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Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei
FEMS Microbiology Letters 220 (2003) 197^206
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Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei Eloise Cedro Fernandes a; , Jose¤ Mauro Granjeiro b , Eula¤zio Mikio Taga b , Jose¤ Roberto Meyer-Fernandes c , Hiroshi Aoyama d a Departamento de Bioqu|¤mica, Universidade Federal de Sa‹o Paulo (UNIFESP), 04023900 Sa‹o Paulo, SP, Brazil Departamento de Cie“ncias Biolo¤gicas, Faculdade de Odontologia de Bauru, Universidade de Sa‹o Paulo (USP), Bauru, SP, Brazil Departamento de Bioqu|¤mica, Instituto de Ciencias Biologicas, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil d Departamento de Bioqu|¤mica, Instituto de Biologia, Universidade Estadual de Campinas (Unicamp), 13083-970 Campinas, SP, Brazil b
c
Received 1 November 2002 ; accepted 24 January 2003 First published online 15 February 2003
Abstract Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [32 P]phosphocasein, is inhibited weakly by NaF and tartrate but strongly by vanadate. In this work, we describe the presence of an external phosphatase activity in intact bloodstream forms of T. brucei. With p-nitrophenyl phosphate (pNPP) as substrate, these intact cells produced 3^5 nmol pNP min31 mg31 , linearly for up to at least 30 min. The activity was not significantly increased by Mg2þ , Mn2þ , Ca2þ and Co2þ , but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn2þ and was insensitive to okadaic acid. Membrane-enriched fractions of parasites contained an acid phosphatase activity, with a pH optimum in the range of 4.5^5.5. This activity hydrolyzed phosphotyrosine (40 nmol phosphate min31 mg31 ) better than phosphothreonine or phosphoserine. Partial purification of this phosphatase yielded a single activity band following gel electrophoresis, a Km value of 0.29 mM with pNPP and was insensitive to the Fe2þ /H2 O2 /ascorbate system. < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Phosphotyrosine phosphatase ; Bloodstream forms ; Trypanosoma brucei
1. Introduction Phosphorylation and dephosphorylation are fundamental pathways that regulate a wide variety of cellular events [1]. Tyrosine phosphorylation in vivo is regulated by protein tyrosine kinases, which catalyze tyrosine phosphorylation, and protein tyrosine phosphatases (PTPs), which are responsible for dephosphorylation [2]. Cell membrane enzymes in which the active site faces the external medium rather than the cytoplasm can be assayed using live cells
* Corresponding author. Present address: Centro de Gene¤tica, Biologia Molecular e Fitoqu|¤mica, Instituto Agrono“mico de Campinas, Av. Theodureto de A. Camargo 1500, 13.075-630, Campinas, SP, Brazil. Tel. : +55 (19) 32415188; Fax: +55 (19) 32423602. E-mail address : [email protected] (E. Cedro Fernandes). Abbreviations : EDTA, ethylenediaminetetraacetic acid ; CDTA, trans1,2-diaminocyclohexane-N,N,NP,NP-tetraacetic acid ; pCMB, p-chloromercuribenzoate; pNPP, p-nitrophenyl phosphate ; pNP, p-nitrophenol
(reviewed in [3]). Of particular interest are ecto-enzymes present on the outer surface of the membrane of bloodstream parasites since they can mediate parasite^host interactions. Trypanosoma brucei is a protozoan parasite that passes through several extracellular cycles in its mammalian hosts and tsetse £y vector. These cycles involve changes in the parasite’s metabolism, morphology and membrane composition. Several protein kinases have been described in T. brucei [4^7] with the appearance of the protein phosphorylation networks probably representing an ancient event in trypanosomes [8]. The di¡erentiation of the parasite from bloodforms to procyclic forms was apparently accompanied by increased tyrosine phosphorylation [9]. In contrast, little is known about protein phosphatases and their function in the life-cycle of T. brucei [10^12]. In other trypanosomatids, such as Leishmania donovani [13], phosphatase activity is uniformly distributed in the plasma membrane and in the vicinity of the £agellar pocket. Some of these enzymes have been puri¢ed [14] and because
0378-1097 / 03 / $22.00 < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00091-0
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they are excreted, it has been suggested that they may be useful as a marker of virulence [15]. The cloning and characterization of a protein phosphatase from Leishmania chagasi showed that this protein was conserved among Leishmania and eukaryotic serine/threonine protein phosphatases [16]. Since most of studies of Trypanosoma phosphatases have been done either with crude trypanosome lysates or with puri¢ed enzymes, we have investigated the behavior of these enzymes in intact cells. The results presented here indicate that intact and alive bloodstream forms of T. brucei possess ecto-phosphatase activities able to hydrolyze the phosphotyrosine analog p-nitrophenyl phosphate (pNPP) and phosphotyrosine. In addition, this membraneassociated acid phosphatase was found to be resistant to inactivation by oxygen metabolites, as also reported for Leishmania phosphatase [17].
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under positive pressure with nitrogen in an Amicon ultra¢ltration system using PM-10 membranes. Enzyme activity was assayed by pNPP hydrolysis and purity was assessed by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE). 2.4. Phosphatase assay
Bloodstream forms of the monomorphic clone T. brucei brucei MITat 1.4 were harvested at mid-exponential phase from infected rats and isolated by DEAE-cellulose (Whatman DE-52) chromatography [18]. T. brucei brucei procyclic forms ILTar 2 were grown at 28‡C in SDM-79 medium. The parasites were collected, washed and kept in 50 mM Tris^HCl, pH 7.2, containing 20 mM KCl and 100 mM sucrose.
Unless otherwise speci¢ed, pNPP hydrolysis was assayed as previously described [21]. Reactions were initiated by the addition of intact cells, membrane-enriched fractions or partially puri¢ed enzyme and stopped by the addition of 1 ml of 1 M NaOH. trans-1,2-Diaminocyclohexane-N,N,NP,NP-tetraacetic acid (CDTA) or ethylenediaminetetraacetic acid (EDTA) was used to measure activity in the absence of divalent cations. The p-nitrophenol (pNP) released was measured based on the increase in absorbance at 425 nm (the extinction coe⁄cient used for the p-nitrophenolate ion was 1.75U104 M31 cm31 ). Speci¢c activity was expressed as nmol of pNP released min31 mg31 of protein. The hydrolysis of phosphorylated amino acids was measured under the same conditions as for pNPP. The free inorganic phosphate released was determined according to Lowry and Lopez [22]. Phosphatase activity was assayed in the absence of metal ions by including 1 mM CDTA. Phosphatase activity was also detected in gels as described by Gomez [23]. Brie£y, after electrophoresis in non-denaturing conditions, any activity was detected by incubating the gels with 50 mM L-naphtylphosphate and 1 mg Fast-blue BB ml31 in 100 mM acetate bu¡er, pH 5.0, at 37‡C.
2.2. Preparation of the membrane fraction
2.5. Chemicals
Parasite membrane-enriched fractions were prepared as described by Seyfang and Duszenko [19] and stored at 3130‡C. Protein concentrations were determined by the biuret method [20] using bovine serum albumin as standard.
All chemicals were obtained from Sigma Chemical (St. Louis, MO, USA).
2.3. Partial puri¢cation of the membrane phosphatase
A phosphatase activity not dependent on divalent cations has been described on the external surface of intact, live procyclic forms of T. brucei (0.67 nmol pNP min31 mg31 )[21]. As shown here, infectious, intact and live bloodstream forms of this parasite also have external phosphatase activity able to hydrolyze pNPP at pH 8.0 and 37‡C (3^5 nmol pNP min31 mg31 ). Fig. 1A compares the phosphatase activities of intact T. brucei procyclic
2. Materials and methods 2.1. Parasites
Phosphatase was puri¢ed from cell concentrates as described by Seyfang and Duszenko [19]. Brie£y, a ghost fraction was obtained by hypotonic lysis, washed several times and the protein fraction was homogenized in acetate bu¡er, pH 5.0, and Triton X-114. After di¡erential centrifugation, the supernatant was collected and concentrated
3. Results
6 Fig. 1. Comparison of the phosphatase activity in procyclic and bloodstream forms of T. brucei. A: Time-dependent hydrolysis of pNPP by intact parasites of procyclic forms at pH 7.2 and 30‡C without metals (line a) and bloodstream forms at pH 8.0 and 37‡C (line b), assayed as described in Section 2. B: Phospho-amino acid hydrolysis by bloodstream forms of T. brucei. 1 mg of bloodstream membrane fraction was incubated with 60 mM sodium citrate, pH 5.5, containing 44 mM NaCl, 20 mM KCl, 55 mM glucose and 10 mM of phospho-amino acids, at 37‡C. Dephosphorylation of 10 mM P-Tyr was measured at pH 5.5 or at pH 8.0; higher activity was observed at pH 5.5 (inset). C: Phosphatase activity towards pNPP (0.01^30 mM) was measured during 30 min using 1 mg of protein. Line a, intact bloodstream forms at 37‡C ; line b, membrane fraction from bloodstream forms, 37‡C ; line c, intact procyclic forms without metals at 30‡C. Inset shows the double reciprocal plot for line b.
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Fig. 2. Characterization of phosphatases in intact T. brucei. A: E¡ect of pH on T. brucei phosphatase activity. 1 mg of membrane fraction of bloodstream forms was incubated for 30 min at 37‡C with 10 mM pNPP at di¡erent pH values, as described in Section 2. B: E¡ect of divalent cations on phosphatase activity of intact procyclic and bloodstream forms of T. brucei. The assays were done at pH 7.2 and 30‡C (procyclics) or pH 8.0 and 37‡C (bloodstreams) using 1 mg of T. brucei protein ml31 and 10 mM of pNPP. C: E¡ect of classic phosphatase inhibitors on phosphatase activity of intact procyclic and bloodstream forms. Intact procyclic and bloodstream forms (1 mg ml31 ) were incubated at 37‡C in the presence of 1 mM of di¡erent phosphatase inhibitors as described in Section 2. Other concentrations are indicated in the ¢gure.
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forms and intact, infective bloodstream forms of T. brucei toward pNPP. Fig. 1B shows the ability of membrane fractions to hydrolyze phosphothreonine (1.3 V 0.1 nmol phosphate min31 mg31 ), phosphoserine (4.0 V 1.3 nmol phosphate min31 mg31 ) and especially phosphotyrosine (20.0 V 0.7 nmol phosphate min31 mg31 ) at pH 5.5. The phosphatase activity towards phospho-amino acids could not be measured using intact infectious cells (see Section 4). To con¢rm the acidic nature of the enzyme(s) towards phosphotyrosine, this activity was tested at acidic and alkaline pH and was found to be greater at pH 5.5 (Fig. 1B, inset). The phosphatase activity of intact, infectious bloodstream forms showed no saturation with pNPP up to at least 30 mM pNPP (Fig. 1C, line a). The bloodstream membrane fractions showed a Michaelis^Menten behavior for pNPP hydrolysis, with a Vmax of 3.5 nmol pNP mg31 min31 and a Km of 1.5 V 0.4 mM, at pH 8.0 (Fig. 1C, line b). When tested at pH 5.5, Km was 0.40 V 0.12 mM pNPP (curves not shown). Line c represents the phosphatase activity of intact procyclic forms in the presence of increasing amounts of substrate. The inset shows the double reciprocal transformation for line b. To avoid the possibility that secreted phosphatases or cytoplasmic phosphatases released from dead parasites could be responsible for the pNPP hydrolysis, the same reactions were run with pNPP in reaction medium in which live cells had been pre-incubated for 60 min and then removed by centrifugation. No pNPP hydrolysis was detected in the resulting supernatants (data not shown). The viability of bloodstream cells was assessed by incubating parasites at the same concentration in medium for the same time and then counting the cells in a Neubauer chamber. All cells remained viable and 80% retained their mobility. Rats inoculated with these cells developed sleeping sickness within 3 days, con¢rming that the parasite virulence was also retained. Since the bloodstream forms of T. brucei cannot survive
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at pH values below 7.4, it was necessary to use membraneenriched fractions to examine the e¡ect of pH values on the phosphatase activity. Fig. 2A shows high bloodstream phosphatase activity at pH 4.5^5.5 and an abrupt fall in activity above or below this range. Fig. 2B compares the e¡ect of divalent cations that caused an opposed e¡ect on the phosphatase activity of intact bloodstream and procyclic forms. Since the e¡ects of Zn2þ have been described for other membrane phosphatases [24], we examined the inhibition by Zn2þ in more detail: the activity in intact bloodstream parasites was inhibited by 50% by 0.05 mM Zn2þ whereas 3 mM Zn2þ inhibited the activity by 90%. Table 1 shows the in£uence of other divalent cations on the phosphatase activity of intact procyclic and bloodstream forms of T. brucei. CDTA and EDTA (1 mM) were ¢rst tested in the control assays. Mn2þ and Co2þ increased the phosphatase activity of intact procyclic forms more than in bloodstream intact forms. Ca2þ , an essential divalent cation for Ca-calmodulin phosphatases, stimulated the phosphatase activity of intact bloodstream forms by 50% but had no e¡ect on that of intact procyclic forms up to a concentration of 3 mM. Fe2þ , Sr2þ , Ni2þ and Ba2þ did not cause signi¢cant alterations ( 6 1%) in the control activity of both bloodstream and procyclic forms, even at a concentration of 5 mM (data not shown). Fig. 2C compares the e¡ect of phosphatase inhibitors on the phosphatase activities of live procyclic forms in the absence of divalent metals and of live bloodstream forms without the addition of cations to the assay. At 1 mM, NaF inhibited 40% of the enzyme activity in intact procyclic forms and 50% of the control phosphatase activity of the bloodstream forms. Levamizole (1 mM), a well-known inhibitor of alkaline phosphatase, had little e¡ect on the phosphatase activity of intact procyclic or bloodstream forms. A higher concentration of levamizole increased the phosphatase activities of both forms (data not shown). Tetramizole (1 mM) inhibited 40% of the procyclic phos-
Table 1 E¡ect of divalent cations on phosphatase activity present in intact procyclic and bloodstream forms of T. brucei Additions
None CDTA EDTA Mg2þ Mn2þ Co2þ Ca2þ Fe2þ
Final concentration
Percentage of control activity
(mM)
Intact procyclic forms
Intact bloodstream forms
^ 1 1 1 3 1 5 1 3 1 5 1 3
100 50 50 250 300 300 400 200 270 100 150 100 100
100 80 80 110 120 150 150 110 140 130 145 90 85
The assays were done as described for each form in Section 2, using 1 mg of intact parasites. The results are the mean of three experiments, corrected for appropriate blanks.
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phatase activity but had no e¡ect on the phosphatase activity of bloodstream forms; at higher concentrations, tetramizole activated the enzyme (data not shown). Tartrate, an inhibitor of the secreted phosphatase of L. donovani [25], did not a¡ect the enzyme in intact procyclic forms but decreased the activity in intact bloodstream forms to 70% of the control activity. p-chloromercuribenzoate (pCMB ; 1 mM) an inhibitor of SH group-dependent enzymes, decreased the procyclic phosphatase activity to 50% and, at the same concentration, lowered the enzyme activity of bloodstream forms to less than 10% of the
control activity (50% inhibition was achieved with 1 WM pCMB; data not shown). Okadaic acid (10 WM), a toxin used to identify serine^threonine phosphatase, did not affect this phosphatase activity. Vanadate, a potent inhibitor of acid phosphatases and phosphotyrosine phosphatases, caused 90% inhibition of the phosphatase activity in both forms of intact parasite. The IC50 for enzyme inhibition was 0.5 WM for the procyclic forms and 0.7 WM for the bloodstream forms. After puri¢cation, the membrane-bound acid phosphatase was subjected to gel electrophoresis with subsequent
a b c d e kDa 94 68 43 30 24
Fig. 3. Phosphatase activity and kinetic parameters of the enzyme puri¢ed from T. brucei bloodstream forms. A: Non-denaturating PAGE ; lane a, gel slice with 30 Wg of protein was incubated with L-naphtylphosphate as substrate at pH 5.0 [23]. Under the same non-denaturing conditions, protein staining revealed two bands at pH 5.0 (lane b, 30 Wg of protein ; lane c, 50 Wg of protein) and pH 7.5 (lane d, 50 Wg of protein). Lane e, SDS^PAGE showing a major band at 95^100 kDa. B: E¡ect of incubation time on the release of pNP. The activity was assayed using 10 mM pNPP at 37‡C, as described in Section 2. C: E¡ect of pH on the enzyme puri¢ed from T. brucei, bloodstream forms. The activity was assayed using 10 mM pNPP at 37‡C, as described in Section 2. D : E¡ect of pNPP concentration on enzyme behavior, showing the Michaelis^Menten behavior. Inset : double reciprocal plot, with a Km of 0.29 mM for pNPP.
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detection of activity in situ [23] : under non-denaturing conditions, the puri¢ed enzyme showed a single band of phosphatase activity (Fig. 3A, lane a) coincident with the band detected by Coomassie blue staining (lanes b and c with 30 and 50Wg of protein, respectively). Electrophoresis under the same conditions, but at pH 8.0, revealed two main bands, suggesting that this heterogeneity may be due to protein glycosylation (lane d, 30 Wg of protein). SDS^ PAGE showed a major band of approximately 95^100 kDa (lane e, 50 Wg of protein). Fig. 3B shows that the enzymatic reaction was linear with time, at least up to 60 min. The activity was also linearly dependent on the amount of protein used (data not shown). The puri¢ed enzyme had a pH optimum between 4.5 and 5.5 (Fig. 3C) with an apparent Km of 0.29 V 0.03 mM pNPP (Fig. 3D) and a Vmax of 0.74 Wmol pNPP min31 mg31 (Fig. 3D, inset). As with the intact parasites, the puri¢ed enzyme was not a¡ected by Ca2þ , Mg2þ and Co2þ (Fig. 4A) or by 5 mM EDTA or CDTA (data not shown). However, Zn2þ and Cu2þ (1.0 mM) inhibited the activity by 40% and 60%, respectively. Of the inhibitors tested (Fig. 4B), sodium vanadate, NaF, molybdate and, to a lesser extent, pCMB were the strongest inhibitors. High concentrations of tartrate, cysteine, dithiothreitol and guanosine, described as activators of low molecular mass PTPs, did not a¡ect the enzyme activity. The IC50 for vanadate at pH 5.0 was 0.8 mM (Fig. 5B, inset). Among the other phosphatase substrates tested (Fig. 4C), pyrophosphate and phosphotyrosine provided the best results; glycerophosphate and phosphoserine were also hydrolyzed at a lesser extent; no hydrolysis was observed when 6-phosphoglucose was tested (data not shown). To test the hypothesis proposed by Gottlieb et al. [13], we examined the resistance of puri¢ed enzyme to toxic oxygen metabolites produced by a Fe2þ /H2 O2 /ascorbate system. Fig. 5 shows that ectophosphatase puri¢ed from bloodstream forms of T. brucei remained active towards pNPP after a 15 min exposure. Under the same conditions, a low molecular mass PTP, extracted from bovine kidney [26], used as a control, lost 50% of its activity.
4. Discussion To our knowledge, this is the ¢rst demonstration of a phosphatase activity on the surface of intact procyclic and bloodstream forms of T. brucei. The present results maintained their attention on the behavior of the phosphatase activity of the entire and live parasites because we were interested in the possible role of this enzyme in the cell^cell interactions. That phosphatase activity could not be measured in the supernatants of cells, even after a 60 min incubation, indicates that the pNPP hydrolysis seen was not caused by broken cells. As also shown by phase contrast microscopy, control cells retained their motility. Sim-
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ilarly, the phosphatase activity detected in live procyclic and bloodstream forms showed a linear increase in product formation with time, during at least 30 min. If cell lysis were occurring, the increase in pNP-formation would probably have been non-linear since more and more cells would be dying during the assay. Comparison of the kinetic parameters of the procyclic and bloodstream forms indicated that: (i) the phosphatase activity was more intense in live bloodstream forms (3^5 nmol of pNP min31 mg31 ) than in live procyclic forms in the absence of metals (0.67 nmol of pNP min31 mg31 ) or in the presence of Mg2þ 3 mM (1.5 nmol of pNP min31 mg31 ); (ii) in both forms, the phosphatase activity was unequivocally acid, although the activities in (i) were measured at pH 7.2^8.0; (iii) in contrast to live bloodstream forms, the phosphatase activity in intact procyclic forms and in the membrane-enriched fraction showed a Michaelis^Menten behavior, with a Km of 0.40 mM pNPP at pH 5.5 and 2.4 mM pNPP at pH 8.0; (iv) the bloodstream form phosphatase activity was resistant to inactivation by oxygen metabolites; and (v) the greatest distinction between these phosphatase activities was the capacity of bloodstream forms to hydrolyze P-Tyr whereas no activity toward P-amino acids was detected in the procyclic forms. Some of the parameters described here were similar to those reported by Seyfang and Duszenko [19], including the acidic nature of the enzyme and the sensitivity to inhibition by tartrate, NaF and vanadate. Other parameters, like the metal interference, could not be discussed because there were no data about metal in that work. This is not the ¢rst time that acid phosphatases have been described attached to the external surface of the membrane of a bloodstream parasite [11,27,28]. Tosomba et al. [28] used cytochemical procedures to demonstrate the presence of an acid phosphatase on the surface membrane of T. congolense. The low optimum pH and the surface location of these enzymes suggest a role in an acidic microenvironment and/or a close relationship with lysosomal digestion and the £agellar pocket membrane. Indeed, procyclic forms cause a marked acidi¢cation of the medium after 36 h in culture medium. To emphasize the stage speci¢city of the phosphatase in bloodstream forms, the experiments were done simultaneously with live procyclic and bloodstream forms. The procyclic forms were unable to hydrolyze phosphotyrosine and the procyclic phosphatase was dependent on divalent metals, particularly Mg2þ , in a manner similar to type PP2C tyrosine phosphatase [1]. On the other hand, the phosphatase of bloodstream forms was unable to hydrolyze phosphoserine and phosphothreonine and was inhibited by divalent cations such as Cu2þ and Cd2þ , a ¢nding con¢rmed by using puri¢ed enzyme (Fig. 3A). The addition of 1 mM EDTA decreased the phosphatase activity by 50% in the procyclic forms and by 80% in intact bloodstream forms. The Km (0.4 mM) for the phosphotyrosine analog
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A
B
C
Fig. 4. Dephosphorylation of 10 mM pNPP by puri¢ed enzyme in the presence of metals and phosphatase inhibitors. A: 1 mg of protein was incubated for 15 min at 37‡C as described in Section 2. The concentrations of the metals used are indicated in the legend. B: 1 mg of protein was incubated for 15 min at 37‡C in the presence of di¡erent inhibitors, as described in Section 2. The inhibitor concentrations are indicated in the legend. The e¡ect of di¡erent concentrations of vanadate on the puri¢ed phosphatase is indicated in the inset. C: Potential substrates for the puri¢ed enzyme. The enzyme activity was determined using 10 mM of each substrate and an incubation time of 60 min. The inset shows the Michaelis^Menten behavior of the enzyme towards P-Tyr.
pNPP, obtained using bloodstream membrane fractions at pH 5.5, was the same as that described by Schell [29] for the £agellum bag of T. brucei. The speci¢c activities of these two enzymes were very similar: 43.4 mmol pNPP
min31 mg31 [29] and 43.6 mmol pNPP min31 mg31 (this work). Trypanosomes do not secrete acid phosphatases, although exceptions have been reported for T. cruzi [30]
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oxidative stress. Such a resistance could help to explain the survival of the parasite in the host circulatory system without a need to invade host cells to escape the host’s immune system. Just as the parasite developed an alternative system of escape, the variant surface glycoproteins, it is possible that the escape mechanism of the oxidative stress also brings us new surprises.
Acknowledgements The authors thank Dr. Roberto Docampo for the trypanosomes and Dr. Silvia Moreno for helpful comments. This work was supported by FAPESP (FundacXa‹o de Amparo a' Pesquisa do Estado de Sa‹o Paulo) and CNPq (Conselho Nacional de Pesquisa e Desenvolvimento Tecnolo¤gico). Fig. 5. Tolerance of the puri¢ed phosphatase to oxidative stress. E¡ect of the Fe2þ /H2 O2 /ascorbate system on the activity of acid phosphatase puri¢ed from bloodstream forms and on bovine kidney low-molecularmass PTP. The enzyme was pre-incubated for di¡erent periods with 0.2 mM Fe2þ and 10 mM ascorbate, in 0.1 M acetate bu¡er, pH 5.0 at 37‡C. Oxidation was initiated by adding 14.5 mM H2 O2 . At the indicated times, the reaction was stopped by adding 1 mM EDTA. The residual activity was determined using pNPP as substrate, as described in Section 2 and a reaction time of 60 min. Control activity (100%) corresponded to activity in the absence of oxidation.
and L. donovani [13]. The phosphatase of intact bloodstream forms showed a pattern of general inhibition not very di¡erent from that described for the procyclic forms, i.e. inhibition by NaF, vanadate, and tartrate, but not by levamizole and tetramizole. pCMB markedly inhibited the phosphatase of bloodstream forms, indicating that this enzyme was SH-dependent, with a cysteine residue essential for enzyme activity and/or stability. Using the Lowry and Lopez method [22] and phosphotyrosine as substrate, the free phosphate released by live bloodstream parasites was observed to disappear quickly in the reaction medium, giving negative results when compared to the assay blank. On the other hand, with increasing phosphate concentrations, the live bloodstream forms reduced the inorganic phosphate concentration at a rate of 5 pmol phosphate min31 (data not shown). This behavior was not observed with the procyclic forms. Gottlieb and Dwyer [13] and Saha et al. [17] tested the resistance of Leishmania phosphatase to inactivation by oxygen metabolites. The toxicity of H2 O2 to bloodstream forms of T. brucei and the inhibitory e¡ect on parasite oxygen consumption and pyruvate output have been described [31]. Since T. brucei bloodstream forms lack catalase and glutathione peroxidase (for review, see [31]), an alternative H2 O2 -detoxi¢cation pathway has been proposed [32,33]. Because hemo£agellate parasites are exposed to toxic oxygen metabolites derived from drug metabolism or immune mechanisms, it is necessary to determine whether the parasite enzymes are resistant to
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[14] Remaley, A.T., Das, S., Campbell, P.I., LaRocca, G.M., Pope, M.T. and Glew, R.H. (1985) Characterization of L. donovani acid phosphatases. J. Biol. Chem. 260, 880^886. [15] Singla, N., Khuller, G.K. and Vinayak, V.K. (1992) Acid phosphatase activity of promastigotes of L. donovani a marker of virulence. FEMS Microbiol. Lett. 94, 221^226. [16] Burns, J.M., Parsons, M., Rosman, D.E. and Reed, S.G. (1993) Molecular cloning and characterization of a 42-kDa protein phosphatase of L. chagasi. J. Biol. Chem. 268, 17155^17161. [17] Saha, A.K., Das, S., Glew, R.H. and Gottlieb, M. (1985) Resistence of leishmanial phosphatases to inactivation by oxygen metabolites. J. Clin. Microbiol. 22, 329^332. [18] Cross, G.A.M. (1984) Structure of the variant glycoproteins and surface coat of T. brucei. Proc. R. Soc. Lond. 307, 3^12. [19] Seyfang, A. and Duszenko, M. (1993) Functional reconstitution of the T. brucei plasma-membrane D-glucose transporter. Eur. J. Biochem. 214, 593^597. [20] Gornall, A.G., Bardawill, C.J. and David, M.M. (1949) Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751^766. [21] Fernandes, E.C., Meyer-Fernandes, J.R., Silva-Neto, M.A.C. and Vercesi, A.E. (1997) Trypanosoma brucei : ecto-phosphatase present on the surface of intact procyclic forms. Z. Naturforsch. C 52, 351^ 358. [22] Lowry, H.O. and Lopez, J.A. (1946) The determination of inorganic phosphate in the presence of labile phosphate esters. J. Biol. Chem. 162, 421^428. [23] Gomez, J.J. (1978) An improved method for phosphatase detection. Ann. Enzymol. 32, 44^47. [24] Cherno¡, J. and Li, H.-C. (1985) A major phosphotyrosyl-protein
[25]
[26]
[27]
[28]
[29]
[30]
[31] [32]
[33]
phosphatase from bovine heart is associated with a low molecularweight acid phosphatase. Arch. Biochem. Biophys. 240, 135^145. Lovelace, J.K. and Gottlieb, M. (1986) Comparison of extracellular acid phosphatases from various isolates of Leishmania. Ame J. Trop. Med. Hyg. 35, 1121^1128. Granjeiro, J.M., Taga, E.M. and Aoyama, H. (1997) Puri¢cation and characterization of a low-molecular-weight bovine kidney acid phosphatase. Ann. Acad. Bras. Cien. 69, 451^460. McLaughlin, J. (1986) The association of distinct acid phosphatases with the £agella pocket and surface membrane fractions obtained from bloodstream forms of T. rhodesiense. Mol. Cell. Biochem. 70, 177^184. Tosomba, O.M., Coetzer, T.H.T. and Londale-Eccles, D. (1996) Localisation of acid phosphatase activity on the surface of bloodstream forms of T. congolense. Exp. Parasitol. 84, 429^438. Schell, D., Stierhof, Y.D. and Overath, P. (1990) Puri¢cation and characterization of a tartrate-sensitive acid phosphatase of Trypanosoma brucei. FEBS Lett. 271, 67^70. Nakamura, K.H., Tachibana, H. and Kanada, Y. (1985) Alteration on cell surface acid phosphatase concomitant with the morphological transformation in T. cruzi. Comp. Biochem. Physiol. 81B, 815^817. Penketh, P.G. and Klein, R.A. (1986) Hydrogen peroxide metabolism in T. brucei. Mol. Biochem. Parasitol. 20, 111^121. Henderson, G.B., Fairland, A.H. and Cerami, A. (1987) Trypanothione-dependent peroxide mechanism in C. fasciculata and T. brucei. Mol. Biochem. Parasitol. 24, 39^45. El-Sayed, N.M.A., Alarcon, C.M., She⁄eld, V.C. and Donelson, J.E. (1995) cDNA expressed sequence tags of T. rhodesience provide new insights into the biology of the parasite. Mol. Biochem. Parasitol. 44, 116^118.
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Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei Eloise Cedro Fernandes a; , Jose¤ Mauro Granjeiro b , Eula¤zio Mikio Taga b , Jose¤ Roberto Meyer-Fernandes c , Hiroshi Aoyama d a Departamento de Bioqu|¤mica, Universidade Federal de Sa‹o Paulo (UNIFESP), 04023900 Sa‹o Paulo, SP, Brazil Departamento de Cie“ncias Biolo¤gicas, Faculdade de Odontologia de Bauru, Universidade de Sa‹o Paulo (USP), Bauru, SP, Brazil Departamento de Bioqu|¤mica, Instituto de Ciencias Biologicas, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil d Departamento de Bioqu|¤mica, Instituto de Biologia, Universidade Estadual de Campinas (Unicamp), 13083-970 Campinas, SP, Brazil b
c
Received 1 November 2002 ; accepted 24 January 2003 First published online 15 February 2003
Abstract Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [32 P]phosphocasein, is inhibited weakly by NaF and tartrate but strongly by vanadate. In this work, we describe the presence of an external phosphatase activity in intact bloodstream forms of T. brucei. With p-nitrophenyl phosphate (pNPP) as substrate, these intact cells produced 3^5 nmol pNP min31 mg31 , linearly for up to at least 30 min. The activity was not significantly increased by Mg2þ , Mn2þ , Ca2þ and Co2þ , but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn2þ and was insensitive to okadaic acid. Membrane-enriched fractions of parasites contained an acid phosphatase activity, with a pH optimum in the range of 4.5^5.5. This activity hydrolyzed phosphotyrosine (40 nmol phosphate min31 mg31 ) better than phosphothreonine or phosphoserine. Partial purification of this phosphatase yielded a single activity band following gel electrophoresis, a Km value of 0.29 mM with pNPP and was insensitive to the Fe2þ /H2 O2 /ascorbate system. < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Phosphotyrosine phosphatase ; Bloodstream forms ; Trypanosoma brucei
1. Introduction Phosphorylation and dephosphorylation are fundamental pathways that regulate a wide variety of cellular events [1]. Tyrosine phosphorylation in vivo is regulated by protein tyrosine kinases, which catalyze tyrosine phosphorylation, and protein tyrosine phosphatases (PTPs), which are responsible for dephosphorylation [2]. Cell membrane enzymes in which the active site faces the external medium rather than the cytoplasm can be assayed using live cells
* Corresponding author. Present address: Centro de Gene¤tica, Biologia Molecular e Fitoqu|¤mica, Instituto Agrono“mico de Campinas, Av. Theodureto de A. Camargo 1500, 13.075-630, Campinas, SP, Brazil. Tel. : +55 (19) 32415188; Fax: +55 (19) 32423602. E-mail address : [email protected] (E. Cedro Fernandes). Abbreviations : EDTA, ethylenediaminetetraacetic acid ; CDTA, trans1,2-diaminocyclohexane-N,N,NP,NP-tetraacetic acid ; pCMB, p-chloromercuribenzoate; pNPP, p-nitrophenyl phosphate ; pNP, p-nitrophenol
(reviewed in [3]). Of particular interest are ecto-enzymes present on the outer surface of the membrane of bloodstream parasites since they can mediate parasite^host interactions. Trypanosoma brucei is a protozoan parasite that passes through several extracellular cycles in its mammalian hosts and tsetse £y vector. These cycles involve changes in the parasite’s metabolism, morphology and membrane composition. Several protein kinases have been described in T. brucei [4^7] with the appearance of the protein phosphorylation networks probably representing an ancient event in trypanosomes [8]. The di¡erentiation of the parasite from bloodforms to procyclic forms was apparently accompanied by increased tyrosine phosphorylation [9]. In contrast, little is known about protein phosphatases and their function in the life-cycle of T. brucei [10^12]. In other trypanosomatids, such as Leishmania donovani [13], phosphatase activity is uniformly distributed in the plasma membrane and in the vicinity of the £agellar pocket. Some of these enzymes have been puri¢ed [14] and because
0378-1097 / 03 / $22.00 < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00091-0
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they are excreted, it has been suggested that they may be useful as a marker of virulence [15]. The cloning and characterization of a protein phosphatase from Leishmania chagasi showed that this protein was conserved among Leishmania and eukaryotic serine/threonine protein phosphatases [16]. Since most of studies of Trypanosoma phosphatases have been done either with crude trypanosome lysates or with puri¢ed enzymes, we have investigated the behavior of these enzymes in intact cells. The results presented here indicate that intact and alive bloodstream forms of T. brucei possess ecto-phosphatase activities able to hydrolyze the phosphotyrosine analog p-nitrophenyl phosphate (pNPP) and phosphotyrosine. In addition, this membraneassociated acid phosphatase was found to be resistant to inactivation by oxygen metabolites, as also reported for Leishmania phosphatase [17].
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under positive pressure with nitrogen in an Amicon ultra¢ltration system using PM-10 membranes. Enzyme activity was assayed by pNPP hydrolysis and purity was assessed by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE). 2.4. Phosphatase assay
Bloodstream forms of the monomorphic clone T. brucei brucei MITat 1.4 were harvested at mid-exponential phase from infected rats and isolated by DEAE-cellulose (Whatman DE-52) chromatography [18]. T. brucei brucei procyclic forms ILTar 2 were grown at 28‡C in SDM-79 medium. The parasites were collected, washed and kept in 50 mM Tris^HCl, pH 7.2, containing 20 mM KCl and 100 mM sucrose.
Unless otherwise speci¢ed, pNPP hydrolysis was assayed as previously described [21]. Reactions were initiated by the addition of intact cells, membrane-enriched fractions or partially puri¢ed enzyme and stopped by the addition of 1 ml of 1 M NaOH. trans-1,2-Diaminocyclohexane-N,N,NP,NP-tetraacetic acid (CDTA) or ethylenediaminetetraacetic acid (EDTA) was used to measure activity in the absence of divalent cations. The p-nitrophenol (pNP) released was measured based on the increase in absorbance at 425 nm (the extinction coe⁄cient used for the p-nitrophenolate ion was 1.75U104 M31 cm31 ). Speci¢c activity was expressed as nmol of pNP released min31 mg31 of protein. The hydrolysis of phosphorylated amino acids was measured under the same conditions as for pNPP. The free inorganic phosphate released was determined according to Lowry and Lopez [22]. Phosphatase activity was assayed in the absence of metal ions by including 1 mM CDTA. Phosphatase activity was also detected in gels as described by Gomez [23]. Brie£y, after electrophoresis in non-denaturing conditions, any activity was detected by incubating the gels with 50 mM L-naphtylphosphate and 1 mg Fast-blue BB ml31 in 100 mM acetate bu¡er, pH 5.0, at 37‡C.
2.2. Preparation of the membrane fraction
2.5. Chemicals
Parasite membrane-enriched fractions were prepared as described by Seyfang and Duszenko [19] and stored at 3130‡C. Protein concentrations were determined by the biuret method [20] using bovine serum albumin as standard.
All chemicals were obtained from Sigma Chemical (St. Louis, MO, USA).
2.3. Partial puri¢cation of the membrane phosphatase
A phosphatase activity not dependent on divalent cations has been described on the external surface of intact, live procyclic forms of T. brucei (0.67 nmol pNP min31 mg31 )[21]. As shown here, infectious, intact and live bloodstream forms of this parasite also have external phosphatase activity able to hydrolyze pNPP at pH 8.0 and 37‡C (3^5 nmol pNP min31 mg31 ). Fig. 1A compares the phosphatase activities of intact T. brucei procyclic
2. Materials and methods 2.1. Parasites
Phosphatase was puri¢ed from cell concentrates as described by Seyfang and Duszenko [19]. Brie£y, a ghost fraction was obtained by hypotonic lysis, washed several times and the protein fraction was homogenized in acetate bu¡er, pH 5.0, and Triton X-114. After di¡erential centrifugation, the supernatant was collected and concentrated
3. Results
6 Fig. 1. Comparison of the phosphatase activity in procyclic and bloodstream forms of T. brucei. A: Time-dependent hydrolysis of pNPP by intact parasites of procyclic forms at pH 7.2 and 30‡C without metals (line a) and bloodstream forms at pH 8.0 and 37‡C (line b), assayed as described in Section 2. B: Phospho-amino acid hydrolysis by bloodstream forms of T. brucei. 1 mg of bloodstream membrane fraction was incubated with 60 mM sodium citrate, pH 5.5, containing 44 mM NaCl, 20 mM KCl, 55 mM glucose and 10 mM of phospho-amino acids, at 37‡C. Dephosphorylation of 10 mM P-Tyr was measured at pH 5.5 or at pH 8.0; higher activity was observed at pH 5.5 (inset). C: Phosphatase activity towards pNPP (0.01^30 mM) was measured during 30 min using 1 mg of protein. Line a, intact bloodstream forms at 37‡C ; line b, membrane fraction from bloodstream forms, 37‡C ; line c, intact procyclic forms without metals at 30‡C. Inset shows the double reciprocal plot for line b.
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Fig. 2. Characterization of phosphatases in intact T. brucei. A: E¡ect of pH on T. brucei phosphatase activity. 1 mg of membrane fraction of bloodstream forms was incubated for 30 min at 37‡C with 10 mM pNPP at di¡erent pH values, as described in Section 2. B: E¡ect of divalent cations on phosphatase activity of intact procyclic and bloodstream forms of T. brucei. The assays were done at pH 7.2 and 30‡C (procyclics) or pH 8.0 and 37‡C (bloodstreams) using 1 mg of T. brucei protein ml31 and 10 mM of pNPP. C: E¡ect of classic phosphatase inhibitors on phosphatase activity of intact procyclic and bloodstream forms. Intact procyclic and bloodstream forms (1 mg ml31 ) were incubated at 37‡C in the presence of 1 mM of di¡erent phosphatase inhibitors as described in Section 2. Other concentrations are indicated in the ¢gure.
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forms and intact, infective bloodstream forms of T. brucei toward pNPP. Fig. 1B shows the ability of membrane fractions to hydrolyze phosphothreonine (1.3 V 0.1 nmol phosphate min31 mg31 ), phosphoserine (4.0 V 1.3 nmol phosphate min31 mg31 ) and especially phosphotyrosine (20.0 V 0.7 nmol phosphate min31 mg31 ) at pH 5.5. The phosphatase activity towards phospho-amino acids could not be measured using intact infectious cells (see Section 4). To con¢rm the acidic nature of the enzyme(s) towards phosphotyrosine, this activity was tested at acidic and alkaline pH and was found to be greater at pH 5.5 (Fig. 1B, inset). The phosphatase activity of intact, infectious bloodstream forms showed no saturation with pNPP up to at least 30 mM pNPP (Fig. 1C, line a). The bloodstream membrane fractions showed a Michaelis^Menten behavior for pNPP hydrolysis, with a Vmax of 3.5 nmol pNP mg31 min31 and a Km of 1.5 V 0.4 mM, at pH 8.0 (Fig. 1C, line b). When tested at pH 5.5, Km was 0.40 V 0.12 mM pNPP (curves not shown). Line c represents the phosphatase activity of intact procyclic forms in the presence of increasing amounts of substrate. The inset shows the double reciprocal transformation for line b. To avoid the possibility that secreted phosphatases or cytoplasmic phosphatases released from dead parasites could be responsible for the pNPP hydrolysis, the same reactions were run with pNPP in reaction medium in which live cells had been pre-incubated for 60 min and then removed by centrifugation. No pNPP hydrolysis was detected in the resulting supernatants (data not shown). The viability of bloodstream cells was assessed by incubating parasites at the same concentration in medium for the same time and then counting the cells in a Neubauer chamber. All cells remained viable and 80% retained their mobility. Rats inoculated with these cells developed sleeping sickness within 3 days, con¢rming that the parasite virulence was also retained. Since the bloodstream forms of T. brucei cannot survive
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at pH values below 7.4, it was necessary to use membraneenriched fractions to examine the e¡ect of pH values on the phosphatase activity. Fig. 2A shows high bloodstream phosphatase activity at pH 4.5^5.5 and an abrupt fall in activity above or below this range. Fig. 2B compares the e¡ect of divalent cations that caused an opposed e¡ect on the phosphatase activity of intact bloodstream and procyclic forms. Since the e¡ects of Zn2þ have been described for other membrane phosphatases [24], we examined the inhibition by Zn2þ in more detail: the activity in intact bloodstream parasites was inhibited by 50% by 0.05 mM Zn2þ whereas 3 mM Zn2þ inhibited the activity by 90%. Table 1 shows the in£uence of other divalent cations on the phosphatase activity of intact procyclic and bloodstream forms of T. brucei. CDTA and EDTA (1 mM) were ¢rst tested in the control assays. Mn2þ and Co2þ increased the phosphatase activity of intact procyclic forms more than in bloodstream intact forms. Ca2þ , an essential divalent cation for Ca-calmodulin phosphatases, stimulated the phosphatase activity of intact bloodstream forms by 50% but had no e¡ect on that of intact procyclic forms up to a concentration of 3 mM. Fe2þ , Sr2þ , Ni2þ and Ba2þ did not cause signi¢cant alterations ( 6 1%) in the control activity of both bloodstream and procyclic forms, even at a concentration of 5 mM (data not shown). Fig. 2C compares the e¡ect of phosphatase inhibitors on the phosphatase activities of live procyclic forms in the absence of divalent metals and of live bloodstream forms without the addition of cations to the assay. At 1 mM, NaF inhibited 40% of the enzyme activity in intact procyclic forms and 50% of the control phosphatase activity of the bloodstream forms. Levamizole (1 mM), a well-known inhibitor of alkaline phosphatase, had little e¡ect on the phosphatase activity of intact procyclic or bloodstream forms. A higher concentration of levamizole increased the phosphatase activities of both forms (data not shown). Tetramizole (1 mM) inhibited 40% of the procyclic phos-
Table 1 E¡ect of divalent cations on phosphatase activity present in intact procyclic and bloodstream forms of T. brucei Additions
None CDTA EDTA Mg2þ Mn2þ Co2þ Ca2þ Fe2þ
Final concentration
Percentage of control activity
(mM)
Intact procyclic forms
Intact bloodstream forms
^ 1 1 1 3 1 5 1 3 1 5 1 3
100 50 50 250 300 300 400 200 270 100 150 100 100
100 80 80 110 120 150 150 110 140 130 145 90 85
The assays were done as described for each form in Section 2, using 1 mg of intact parasites. The results are the mean of three experiments, corrected for appropriate blanks.
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phatase activity but had no e¡ect on the phosphatase activity of bloodstream forms; at higher concentrations, tetramizole activated the enzyme (data not shown). Tartrate, an inhibitor of the secreted phosphatase of L. donovani [25], did not a¡ect the enzyme in intact procyclic forms but decreased the activity in intact bloodstream forms to 70% of the control activity. p-chloromercuribenzoate (pCMB ; 1 mM) an inhibitor of SH group-dependent enzymes, decreased the procyclic phosphatase activity to 50% and, at the same concentration, lowered the enzyme activity of bloodstream forms to less than 10% of the
control activity (50% inhibition was achieved with 1 WM pCMB; data not shown). Okadaic acid (10 WM), a toxin used to identify serine^threonine phosphatase, did not affect this phosphatase activity. Vanadate, a potent inhibitor of acid phosphatases and phosphotyrosine phosphatases, caused 90% inhibition of the phosphatase activity in both forms of intact parasite. The IC50 for enzyme inhibition was 0.5 WM for the procyclic forms and 0.7 WM for the bloodstream forms. After puri¢cation, the membrane-bound acid phosphatase was subjected to gel electrophoresis with subsequent
a b c d e kDa 94 68 43 30 24
Fig. 3. Phosphatase activity and kinetic parameters of the enzyme puri¢ed from T. brucei bloodstream forms. A: Non-denaturating PAGE ; lane a, gel slice with 30 Wg of protein was incubated with L-naphtylphosphate as substrate at pH 5.0 [23]. Under the same non-denaturing conditions, protein staining revealed two bands at pH 5.0 (lane b, 30 Wg of protein ; lane c, 50 Wg of protein) and pH 7.5 (lane d, 50 Wg of protein). Lane e, SDS^PAGE showing a major band at 95^100 kDa. B: E¡ect of incubation time on the release of pNP. The activity was assayed using 10 mM pNPP at 37‡C, as described in Section 2. C: E¡ect of pH on the enzyme puri¢ed from T. brucei, bloodstream forms. The activity was assayed using 10 mM pNPP at 37‡C, as described in Section 2. D : E¡ect of pNPP concentration on enzyme behavior, showing the Michaelis^Menten behavior. Inset : double reciprocal plot, with a Km of 0.29 mM for pNPP.
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detection of activity in situ [23] : under non-denaturing conditions, the puri¢ed enzyme showed a single band of phosphatase activity (Fig. 3A, lane a) coincident with the band detected by Coomassie blue staining (lanes b and c with 30 and 50Wg of protein, respectively). Electrophoresis under the same conditions, but at pH 8.0, revealed two main bands, suggesting that this heterogeneity may be due to protein glycosylation (lane d, 30 Wg of protein). SDS^ PAGE showed a major band of approximately 95^100 kDa (lane e, 50 Wg of protein). Fig. 3B shows that the enzymatic reaction was linear with time, at least up to 60 min. The activity was also linearly dependent on the amount of protein used (data not shown). The puri¢ed enzyme had a pH optimum between 4.5 and 5.5 (Fig. 3C) with an apparent Km of 0.29 V 0.03 mM pNPP (Fig. 3D) and a Vmax of 0.74 Wmol pNPP min31 mg31 (Fig. 3D, inset). As with the intact parasites, the puri¢ed enzyme was not a¡ected by Ca2þ , Mg2þ and Co2þ (Fig. 4A) or by 5 mM EDTA or CDTA (data not shown). However, Zn2þ and Cu2þ (1.0 mM) inhibited the activity by 40% and 60%, respectively. Of the inhibitors tested (Fig. 4B), sodium vanadate, NaF, molybdate and, to a lesser extent, pCMB were the strongest inhibitors. High concentrations of tartrate, cysteine, dithiothreitol and guanosine, described as activators of low molecular mass PTPs, did not a¡ect the enzyme activity. The IC50 for vanadate at pH 5.0 was 0.8 mM (Fig. 5B, inset). Among the other phosphatase substrates tested (Fig. 4C), pyrophosphate and phosphotyrosine provided the best results; glycerophosphate and phosphoserine were also hydrolyzed at a lesser extent; no hydrolysis was observed when 6-phosphoglucose was tested (data not shown). To test the hypothesis proposed by Gottlieb et al. [13], we examined the resistance of puri¢ed enzyme to toxic oxygen metabolites produced by a Fe2þ /H2 O2 /ascorbate system. Fig. 5 shows that ectophosphatase puri¢ed from bloodstream forms of T. brucei remained active towards pNPP after a 15 min exposure. Under the same conditions, a low molecular mass PTP, extracted from bovine kidney [26], used as a control, lost 50% of its activity.
4. Discussion To our knowledge, this is the ¢rst demonstration of a phosphatase activity on the surface of intact procyclic and bloodstream forms of T. brucei. The present results maintained their attention on the behavior of the phosphatase activity of the entire and live parasites because we were interested in the possible role of this enzyme in the cell^cell interactions. That phosphatase activity could not be measured in the supernatants of cells, even after a 60 min incubation, indicates that the pNPP hydrolysis seen was not caused by broken cells. As also shown by phase contrast microscopy, control cells retained their motility. Sim-
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ilarly, the phosphatase activity detected in live procyclic and bloodstream forms showed a linear increase in product formation with time, during at least 30 min. If cell lysis were occurring, the increase in pNP-formation would probably have been non-linear since more and more cells would be dying during the assay. Comparison of the kinetic parameters of the procyclic and bloodstream forms indicated that: (i) the phosphatase activity was more intense in live bloodstream forms (3^5 nmol of pNP min31 mg31 ) than in live procyclic forms in the absence of metals (0.67 nmol of pNP min31 mg31 ) or in the presence of Mg2þ 3 mM (1.5 nmol of pNP min31 mg31 ); (ii) in both forms, the phosphatase activity was unequivocally acid, although the activities in (i) were measured at pH 7.2^8.0; (iii) in contrast to live bloodstream forms, the phosphatase activity in intact procyclic forms and in the membrane-enriched fraction showed a Michaelis^Menten behavior, with a Km of 0.40 mM pNPP at pH 5.5 and 2.4 mM pNPP at pH 8.0; (iv) the bloodstream form phosphatase activity was resistant to inactivation by oxygen metabolites; and (v) the greatest distinction between these phosphatase activities was the capacity of bloodstream forms to hydrolyze P-Tyr whereas no activity toward P-amino acids was detected in the procyclic forms. Some of the parameters described here were similar to those reported by Seyfang and Duszenko [19], including the acidic nature of the enzyme and the sensitivity to inhibition by tartrate, NaF and vanadate. Other parameters, like the metal interference, could not be discussed because there were no data about metal in that work. This is not the ¢rst time that acid phosphatases have been described attached to the external surface of the membrane of a bloodstream parasite [11,27,28]. Tosomba et al. [28] used cytochemical procedures to demonstrate the presence of an acid phosphatase on the surface membrane of T. congolense. The low optimum pH and the surface location of these enzymes suggest a role in an acidic microenvironment and/or a close relationship with lysosomal digestion and the £agellar pocket membrane. Indeed, procyclic forms cause a marked acidi¢cation of the medium after 36 h in culture medium. To emphasize the stage speci¢city of the phosphatase in bloodstream forms, the experiments were done simultaneously with live procyclic and bloodstream forms. The procyclic forms were unable to hydrolyze phosphotyrosine and the procyclic phosphatase was dependent on divalent metals, particularly Mg2þ , in a manner similar to type PP2C tyrosine phosphatase [1]. On the other hand, the phosphatase of bloodstream forms was unable to hydrolyze phosphoserine and phosphothreonine and was inhibited by divalent cations such as Cu2þ and Cd2þ , a ¢nding con¢rmed by using puri¢ed enzyme (Fig. 3A). The addition of 1 mM EDTA decreased the phosphatase activity by 50% in the procyclic forms and by 80% in intact bloodstream forms. The Km (0.4 mM) for the phosphotyrosine analog
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A
B
C
Fig. 4. Dephosphorylation of 10 mM pNPP by puri¢ed enzyme in the presence of metals and phosphatase inhibitors. A: 1 mg of protein was incubated for 15 min at 37‡C as described in Section 2. The concentrations of the metals used are indicated in the legend. B: 1 mg of protein was incubated for 15 min at 37‡C in the presence of di¡erent inhibitors, as described in Section 2. The inhibitor concentrations are indicated in the legend. The e¡ect of di¡erent concentrations of vanadate on the puri¢ed phosphatase is indicated in the inset. C: Potential substrates for the puri¢ed enzyme. The enzyme activity was determined using 10 mM of each substrate and an incubation time of 60 min. The inset shows the Michaelis^Menten behavior of the enzyme towards P-Tyr.
pNPP, obtained using bloodstream membrane fractions at pH 5.5, was the same as that described by Schell [29] for the £agellum bag of T. brucei. The speci¢c activities of these two enzymes were very similar: 43.4 mmol pNPP
min31 mg31 [29] and 43.6 mmol pNPP min31 mg31 (this work). Trypanosomes do not secrete acid phosphatases, although exceptions have been reported for T. cruzi [30]
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oxidative stress. Such a resistance could help to explain the survival of the parasite in the host circulatory system without a need to invade host cells to escape the host’s immune system. Just as the parasite developed an alternative system of escape, the variant surface glycoproteins, it is possible that the escape mechanism of the oxidative stress also brings us new surprises.
Acknowledgements The authors thank Dr. Roberto Docampo for the trypanosomes and Dr. Silvia Moreno for helpful comments. This work was supported by FAPESP (FundacXa‹o de Amparo a' Pesquisa do Estado de Sa‹o Paulo) and CNPq (Conselho Nacional de Pesquisa e Desenvolvimento Tecnolo¤gico). Fig. 5. Tolerance of the puri¢ed phosphatase to oxidative stress. E¡ect of the Fe2þ /H2 O2 /ascorbate system on the activity of acid phosphatase puri¢ed from bloodstream forms and on bovine kidney low-molecularmass PTP. The enzyme was pre-incubated for di¡erent periods with 0.2 mM Fe2þ and 10 mM ascorbate, in 0.1 M acetate bu¡er, pH 5.0 at 37‡C. Oxidation was initiated by adding 14.5 mM H2 O2 . At the indicated times, the reaction was stopped by adding 1 mM EDTA. The residual activity was determined using pNPP as substrate, as described in Section 2 and a reaction time of 60 min. Control activity (100%) corresponded to activity in the absence of oxidation.
and L. donovani [13]. The phosphatase of intact bloodstream forms showed a pattern of general inhibition not very di¡erent from that described for the procyclic forms, i.e. inhibition by NaF, vanadate, and tartrate, but not by levamizole and tetramizole. pCMB markedly inhibited the phosphatase of bloodstream forms, indicating that this enzyme was SH-dependent, with a cysteine residue essential for enzyme activity and/or stability. Using the Lowry and Lopez method [22] and phosphotyrosine as substrate, the free phosphate released by live bloodstream parasites was observed to disappear quickly in the reaction medium, giving negative results when compared to the assay blank. On the other hand, with increasing phosphate concentrations, the live bloodstream forms reduced the inorganic phosphate concentration at a rate of 5 pmol phosphate min31 (data not shown). This behavior was not observed with the procyclic forms. Gottlieb and Dwyer [13] and Saha et al. [17] tested the resistance of Leishmania phosphatase to inactivation by oxygen metabolites. The toxicity of H2 O2 to bloodstream forms of T. brucei and the inhibitory e¡ect on parasite oxygen consumption and pyruvate output have been described [31]. Since T. brucei bloodstream forms lack catalase and glutathione peroxidase (for review, see [31]), an alternative H2 O2 -detoxi¢cation pathway has been proposed [32,33]. Because hemo£agellate parasites are exposed to toxic oxygen metabolites derived from drug metabolism or immune mechanisms, it is necessary to determine whether the parasite enzymes are resistant to
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