From the Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
Received for publication, March 18, 2003 , and in revised form, May 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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The biosynthesis of trypanothione represents the convergence of two distinct metabolic pathways (glutathione and polyamine biosynthesis) (23). Although all trypanosomatids studied so far are able to synthesize glutathione de novo (24, 25), there are marked differences in polyamine metabolism between T. cruzi and other species (19, 26). Notably, T. brucei and Leishmania spp. can synthesize polyamines de novo, whereas T. cruzi epimastigotes cannot (24). In contrast, T. cruzi relies on inducible high affinity diamine transporters to salvage exogenous putrescine and cadaverine (27), which can be converted to spermidine and aminopropylcadaverine, respectively (24). T. cruzi can further convert these metabolites into spermine and bis(aminopropyl)cadaverine, respectively (24), which is unusual for a trypanosomatid.
In T. brucei, spermidine is conjugated with glutathione to form glutathionylspermidine and then trypanothione (26). In addition to synthesizing glutathionylspermidine and trypanothione, T. cruzi is uniquely able to conjugate aminopropylcadaverine with glutathione to form glutathionylaminopropylcadaverine and homotrypanothione (N1,N9-bis(glutathionyl)aminopropylcadaverine) (24).
In Crithidia fasciculata, the biosynthesis of trypanothione from glutathione and spermidine is catalyzed by two distinct ATP-dependant enzymes (21, 28). Glutathionylspermidine synthetase (EC 6.3.1.8 [EC] ) forms both the N1- and N8-isomers of glutathionylspermidine; trypanothione synthetase (EC 6.3.1.9 [EC] ) subsequently catalyzes the addition of the second glutathione moiety to glutathionylspermidine. However, T. cruzi trypanothione synthetase (TcTryS)1 can catalyze both biosynthetic steps (29). It can also synthesize glutathionylaminopropylcadaverine and homotrypanothione with kinetic parameters equivalent to those of trypanothione (29). All known glutathionylspermidine synthetase (Escherichia coli and C. fasciculata) and trypanothione synthetase (C. fasciculata and T. cruzi) sequences possess an "amidase domain" located in the N-terminal region of the protein (2831). C. fasciculata glutathionylspermidine synthetase preferentially hydrolyzes glutathionylspermidine to GSH and spermidine (31), but can also hydrolyze trypanothione and homotrypanothione at 510% of the rate with glutathionylspermidine when assayed under identical conditions (29). In contrast, TcTryS possesses a weaker amidase activity, but hydrolyzes all the above-mentioned substrates (both mono- and bis-glutathionyl-polyamine adducts) at equivalent rates to each other (29).
Therefore, unlike C. fasciculata and E. coli glutathionylspermidine synthetases, TcTryS catalyzes all biosynthetic steps from glutathione and spermidine to trypanothione and all the subsequent reverse hydrolytic steps. An enzyme with wider polyamine substrate specificity could be more favorable to an organism lacking de novo diamine synthesis. Our previous HPLC studies have identified other unknown thiol species in epimastigotes (24, 32). In this study, we have identified and characterized several novel trypanothione analogues derived from spermine or other physiological polyamines. We show that TcTryS is able to catalyze both forward and reverse reactions to and from these metabolites. The physiological relevance of these novel thiols in all three life cycle stages of this parasite is discussed.
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EXPERIMENTAL PROCEDURES |
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Metabolite DeterminationIntracellular thiols were derivatized with the fluorescent label monobromobimane (34) and analyzed by HPLC as described (35, 36). Authentic thiol standards were prepared by reduction of disulfides either enzymatically with trypanothione reductase and required cofactors (24) or chemically with tris(carboxyethyl)phosphine, followed by derivatization with monobromobimane and purification by preparative HPLC. Total intracellular polyamines were extracted, derivatized with dansyl chloride, and analyzed by HPLC (37). Polyamines were quantified with authentic standards using 1,7-diaminoheptane as the internal control.
Purification of Thiol-Bimane Adducts and Mass Spectrometry
AnalysisT. cruzi epimastigotes grown for 5 days in RTH/CS were
supplemented with polyamines and harvested 1 day later. Approximately 8
x 108 cells were derivatized with excess monobromobimane,
assuming that 108 cells contained 15 nmol of total low molecular
mass thiols (38). Thiols were
separated on an UltraTechsphere reverse-phase C18 column (250
x 10 mm; HPLC Technology Ltd.) using the gradient specified
(34), but at a flow rate of 4
ml/min. Required fractions (1 min) were collected, dried down by rotary
evaporation, and resuspended in 100 µl of 40 mM Li-HEPPS and 4
mM DTPA (pH 8.0). Thiols were desalted using a
methanol/water/trifluoroacetic acid gradient
(39) on an Ultra-Techsphere
reverse-phase ion-paired C18 column (250 x 4.6 mm; HPLC
Technology Ltd). Required fractions (0.5 or 1.0 min) were collected, dried
down by rotary evaporation, and redissolved in acetonitrile/water to give a
concentration of 50100 pmol/µl thiol. MALDI-TOF mass spectrometry
analysis was carried out on a Voyager DE-STR instrument (PerSeptive
Biosystems) equipped with a nitrogen laser using
-cyano-4-hydroxycinnaminic acid as the matrix. Spectra were obtained
from 15 µl of sample, each averaging 40100 laser shots.
Kinetic StudiesApparent Michaelis constants (Km) for polyamine analogues were determined spectrophotometrically using purified recombinant TcTryS (29). TcTryS amidase was determined by HPLC (29). The kinetic parameters of bis(glutathionyl)spermine disulfide with recombinant trypanothione reductase from T. cruzi were measured in 40 mM HEPES (pH 7.5), 1 mM EDTA, and 100 µM NADPH (40). Kinetic data were fitted to the Michaelis-Menten equation by nonlinear regression with the program GraFit.
Time Course of End Product FormationPolyamines (10
mM final concentration, 10 x
Km) were added to a prewarmed (25 °C) assay
mixture (total of 0.1 ml) containing recombinant TcTryS (1
µM final concentration), 100 mM K-HEPPS (pH 8.0), 1
mM EDTA, 10 mM MgSO4, 2 mM ATP, 1
mM GSH, 6 mM tris(carboxyethyl)phosphine, 1
mM phosphoenolpyruvate, and 2 units/ml pyruvate kinase. Aliquots of
10 µl were removed at the specified time points, added to 40 µl of 40
mM Li-HEPPS and 4 mM DTPA (pH 8.0), derivatized with
monobromobimane, and analyzed by HPLC. To inactivate the amidase activity,
aliquots of TcTryS (3 mg/ml) were incubated with 1 mM
iodoacetamide for 20 min at 4 °C and extensively dialyzed against 25
mM K-HEPPS (pH 8.0) and 10% glycerol prior to use in the above
assay.
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RESULTS |
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Because epimastigotes are natural auxotrophs for polyamines, amines, we investigated whether T. cruzi would also conjugate glutathione with naturally occurring acetylated polyamines (N-acetylputrescine, N1-acetylspermidine, N8-acetylspermidine, and N-acetylspermine). Thiol analysis of epimastigote cultures supplemented with the above-mentioned polyamines showed that only the latter two gave prominent novel fluorescent peaks (peak U3 in Fig. 2A and peak U4 in Fig. 3A). Cultures supplemented with N1-acetylspermidine also contained a minor peak that coeluted with peak U3 (2% of peak U3 in N8-acetylspermidine cultures). To isolate sufficient material for structural analysis, epimastigote cultures were supplemented with N8-acetylspermidine or N-acetylspermine and the bimane derivatives purified as described above.
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Purified peak U3 gave peaks corresponding to the predicted masses of protonated glutathionyl-N8-acetylspermidine-bimane and glutathionyl-N8-acetylspermidine (m/z 667.43 and 477.33, respectively; peak U3) (Fig. 2B). The two peaks at m/z 689.43 and 705.40 correspond to the Na+ and K+ ions of M + H+, respectively. The loss of a bimane and sulfur from M + H+ may give a peak at m/z 445.14 (predicted m/z 444.27). Loss of the acetyl moiety would be consistent with the fragment at m/z 624.21. Two other minor peaks (m/z 640.20 and 656.17) could not be unambiguously assigned, although they could represent the sulfoxide and sulfone derivatives of m/z 624.21; the equivalent peaks for the parent ion at m/z 667.43 are not visible.
The retention time of peak U4 is similar to that of trypanothione (Fig. 3A). However, polyamine analysis of epimastigotes grown with N-acetylspermine as supplement in RTH/FCS or RTH/CS showed no significant increase in total spermidine content (data not shown); and therefore, peak U4 could not be trypanothione. MALDI-TOF analysis of purified peak U4 gave a peak at m/z 724.38, which corresponds exactly with the predicted mass of protonated glutathionyl-N-acetylspermine-bimane (peak U4) (Fig. 3, B and C). Peaks at m/z 746.35 and 762.33 correspond to the Na+ and K+ ions of M + H+, respectively. A minor unassigned peak appeared at m/z 768.33. Peaks corresponding to the predicted masses of the protonated and sodium ions of glutathionyl-N-acetylspermine (M-bimane) appeared at m/z 534.29 and 556.29, respectively. Another peak at m/z 549.29 (with an associated Na+ ion at m/z 571.26) may represent the sulfoxide of m/z 534.29. Some minor peaks in the region of m/z 600700 could not be assigned (<8% of the intensity of M + H+) and may be low level impurities. No peaks corresponding to the protonated ions of trypanothione-(bimane)2 (m/z 1104.25), trypanothione-bimane (m/z 914.22), and trypanothione (m/z 722.17) appeared in this sample.
Thiol and Polyamine Content of the Different Life Cycle Stages of T. cruziThe above experiments indicate that the insect (epimastigote) form is able to take up spermidine and spermine and their mono-N-acetyl derivatives and to conjugate them with glutathione. Because amastigotes reside in the cytosol of mammalian cells and are presumed to be incapable of de novo polyamine biosynthesis, we decided to compare the thiol and polyamine content of each of the life cycle stages (Table I).
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Epimastigotes grown to late exponential phase in RTH/FCS containing limiting amounts of polyamines (0.22 µM putrescine and 0.63 µM spermidine) (32) have a markedly lower polyamine content than amastigotes cultured in the polyamine-rich environment of human foreskin fibroblasts (5 µM putrescine, 11 µM diaminopropane, 700 µM spermidine, and 1050 µM spermine; calculated assuming that the fibroblast cell volume is 1.78 µl/106 cells) (41). The total spermidine and spermine content was 12- and 3-fold lower in epimastigotes compared with amastigotes, respectively (Table I). Non-dividing trypomastigotes that transformed from amastigotes and escaped into the culture medium contained similar amounts of spermidine, but 4-fold less spermine. Total polyamine content was highest in amastigotes (5.9 nmol/108 cells), followed by trypomastigotes (3.6 nmol/108 cells) and epimastigotes (1.1 nmol/108 cells). The most abundant thiols in all three life cycle stages were cysteine, glutathione, and trypanothione. Ovothiol A (N1-methyl-4-mercaptohistidine) was reduced 5-fold in amastigotes compared with amastigotes and trypomastigotes. This is similar to the situation in Leishmania donovani, where ovothiol A is reduced from 35% of total SH groups in late exponential phase promastigotes (insect stage) to <3% in amastigotes (intracellular mammalian stage) (38). Both mammalian stages contained trace amounts of monoglutathionylacetylspermidine, presumably the N1-glutathionyl-N8-acetylspermidine isomer. However, despite containing levels of spermine equivalent to those in epimastigotes, amastigotes and trypomastigotes had levels of glutathionylspermine conjugates that were at or below the limits of detection. Total thiol content (SH groups) was highest in epimastigotes (3.0 nmol/108 cells), with similar amounts in amastigotes and trypomastigotes (1.4 and 1.5 nmol/108 cells, respectively).
The proportion of free GSH in epimastigotes reflects the availability of diamines and polyamines in the medium (24, 32, 38). To determine whether spermine exerts a similar effect, epimastigotes were cultured in RTH/FCS supplemented with 10 µM spermine or with a mixture of 5 µM spermine plus spermidine (Fig. 4). Without supplements, the majority of the total glutathione-containing peptide was present as free GSH (80%), with the remainder conjugated to spermidine and spermine (15 and 5%, respectively). Addition of spermine to the medium significantly increased the total spermine content 17-fold from 0.8 ± 0.2 to 14 ± 2 nmol/108 cells, with a smaller 3-fold increase in total spermidine content from 0.29 ± 0.08 to 0.87 ± 0.17 nmol/108 cells (Fig. 4A). Spermine supplementation reduced the free glutathione content, similar to the effects of spermidine supplementation reported previously (32). However, despite the considerable excess of spermine in the parasites, spermidine-containing conjugate levels (33%, mainly as trypanothione) were higher than spermine-containing conjugate levels (23%, mainly as bis(glutathionyl)spermine) (Fig. 4B). When spermidine and spermine were present in equal amounts, the intracellular concentrations of both polyamines were similar (5.3 ± 0.7 and 5.7 ± 0.7 nmol/108 cells, respectively). Despite these equivalent intracellular polyamine concentrations, epimastigotes displayed a pronounced preference to synthesize trypanothione (2.2 ± 0.5 nmol/108 cells, 88% of total GSH pool) over bis(glutathionyl)spermine (0.04 ± 0.01 nmol/108 cells). This is in contrast to epimastigotes grown in the presence of equal amounts of putrescine (or spermidine) and cadaverine (or aminopropylcadaverine), which synthesize trypanothione and homotrypanothione in equal amounts (24, 32). Likewise, trypomastigotes and amastigotes purified from fibroblasts principally conjugated glutathione with spermidine (66 and 43%, respectively) rather than with spermine (<1%) (Fig. 4). Addition of 50 µM spermine and 0.5 mM aminoguanidine (an inhibitor of amine oxidases) to cultures of infected fibroblasts increased glutathionylspermine conjugates in amastigotes only to 4% of the total. Silvio X10-7 epimastigotes also synthesized bis(glutathionyl)spermine and other glutathionyl-polyamine conjugates in equivalent amounts to Y0 epimastigotes (data not shown), indicating that representatives of both major lineages of T. cruzi (42) have a similar metabolic capability to take up polyamines and to conjugate them with glutathione.
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Substrate Specificity of Trypanothione SynthetaseIn T. cruzi, a single enzyme (TcTryS) catalyzes ATP-dependent synthesis of trypanothione and homotrypanothione from glutathione and the appropriate polyamine (29), suggesting that spermine and other analogues may also be substrates. Because the enzyme shows pronounced substrate inhibition with GSH, initial rates were determined with 1 mM GSH, saturating MgATP, and varying concentrations of polyamine. Spermine and all of the analogues tested displayed hyperbolic kinetics and were fitted to the Michaelis-Menten equation to obtain kinetic parameters for apparent Km and kcat (Table II). Comparison of the specificity constants (kcat/Km) indicates that spermine, N-acetylspermine, and N8-acetylspermidine are all efficient substrates for the enzyme compared with spermidine, whereas N1-acetylspermidine, putrescine, and diaminopropane are markedly less so.
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The reaction products of these enzyme assays were derivatized with
monobromobimane and analyzed by HPLC. With N8- or
N1-acetylspermidine as substrate, a single peak with an
elution time identical to that of peak U3 was obtained (see
Fig. 2). Similarly, incubation
with N-acetylspermine yielded peak U4 (see
Fig. 3). However, two products
were obtained with spermine as substrate corresponding to peaks U1 and U2 (see
Fig. 1). Peak U1 corresponds to
bis(glutathionyl)spermine; and thus, peak U2 must be monoglutathionylspermine.
When a time course of the reaction was analyzed, the rate of formation of
monoglutathionylspermine exceeded that of the bis adduct for the first 20 min
until a steady-state concentration (160 µM) was achieved
(Fig. 5A). Thereafter,
formation of bis(glutathionyl)spermine continued until GSH was exhausted after
2 h. Essentially identical results were obtained with glutathione and
spermidine, where the intermediate product, glutathionylspermidine, rose to a
similar steady-state level and preceded formation of trypanothione over the
first 20 min (Fig.
5B). When presented with an equimolar mixture of
spermidine and spermine (5 mM each), formation of spermine
conjugates was 3.4 ± 0.3-fold faster than that of spermidine
conjugates, consistent with the kinetic data presented in
Table II. A second experiment
with TcTryS pretreated with iodoacetamide to inactivate the amidase
activity gave a similar ratio (3.2 ± 0.2-fold), indicating that the
amidase activity does not affect partitioning into the end products.
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Amidase Activity of Trypanothione SynthetaseThe finding that TcTryS preferred spermine to spermidine as substrate was unexpected because the formation of bis(glutathionyl)spermine was not favored compared with trypanothione in cells cultured with spermine (Fig. 4). However, in addition to its biosynthetic activity, TcTryS also displays weak amidase activity, hydrolyzing trypanothione and homotrypanothione to glutathione and the respective free polyamine via the intermediate mono adducts (29). To investigate whether bis(glutathionyl)spermine is a better substrate than trypanothione, 0.5 mM bis(glutathionyl)spermine was incubated with TcTryS in the absence of other substrates, and the products were analyzed by HPLC. Formation of glutathionylspermine and glutathione was linear over the first 10 min (data not shown), with a specific activity of 13.3 ± 2.1 nmol of amide bond hydrolyzed per min/mg, which is four times faster than with the equivalent concentration of trypanothione (3.1 ± 0.8 nmol of amide bond hydrolyzed/min/mg) (29). Because of the low activity of the reverse reaction (<1% of the forward synthetic reaction), it was not possible to perform a full kinetic analysis.
Bis(glutathionyl)spermine Disulfide as Substrate for Trypanothione ReductaseBis(glutathionyl)spermine was present as the free thiol in cells supplemented with spermine (Fig. 1), indicating that it must be maintained as the dithiol form either through thiol disulfide exchange with trypanothione or by direct reduction by trypanothione reductase. Kinetic analysis with recombinant T. cruzi trypanothione reductase yielded an apparent Km of 34 ± 10 µM and a kcat of 403 ± 47 s1 for bis(glutathionyl)spermine disulfide in the presence of saturating NADPH. The specificity constant for bis(glutathionyl)spermine disulfide (kcat/Km = 12.0 x 106 M1 s1) compares favorably with previously determined values for trypanothione disulfide (5.5 x 106 M1 s1), homotrypanothione disulfide (4.1 x 106 M1 s1), and glutathionylspermidine disulfide (1.9 x 106 M1 s1) (24, 40), indicating that this metabolite is a physiological substrate in vivo.
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DISCUSSION |
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Earlier studies on the polyamine substrate specificities of E. coli and C. fasciculata glutathionylspermidine synthetases have shown that the 3-aminopropyl moiety is the preferred site for acylation by glutathione and that the N-4 secondary amine of spermidine is a crucial recognition determinant (21, 30). TcTryS behaves like these previously characterized enzymes in that spermine, N-acetylspermine, and N8-acetylspermidine are all efficient substrates compared with spermidine, whereas N1-acetylspermidine, putrescine, and diaminopropane are markedly less so. Thus, a spacing of three methylene groups between the free amino group and the secondary amine (in polyamines) or the other primary amine (in diamines) is preferred over four (cf. N8-with N1-acetylspermidine or diaminopropane with putrescine in Table II). Spermine, which has two functionally equivalent aminopropyl moieties, has a 5-fold higher specificity constant than spermidine and N8-acetylspermidine, which have only one.
The finding that TcTryS preferred spermine rather than spermidine as substrate was somewhat unexpected. Epimastigotes contained 50-fold less bis(glutathionyl)spermine than trypanothione when cultured with spermine and spermidine, even though the total intracellular concentrations of spermine and spermidine were equivalent. Likewise, amastigotes, which have similar levels of these polyamines, contained >60-fold less bis(glutathionyl)spermine than trypanothione. Yet, based on the ratios of specificity constants for TcTryS, the concentration of bis(glutathionyl)spermine would be predicted to be 5-fold greater than that of trypanothione.
Several factors could account for this discrepancy. First, the free
cytosolic concentration of spermine could be considerably less than that of
spermidine (this would be predicted to be 250-fold to account for the
above observations). Unfortunately, there are no accurate measurements of free
polyamine concentrations in any organism. However, the best available indirect
estimate is that 9599% of spermine and 8592% of spermidine are
bound to macromolecules (DNA, RNA, and phospholipids) and ATP in bovine
lymphocytes and rat liver cells
(43). Assuming that
108 amastigotes have an intracellular volume of 2 µl,
intracellular spermine and spermidine concentrations are
1.1 and 1.8
mM, respectively. These values are comparable to spermine and
spermidine concentrations in the above-mentioned mammalian cell models
(43). However, the ratio of
free spermidine to spermine in mammalian cells is only 2.6 for bovine
lymphocytes and 4.6 for rat liver, which, if applicable to T. cruzi,
would result in formation of approximately equimolar amounts of trypanothione
and bis(glutathionyl)spermidine. Because this was not observed, either the
model does not apply to trypanosomes, or additional selective
compartmentalization of spermine into other unique organelles such as the
mitochondrion (binding to kinetoplast DNA) or the acidocalcisome (binding to
polyphosphate) would have to occur.
A second possibility is that the amidase function of TcTryS could selectively hydrolyze bis(glutathionyl)spermine. However, this was only 4-fold faster than with trypanothione. In addition, the forward synthetic reaction conjugating glutathione with spermine was 2 orders of magnitude greater than the rate of hydrolysis of bis(glutathionyl)spermine. Therefore, the rate of hydrolysis is negligible, in agreement with the time course of product formation shown in Fig. 5. Significantly, inhibition of the amidase activity did not affect the relative incorporation of spermine and spermidine into products, effectively excluding this possibility.
A third explanation could be that rapid oxidation of bis(glutathionyl)spermine occurs in vivo, so it is not detected by the monobromobimane HPLC assay. This possibility is highly unlikely because our kinetic analyses show that bis(glutathionyl-)spermine disulfide is a physiological substrate for the enzyme. Moreover, quantification of amastigote thiols derivatized in the presence or absence of the reducing agent tris(carboxyethyl)phosphine gave identical values (data not shown), indicating that the majority is present as the free dithiol in the cell.
Other possibilities include selective feedback inhibition of the native enzyme in vivo, selective degradation of glutathionylspermine conjugates by an unknown pathway, and selective export of bis(glutathionyl)spermine from the cell. Further experiments are required to examine these possibilities.
Despite the failure to detect bis(glutathionyl)spermine in amastigotes,
measurable amounts of a thiol eluting with the retention time of
glutathionylacetylspermidine were present. The resolution of our analytical
method is insufficient to resolve which isomer was formed, although the
specificity of TcTryS would favor glutathionylation of
N8-acetylspermidine. The concentration of acetylspermidine
in uninfected fibroblasts was below the limits of detection (<3
pmol/106 cells or <1.5 µM), i.e.
1000-fold less than spermine, which raises the interesting question of
how the amastigote obtains acetylspermidine. Under certain stress conditions,
mammalian cells can activate polyamine catabolism through the induction of
spermine/spermidine N1-acetyltransferase, forming
physiologically inert acetylated polyamines that are either excreted or
oxidized by polyamine oxidase isoenzymes, producing
H2O2, 3-acetamidopropanal, and putrescine or spermidine,
depending on the initial substrate
(44,
45). Activation of this
pathway in response to infection does not appear to have been studied.
Mammalian cells can also reversibly form
N8-acetylspermidine, and inhibition of
N8-acetylspermidine deacetylase has been reported to
promote either growth or differentiation, depending on the cell type
(46,
47). Whether T. cruzi
manipulates host cell polyamine levels or responds to changes in polyamine
metabolism induced by the host
(48) is an area worthy of
further investigation.
Even though C. fasciculata glutathionylspermidine synthetase is able to utilize spermine as substrate (21), C. fasciculata does not synthesize spermine, and bis(glutathionyl)spermine is absent in wild-type cells (34). L. donovani promastigotes grown in RTH/FCS (under conditions identical to those used for T. cruzi epimastigotes) supplemented with 10 µM spermine did not contain any detectable spermine or bis(glutathionyl-)spermine (<0.02 and <0.002 nmol/108 cells, respectively). Presumably, the lack of synthesis/uptake of spermine in other trypanosomatids (35, 49, 50) prevents the biosynthesis of glutathionylspermine conjugates, although additional controls on the biosynthetic enzymes may exist. T. cruzi therefore appears to be unique among trypanosomatids in its ability to take up/synthesize spermine (24, 32) and to conjugate it with glutathione. The wide polyamine substrate specificity of Tc-TryS presents an attractive target that can be exploited in the design of polyamine-based compounds that can inhibit polyamine and trypanothione metabolism (5153).
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 44-1382-345155; Fax:
44-1382-345542; E-mail:
a.h.fairlamb{at}dundee.ac.uk.
1 The abbreviations used are: TcTryS, T. cruzi
trypanothione synthetase; HPLC, high pressure liquid chromatography; RTH, RPMI
1640 medium/trypticase/hemin; FCS, fetal calf serum; CS, chicken serum;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; HEPPS,
4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; DTPA,
diethylenetriaminepentaacetic acid; MALDI-TOF, matrix-assisted laser
desorption ionization time-of-flight.
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ACKNOWLEDGMENTS |
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REFERENCES |
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