophoretie mobility identical or very close to that of the purified
enzyme, in most cases with a fainter smear at a higher molecular weight value. This would correspond to the double band observed in the fibrinogen-containing gels (Fig. 1). In the case of the amastigotes, there were two clearly separated bands, of slightly higher molecular weight, in good agreement with the results shown in Fig. 1B and C. The double band observed for the purified enzyme in the Western blots, and very seldom seen in fibrinogen-containing gels (not shown), might be an artifact. Alternatively, it is possible that two proteins very close in molecular weight and with very similar properties, would co-purify in the procedure used; indeed, the elution profile in the Mono Q step indicates mieroheterogeneities [3]. Differential expression of immunological cross-reactive protein variants might be responsible for the different patterns observed in amastigotes and epimastigotes. The results presented in this communication suggest that (a) the major proteinase activities present in the three main developmental forms of T. cruzi, and in epimastigutes of different stocks and clones, belong exclusively to the cysreine proteinase class; (b) the three different experimental approaches followed indicate that epimastigores of different parasite stocks and clones have similar proteinase levels, but that the enzyme is present at lower levels in amastigotes and trypomastigotes. This su~ests that, as recently shown for the 28 kDa cysteine proteinase from T. brueei [12], the expression of the T. cruzi enzyme is developmentally regulated. (c) the pattern of proteinase activities seems to be considerably simpler in T. eruzi, as compared with the African trypanosomes [9]. It is noteworthy that incubation of the gels for up to 18 h, or even 24 h (not shown) failed to detect significant proteinase activity in other regions of the gels. Among the other proteinases purified from T. cruzi [1]1, one has the same molecular weight, and the other, of molecular weight 200 kDa, might become inactive when dissociated. We can not rule out the presence of other proteinases unable to degrade fibrinogen at pH 4 and to cross-react wit hthe antibodies against the purified eysteioc proteinase; (d) the enzyme we have previously purified [2,3] from epimastigotes of the Tul 2 stock, or closely related isocnzymic variants,
149 seems to be the m a i n ¢ysteine proteinase present in different stocks and clones of the parasite.
ACKNOWLEDGEMENTS W e are indebted to Dr. F r a n k AshaU, from the D e p a r t m e n t of M ~ l i c a l Protozoology, London School of Hygiene a n d T r o p i c a l Medicine, for his suggestion o f the use of minigels, and also for his generous gift of a sample of E-64, and to Dr. A.C.C. Frasch for helpful discussions. T h i s w o r k was performed with grants from the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research and T r a i n i n g in T r o p i c a l Diseases, from the Swedisch Agency for Research Cooperation w i t h Developing Countries ( S A R E C ) , and from the Consejo N a c i o n a l de Investigaciones Cientificas y T ~ n i c a s de la Repfiblica A r g e n t i n a ( C O N I C E T ) . JJC is a m e m b e r of the C a r r e r a del Investigador Cienfifico, and O C a Research Fellow, from C O N I C E T , and ,fM holds a student schollarship from the University of Buenos Aires.
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