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J. Biol. Chem., Vol. 279, Issue 26, 27246-27256, June 25, 2004

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Trypanothione S-Transferase Activity in a Trypanosomatid Ribosomal Elongation Factor 1B^*

Tim J. Vickers and Alan H. Fairlamb

From the Division of Biological Chemistry and Molecular Microbiology, The Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom

Received for publication, October 7, 2003 , and in revised form, April 7, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular weight of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine and only used 1-chloro-2,4-dinitrobenzene from a range of glutathione S-transferase substrates. Peptide sequencing revealed that the three components were the , , and subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione, and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4-dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1B genes were cloned from C. fasciculata, but recombinant C. fasciculata eEF1B had no S-transferase activity, suggesting that eEF1B is unstable in the absence of the other subunits.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Infections with parasitic protozoa of the order Kinetoplastida are a common cause of serious illness and death in the tropics. Trypanosoma brucei sp. cause sleeping sickness in Africa, Trypanosoma cruzi is the cause of Chagas' disease in South America, and infections with Leishmania sp. produce a variety of pathologies termed the leishmaniases in Asia, Africa, South America, and Europe. In general, the chemotherapy of these diseases is poor, with the available drugs suffering various drawbacks such as toxicity, limited efficacy, and drug resistance (1, 2). For instance, the majority of cases of visceral leishmaniasis in India do not respond to antimonials, which are the first-line drugs (3). A promising target for the design of new drugs to treat these illnesses involves thiol metabolism. In these parasites, this depends upon trypanothione (T[SH]₂¹ or N¹,N⁸-bis(glutathionyl)spermidine) (4), in contrast to most other organisms (including their mammalian hosts), which use glutathione as the unmodified tripeptide (Fig. 1) (5). The main function of trypanothione is the maintenance of cellular redox state, and consequently, studies have concentrated on its role as an antioxidant (6).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Structures of glutathione (-L-glutamyl-L-cysteinylglycine) and trypanothione (N1,N8-bis(glutathionyl)spermidine).

Although the classical GSTs have been well characterized, the GST fold is also found in functionally unrelated proteins such as plant stress-induced proteins (11), -etherases (12), ion channels (13), and the eukaryotic translation elongation factor 1B (eEF1B) (14, 15). The functions of the GST domain in these proteins remain poorly understood, although the eEF1B from rice has recently been shown to possess a GST activity (16). This result was surprising, because eEF1B was previously thought only to be involved in protein synthesis, and no enzymatic activity had been proposed for the eEF1B subunit. Previously, the other two subunits (eEF1B and ) of eEF1B were shown to function to recycle elongation factor 1A (eEF1A) complexed with GDP back to the active eEF1A·GTP form (17, 18). This eEF1A·GTP complex is then able bind to an aminoacyl tRNA, forming a ternary complex that is able to enter the A-site of the ribosome, with the hydrolysis of GTP.

In contrast to the wealth of information on GSTs and related proteins in other organisms, little is known about thiol-xenobiotic conjugation in trypanosomes. Although there has been one report of low levels of GST activity in T. cruzi (19), this has been disputed (20). In addition, no GST activities have been reported in the related Leishmania sp. or T. brucei. However, because the major low molecular mass thiol in these organisms is trypanothione, it has been proposed that these trypanosomatids instead possess a trypanothione S-transferase or TST (5). This activity may also be involved in the resistance of Leishmania sp. to the metalloid antimony (21), which is currently the front-line drug for the treatment of leishmaniasis. It has been found that the acquisition of high level antimony resistance in L. tarentolae requires the overproduction of trypanothione, the overexpression of metal-thiol conjugate transporters and a third unidentified trypanothione-dependent factor (22). This factor was postulated to be a TST activity involved in the formation of an antimony-trypanothione conjugate. Indeed, in an analogous system, the mechanism of resistance of mammalian cells to arsenite involves the efflux of the metalloid, which is thought to be facilitated by the overexpression of a GST-pi (23, 24).

Here, we report the detection of TST activities in trypanosomatids and the purification and characterization of the Crithidia fasciculata TST. This is identified as the ribosomal elongation factor eEF1B complex and the subunit containing the TST active site defined as eEF1B.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—All reagents were standard commercial products and of the highest available purity. Trypanothione and glutathionylspermidine were obtained from Bachem. Glutathione affinity agaroses were obtained from Sigma, all other chromatographic resins and columns were from Amersham Biosciences. Rat liver GST was purchased as a mixture of isoforms from Sigma.

Cell Culture—C. fasciculata choanomastigotes; T. cruzi epimastigotes; T. brucei bloodstream form; and L. major, L. infantum, and L. tarentolae promastigotes were grown as described previously (25, 26).

Enzyme Assays—GST was assayed at 25 °C in 100 mM (Na⁺) phosphate, pH 6.5, with 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 1 mM GSH as substrates. The rate of formation of the glutathione S-dinitrobenzene conjugate was followed at 340 nm, using the published extinction coefficient of 9.6 mM cm^-1 (27). TST activity was measured under identical conditions with T[SH]₂ and CDNB being added to 400 µM. Assay reactions were initiated with thiol, which was produced immediately before addition to assays by mixing trypanothione disulfide (T[S]₂) with a 2-fold excess of tris(2-carboxyethyl)phosphine and a 5-fold excess of NaOH. The absorbance coefficient for the T[SDNB]₂ conjugate was measured by allowing assays to proceed to completion and found to be 9.2 mM^-1 cm^-1 per millimolar of sulfhydryl group. One unit of TST activity corresponds to 1 µmol of sulfhydryl group conjugated per minute.

Where necessary, in TST assays with alternative electrophilic substrates, the pH and substrate concentrations given for the corresponding GST assay (27) were altered to minimize the rate of the spontaneous reaction. The activities with these substrates were calculated using the absorbance coefficients for the corresponding GSH conjugates. In the case of the peroxidase, dehydroascorbate reductase and thioltransferase assays, tris(2-carboxyethyl)phosphine was omitted and T[S]₂ was reduced by 10 µg ml^-1 trypanothione reductase and 540 µM NADPH, before addition of the second substrate.

For enzyme purification, aliquots of fractions were screened for TST activity using a Molecular Devices Thermomax plate reader. In this assay, samples were diluted in the plate with water to give a final volume of 50 µl, and the reactions were initiated by the addition of 200 µl of a mixture containing buffer, dithiothreitol (DTT), and substrates. The final assay mixture contained 800 µM CDNB, 1 mM DTT, 50 µM T[S]₂, and 100 mM (Na⁺) phosphate, pH 6.5. The average rate of two blank assays was subtracted from these rates. Protein concentrations were measured using the procedure of Bradford (28), using BSA as the analytical standard. All trypanosomatid extracts were produced as described below for C. fasciculata, and mouse liver was extracted by disruption in a Dounce homogenizer and then processed as for C. fasciculata cells.

Purification of the C. fasciculata TST—Unless otherwise stated, all the steps in the following purification were performed at 4 °C. Frozen C. fasciculata cell pellets were thawed on ice and resuspended in an equal volume of ice-cold lysis buffer, giving in the final mixture 75 mM (Na⁺) phosphate, pH 7.2, 2 mM DTT, 1 mM EDTA, 1 mM benzamidine, 1 mM phenanthroline, 3 µg ml^-1 leupeptin, 250 µM 4-(2-aminoethyl-)benzenesulfonyl fluoride, and 1 µM pepstatin A. The cells were lysed by sonication (4 x 30 s pulses at 20-µm amplitude from a 19-mm enddiameter probe) in a Sanyo Soniprep 150 sonicator with cooling to <4 °C in an ice-salt bath between pulses. After centrifugation for 80 min at 40,000 x g, the resulting supernatant was brought to a final concentration of 4% (w/v) polyethylene glycol 6000 (PEG) over 5 min, by the dropwise addition of PEG from a 50% (w/v) stock, stirred for 30 min, and centrifuged for 30 min at 30,000 x g. The supernatant was adjusted to 9% (w/v) PEG and centrifuged as before. The resulting pellet was resuspended in 150 ml of buffer A (25 mM (Na⁺) Bis-tris, 1 mM EDTA, 1 mM DTT, pH 6.5) and then centrifuged to remove insoluble material.

The clarified supernatant was applied at 2 ml min^-1 to a 100-ml (19 x 2.6 cm) Amersham Biosciences Q-Sepharose anion exchange column equilibrated in buffer A. After washing with 2 column volumes of buffer A, bound proteins were eluted at 1 ml min^-1 with a linear gradient of 200–500 mM NaCl in the same buffer, and active fractions were pooled.

Following overnight dialysis against 2 liters of 550 mM (NH₄)₂SO₄ in buffer B (25 mM (Na⁺) HEPES, 1 mM EDTA, 1 mM DTT, pH 7), the sample was applied at 2 ml min^-1 to an 85-ml (16 x 2.6 cm) Amersham Biosciences phenyl-Sepharose (low substitution) column equilibrated with 550 mM (NH₄)₂SO₄ in buffer B. The column was washed with 5 column volumes of the same buffer, and TST activity eluted with 250 ml of buffer B.

Active fractions were pooled and concentrated to 1 ml by vacuum ultrafiltration in a Sartorius collodion bag. The concentrated TST was applied to a 319-ml (2.6 x 60 cm) Superdex 200 26/60 size-exclusion column equilibrated with buffer C (50 mM (Na⁺) HEPES, 300 mM NaCl, 0.01% (w/v) sodium azide, pH 7.5) and eluted at a flow rate of 2 ml min^-1. Fractions with TST activity were pooled and concentrated as before.

Analytical Chromatography—Analytical size-exclusion chromatography was carried out using a 24-ml (1 x 30 cm) Superdex 200 HR 10/30 size-exclusion column equilibrated with buffer C, with separations performed at a flow rate of 0.5 ml min^-1. This procedure was also used to calculate the relative molecular weight (M_r) of proteins, using carbonic anhydrase (29 kDa), BSA (66 kDa), alcohol dehydrogenase (150 kDa), -amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa) as analytical standards.

Analytical anion-exchange chromatography was carried out using a 6-ml (1.6 x 3 cm) Amersham Biosciences Resource Q column under the same buffer conditions as the Q-Sepharose step in the TST purification scheme. The sample was applied at 1 ml min^-1, and the column was washed with 2 column volumes of buffer. The bound proteins were then eluted with a complex gradient that went from 0–200 mM NaCl in 6 ml, 200–500 mM NaCl in 60 ml, and then 500–1000 mM NaCl in 6 ml, all in buffer A.

Analysis of Products of TST Reaction—Assays for mass spectrometric analysis were performed in 200 mM ammonium acetate, pH 6.5, with the other conditions as before. The reactions were followed at 340 nm and after 30 min 2-hydroxyethyl disulfide was added to 10 mM, oxidizing the T[SH]₂ by disulfide exchange and thus quenching the trypanothione reaction. The products were then lyophilized in glass vials, re-dissolved in water and diluted 1:500 with 1:1 acetonitrile and water. Samples were analyzed on a Micromass Ultima electrospray mass spectrometer in positive mode. Several scans were combined, background-subtracted, and smoothed to produce the final spectra.

Cross-linking Analysis—All samples and controls were dialyzed overnight against 55 mM (Na⁺) phosphate, pH 7.5, reaction buffer, protein samples were then diluted to 1 mg ml^-1 in reaction buffer. The amine-reactive homobifunctional reagent bis(sulfosuccinimidyl)suberate (BS³) was added, from a freshly prepared 10 mM stock in 5 mM sodium succinate, pH 5, to the required final concentration. The reactions were incubated at room temperature for 40 min and then quenched by the addition of ethanolamine to a final concentration of 5 mM. Aldolase was used as a positive control and BSA and carbonic anhydrase as negative controls. Samples from the reactions were then analyzed by using either 10% SDS-PAGE gels (29), to examine complexes less than 150 kDa or 6% Weber SDS-PAGE (30) to examine complexes greater than 150 kDa.

Dissociation of Complex—For thiocyanate disassociation, samples of TST in buffer C were incubated on ice with 2 mM DTT for 3 h in the presence or absence of 1.5 M NaSCN. The proteins were then applied to a Superdex 200 HR 10/30 column equilibrated with either buffer C or this buffer plus 500 mM NaSCN, and eluted at 0.5 ml min^-1. Because NaSCN inhibits the TST assay, 200-µl samples from each column fraction were dialyzed against TST assay buffer (100 mM (Na⁺) phosphate, pH 6.5) before analysis by enzyme assay and SDS-PAGE.

Inhibition and Chemical Modification of TST—Modification with N-ethylmaleimide (NEM) was carried out in an 80-µl volume at room temperature in buffer C. Samples of TST (16 µg and 61 milliunits) were incubated with 100 µM DTT for 5 min, and then NEM was added to a final concentration of 400 µM. After a 15-min incubation, the reactions were quenched by the addition of DTT to 625 µM.

Inhibition of TST by Cibacron blue F3G-A was determined under standard assay conditions, in the presence or absence of 0.5% (w/v) BSA. Routinely, TST was preincubated with the inhibitor, but no decrease in inhibition was observed when assays were initiated with enzyme. Dye concentrations were determined spectrophotometrically, using the absorbance coefficients of _m = 11.6 (622 nm) for Cibacron blue (31) and _m = 4.2 (610 nm) for reactive blue 4 (32).

The rate of time-dependent inhibition of TST by chlorotriazine dyes was measured in reaction mixtures of 100 µl containing 70 milliunits (18 µg) samples of TST and varying concentrations of dye in 20 mM (Na⁺) phosphate, pH 7.5. The reactions were allowed to proceed at room temperature, with 5-µl samples being removed at intervals to determine residual TST activity. These samples were assayed by 100-fold dilution into assay mixtures containing 0.5% (w/v) BSA, to prevent inhibition by any unreacted dye. Incubations with CDNB were performed under identical conditions, and samples were assayed in standard assay conditions. T[SH]₂ and GSH were generated in incubations by the addition of 600 µM NADPH, 1 milliunit of trypanothione reductase or glutathione reductase, and 500 µM T[S]₂ or GSSG. The resulting data were fitted by non-linear regression analysis to the single exponential decay function and expressed as a percentage of the activity at zero time.

Samples of proteins for mass spectrometry were desalted by dialysis against one liter of water for 3 h at 4 °C, before analysis by MALDI-TOF mass spectrometry on a PerSeptive Biosystems Voyager-DE STR mass spectrometer.

Cloning of eEF1BG1 and eEF1BG2 from C. fasciculata—A partial clone of the C. fasciculata eEF1BG1 was obtained by RT-PCR from cDNA prepared using the Cells-to-cDNA kit (Ambion). The sense primer (5'-CGCTATATAAGTATCAGTTTCTGTA-3') contained 25 nucleotides of the mini-exon sequence that is trans-spliced onto the 5'-end of all trypanosomatid mRNA transcripts (33, 34). The degenerate antisense primer (5'-CCYTCCCANGCNAGGTAKTC-3') was designed to the tryptic peptide ITDYLA(F/W)EGPTIPLPV. PCR was performed in 50-µl volume reactions containing template cDNA, 20 ng ml^-1 sense and antisense primers, 250 µM dNTPs, 5 units of Taq polymerase (Promega), and Taq buffer plus 1.5 mM MgCl₂. The reactions were placed in a thermocycler that had been preheated to 95 °C and subjected to the following: 95 °C for 5 min, 30 cycles of 95 °C for 1 min, 55 °C for 1 min, then 72 °C for 2 min, and finally the reactions were heated to 72 °C for 10 min. PCR products were then cloned into the pCR-Blunt II-TOPO plasmid using the Zero Blunt TOPO PCR cloning kit (Invitrogen).

Southern blotting was carried out by the standard capillary transfer method (35), using the RT-PCR clone of eEF1BG1 as a probe. The probe was labeled using the Gene Images labeling kit and hybridization detected with the Gene Images dioxetane detection module (Amersham Biosciences).

PCR amplification of C. fasciculata eEF1BG intergenic regions was carried out using sense (5'-GGAGCTGTTTGACTGGGAGGAGAT-3') and antisense primers (5'-GAAGTCCGCCGTCTCGTTGTC-3') as described above, with the substitution of Pfu polymerase and Pfu buffer (Promega) for Taq polymerase. Four intergenic regions were amplified, cloned, and sequenced. Multiple primers were then designed to their 5' and 3' regions. One pair of these primers (sense 5'-GCACCGGCGTACCTGATGACTT-3' and antisense 5'-TTAGTGGCCACTGATGCGACAGC-3') produced a product that was cloned, sequenced, and identified as an eEF1B gene (eEF1BG2). This gene was then cloned into the expression vector pET15b (Novagen), and recombinant protein was expressed and purified according to the manufacturer's instructions.

Nomenclature—The terminology of eukaryotic elongation factors is somewhat confusing. In this report we have used the IUBMB nomenclature recommendations (available www.chem.qmul.ac.uk/iubmb/misc/trans.html), with the elongation factor 1B complex, previously named eEF-1 being referred to as eEF1B. The complex's subunits are, in order of increasing size, elongation factor 1B (previously eEF-1), now referred to as eEF1B; elongation factor 1B (previously eEF-1) referred to as eEF1B and elongation factor 1B (previously eEF-1) referred to as eEF1B. The SWISS-PROT identifier is used when referring to previously characterized elongation factors.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Detection and Initial Characterization of Trypanothione S-Transferase Activities in Trypanosomatids—Clarified extracts of various organisms and cell types were assayed for S-transferase activities (Table I). L. tarentolae (a lizard parasite) was included, because this organism has been frequently used in studies on Leishmania antimony resistance. Mouse liver extract was assayed as a positive control for the GST assay and trypanothione reductase activity was used to confirm adequate extraction of the parasites. No GST activity was detected in the trypanosomatids. Instead a trypanothione S-transferase (TST) activity was detected in C. fasciculata, all the Leishmania sp. and T. brucei. The TST activity in mouse liver extracts is probably due to the ability of GSTs to use T[SH]₂ as an alternative substrate, although with a specific activity 100-fold less than that given with GSH. Because TST activity was highest in C. fasciculata and L. major, these activities were further characterized. Activity in these extracts was proportional to the amount of protein added, heat labile, and, when analyzed by size-exclusion chromatography the activity eluted close to the void volume of the column, showing M_r values greater than 400,000 (data not shown). This is in contrast to the GST activity in the mouse liver extract, which eluted with an M_r of 45,000 (this value being identical to the reported M_r of the three major mouse glutathione S-transferases) (36).

Purification of C. fasciculata TST—Because C. fasciculata had the highest TST activity, it was used in further studies. The C. fasciculata TST was concentrated by PEG 6000 precipitation and then purified to constant specific activity by a combination of anion-exchange, hydrophobic interaction, and size exclusion chromatography (Table II). Typically, TST activity can be purified 200-fold with about 10% recovery. Surprisingly, when the purification fractions were analyzed by SDS-PAGE, three major polypeptides of mass 31, 38, and 45 kDa and one minor polypeptide of 65 kDa were visible. These proteins did not alter in relative abundance in the last two purification steps (Fig. 2). To determine if these polypeptides were associated through disulfide bonds, samples of the final preparation were separated in the presence and absence of DTT. No association between the four polypeptides was seen in the oxidized sample, showing that any complex is formed by noncovalent interactions.

View larger version (67K): [in this window] [in a new window]   FIG. 2. SDS-PAGE of C. fasciculata TST purification fractions. Samples (10 µg of protein in each lane) from the purification were separated in a 10% SDS-PAGE gel. Lane 1, clarified cell extract; lane 2, 2–10% redissolved PEG 6000 precipitate; lane 3, Q-Sepharose eluant; lane 4, phenyl-Sepharose eluant; and lane 5, Superdex 200 eluant. Lanes 6 and 7 were additional samples of the Superdex 200 pool with 1 mM DTT being added to the sample in lane 6 and the sample in lane 7 containing no reducing agent.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Analytical size-exclusion chromatography of purified C. fasciculata TST. A, elution profiles of TST activity and protein from a Superdex 200 HR analytical size exclusion column. The absorbance at 280 nm is shown with a solid line, the fraction numbers are shown above this trace and are corrected for the delay volume between the fraction collector and the UV flowcell. The TST activity of fractions was measured using the microtiter plate assay and shown in filled circles joined with a dotted line. B, determination of the relative molecular mass of this species, with the sample () shown relative to a set of analytical standards (•). C, SDS-PAGE analysis of 10-µl samples taken from the 0.5-ml fractions collected during this separation. The four visible bands, subsequently identified as the eEF1B, , and subunits of the eEF1B complex and an aminoacyl-tRNA synthetase are labeled at the right-hand side of the gel.

Quaternary Structure of TST Complex—The purified TST preparation contained three major polypeptides, and the relative intensities of the protein bands were not altered in the last three purification steps, suggesting that these three proteins associate to form the high molecular mass TST complex. The association of these proteins was therefore investigated by chemical cross-linking. As seen in Fig. 4, addition of BS³ to purified TST results in cross-linking of the eEF1B subunits, producing high molecular mass species (Fig. 4A, lanes 2–4). In the positive control, the tetrameric enzyme aldolase is cross-linked into dimers, trimers, and tetramers (Fig. 4A, lane 7), whereas in the negative control, the monomeric proteins, BSA (68 kDa), and carbonic anhydrase (29 kDa) are unaffected (Fig. 4A, lane 6). Significantly, the product formed in the TST cross-linking reaction (Fig. 4B, lane 4) has a mass between 150 and 200 kDa, which is in the range of the 160-kDa mass given by the sum of the SDS-PAGE masses of the , , and subunits in a 1:1:2 ratio obtained by image analysis. In addition, 170 kDa is also one quarter of the molecular mass given by gel filtration. These results suggest that the three subunits associate in a ₂ tetramer and then this tetramer itself tetramerizes to form a hexadecamer. Indeed the (₂)₄ hexadecameric species may have been formed in the cross-linking reaction, because, despite equal amounts of protein being loaded in each lane, the total amount of protein visible in the gel decreases with increasing degrees of cross-linking. This effect may be due to species of more than 400 kDa being unable to enter these gels. These results are in good agreement with previous studies on the mammalian eEF1B, which was shown to be a M_r 670,000 complex containing the eEF1B, , and subunits in a 1:1:2 ratio (38).

View larger version (93K): [in this window] [in a new window]   FIG. 4. Cross-linking of the TST complex. A, 10% Laemmli SDS-PAGE analysis of the products of the reaction between increasing concentrations of BS3 and TST (lane 1,noBS3; lane 2,75 µM; lane 3, 200 µM; and lane 4, 1 mM). The negative control used the monomeric proteins BSA (66 kDa) and carbonic anhydrase (29 kDa), which were treated with 1 mM BS3 in the presence (lane 5) or absence (lane 6) of TST. The positive control in lane 7 shows the products from treatment of the tetrameric protein aldolase with 75 µM BS3, with the masses of the products formed indicated on the right-hand side of the gel. B, the same samples were analyzed in a 6% Weber SDS-PAGE gel. The masses on the left of the gel are from a cross-linked phosphorylase b marker, and again the masses of the aldolase products are indicated on the right-hand side of the gel. Samples as described in panel A.

Denaturation of the TST Complex and Separation of Subunits—To identify which subunit is responsible for the TST activity in the eEF1B complex, samples of TST were partially denatured, and the subunits were separated and assayed for activity. This was achieved by treatment with the chaotropic agent sodium thiocyanate (NaSCN). TST was incubated on ice in the presence or absence of NaSCN, and the proteins were then separated by size exclusion chromatography, again in the presence or absence of thiocyanate. As expected, a sample of the eEF1B complex that had not been exposed to NaSCN eluted as a single species (Fig. 5A), with a protein peak in fraction 14 and all the subunits present in a 1:1:2 (, , and respectively) ratio (Fig. 5C). In contrast, when the TST was preincubated for 3 h with NaSCN and then chromatographed in the presence of NaSCN, two partially resolved protein species were observed to elute from the column (Fig. 5A). Due to the incomplete separation of these complexes, their subunit compositions could not be clearly defined. However, when the amounts of each subunit present in these fractions (Fig. 5D) is compared with the level of enzyme activity, a clear correlation between the levels of eEF1B and TST activity can be observed. Significant TST activity is present in fractions 13–17, which corresponds well with the elution profile of eEF1B but not with that of eEF1B or , which elute mainly in fractions 16 and 17.

View larger version (28K): [in this window] [in a new window]   FIG. 5. Separation of the subunits of the TST complex using thiocyanate. Samples of TST were incubated in the presence and absence of 1.5 M NaSCN and then applied to a size-exclusion column. A, elution profiles of these samples; the untreated control is shown in red, and the NaSCN-treated sample is blue. The elution positions and masses of protein standards are indicated with arrows. B, TST activity profiles of the fractions collected from this separation; the untreated control is indicated with red triangles, and the NaSCN-treated sample is indicated with blue squares. C and D, SDS-PAGE analysis of these column fractions, with Panel C corresponding to the untreated control and Panel D to the NaSCN-treated sample.

Cibacron blue F3G-A, a monochlorotriazine dye, was found to be an extremely potent inhibitor of the C. fasciculata TST, with an IC₅₀ of 13 ± 0.8 nM (Fig. 6). Inhibition constants in this range can indicate an irreversible active site titration, and indeed no recovery of activity was observed upon prolonged dialysis of Cibacron blue-inhibited TST. However, this result was found to be due to the inability of Cibacron blue to pass through dialysis membranes, possibly as a result of dye aggregation (40). In light of the known affinity of this dye for serum albumin (41), TST inhibition was therefore determined in the presence of 0.5% (w/v) BSA. The addition of this protein caused a large shift in the IC₅₀ to 12 ± 0.9 µM, most probably due to the sequestration of the inhibitor from the assay solution (Fig. 6). This modified assay allows the accurate determination of TST activity in the presence of micromolar concentrations of Cibacron blue. Using this procedure, samples of TST were incubated with 100 µM Cibacron blue, and then samples were diluted into assays containing 0.5% (w/v) BSA. No change in TST activity was observed over a 30-min period, indicating that Cibacron blue does not irreversibly inactivate the enzyme (Fig. 7A, inverted triangles).

View larger version (17K): [in this window] [in a new window]   FIG. 6. Inhibition of TST by Cibacron blue. Inhibition of TST by Cibacron blue in the presence () and absence (•) of 0.5% (w/v) BSA in the assay buffer.

View larger version (13K): [in this window] [in a new window]   FIG. 7. Time-dependent inhibition of TST by reactive blue 4. A, time-dependent inactivation of TST by reactive blue 4. TST was incubated with the inhibitors in 20 mM (Na+) phosphate, pH 7.5, at 25 °C, and the residual activity was determined at intervals, as described under "Experimental Procedures." The incubations contained either 100 µM Cibacron blue () or reactive blue 4 at 2 µM (), 10 µM (•), 20 µM (), 50 µM (), 75 µM (), and 100 µM (). B, observed rates of TST inactivation by reactive blue 4 as a function of the concentration of inhibitor.

View larger version (15K): [in this window] [in a new window]   FIG. 8. Covalent modification of eEF1B by reactive blue 4. Reactive blue 4-inactivated TST was analyzed by MALDI-TOF mass spectrometry. The two samples were prepared as described in the text, with the TST sample shown in panel A being an untreated negative control and the TST sample shown in panel B being inactivated by incubation with 50 µM reactive blue 4 for 1 h.

View larger version (10K): [in this window] [in a new window]   FIG. 9. Interactions between TST substrates and triazine dyes. Incubations were performed in 20 mM (Na+) phosphate, pH 7.5, at 25 °C, and the residual activity was determined at intervals, as described under "Experimental Procedures." A, time-dependent inactivation of TST by CDNB. The incubations contained no CDNB (), 500 µM CDNB (•), 500 µM CDNB and 500 µM T[SH]2 (), and 500 µM CDNB and 100 µM Cibacron blue (). B, effects of thiols on the inactivation of TST by reactive blue 4. The incubations contained no reactive blue 4 (), 50 µM reactive blue 4 (•), 50 µM reactive blue 4 and 1 mM GSH (), 50 µM reactive blue 4 and 500 µM T[SH]2 (), and 50 µM reactive blue 4 and 500 µM DTT ().

Cloning of C. fasciculata eEF1B Genes—

eEF1B2

C. fasciculata eEF1B1

eEF1B2

eEF1B2

C. fasciculata eEF1B1

eEF1B2

eEF1B2

C. fasciculata eEF1B2

View larger version (60K): [in this window] [in a new window]   FIG. 10. Alignment of the sequences of trypanosomatid eEF1B proteins. The predicted amino acid sequences of the C. fasciculata eEF1B genes isolated by RT-PCR (eEF1B1 or RT-PCR C.f.) and intergenic PCR (eEF1B2 or Full-length C.f.) are shown aligned with the sequences of the Homo sapiens (P26641 [GenBank] ), T. brucei (temporary name: TRYPtp_ends-17b11.p1k_188), L. infantum (Q9BHZ6), and T. cruzi (P34827 [GenBank] ) proteins. Gaps are indicated with dashes, and the alignments are shaded according to similarity. The colors indicate: black background, white text, over 80% identity; dark gray background, white text, over 60% identity; and light gray background, black text, over 40% identity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The assignation of the TST activity of the C. fasciculata eEF1B to eEF1B is supported by several independent studies. From sequence analysis and homology modeling Koonin et al. (15) predicted that the N-terminal domain found in eEF1B proteins would form a GST-like fold, which was expected to possess GST activity. Subsequent structural studies on the Saccharomyces cerevisiae eEF1B-1 (P29547 [GenBank] ) N-terminal domain confirmed that the fold of this domain is remarkably similar to that of the GSTs, but no GST activity was detected in this truncated protein (14). However, an unusual 50-kDa GST from the yeast Yarrowia lipolytica has been purified (43), and we note that the published N-terminal sequence shows high similarity to the S. cerevisiae eEF1B-2 (P36008 [GenBank] ; 61% identity and 80% similarity in 29 residues). In common with the C. fasciculata TST, this unusual GST was highly specific for CDNB as an electrophilic substrate and did not bind to glutathione-affinity resins. These data suggest that native forms of yeast eEF1B proteins should be examined for S-transferase activities.

More recently, GST activity was detected in recombinant rice eEF1B (Q9ZRI7) and preparations of the native eEF1' complex (16). The eEF1B had a low specific activity, with a k_cat value of approximately one-fiftieth of a classic rice GST. Moreover, unlike classical GSTs from rice, this eEF1B lacked glutathione peroxidase activity. The S-transferase activity of the C. fasciculata eEF1B therefore appears to be similar to that of the rice subunit, with no activity toward organic peroxides and a comparatively low specific activity with CDNB. Interestingly, in common with the T. cruzi and Bombyx mori (Q9BPS3) eEF1B subunits (44, 45), and in contrast to the C. fasciculata protein, the rice eEF1B can be purified by glutathione affinity chromatography. This indicates that the eEF1B of these organisms can bind GSH and may indicate that the T. cruzi eEF1B has a different substrate specificity to those of the other kinetoplastids.

We were unable to detect TST or GST activity in crude extracts of T. cruzi, in agreement with another report (20). However, another study did detect trace amounts of GST activity (70 milliunits mg^-1) in T. cruzi proteins purified on a glutathione affinity resin (46). Interestingly, the major components of this preparation were later identified as the three subunits of the T. cruzi eEF1B complex (47, 48). Purified recombinant T. cruzi eEF1B was found to be devoid of GST activity (49), and activity with trypanothione was not examined in either study (46, 49). However, the authors proposed that the GST activity of the subunit may require association with the other subunits of the eEF1B complex. This hypothesis is consistent with the instability of the isolated rice eEF1B protein, in comparison to the native complex (16) and our failure to detect TST activity in the recombinant C. fasciculata eEF1B2. Indeed, further studies in our laboratory have shown that no S-transferase activity is detectable in the recombinant L. major eEF1B protein either, whereas the L. major eEF1B holocomplex possesses TST activity. These findings will be described in a subsequent paper.

The diseases caused by Leishmania sp. are usually treated by antimonial compounds, and Grondin et al. (22) have hypothesized that a TST activity may be important in the development of drug resistance. This was suggested by their finding that the overexpression of thiol-metal conjugate transporters and increased T[SH]₂ levels are necessary but not sufficient for high-level metalloid resistance in L. tarentolae. They therefore proposed that overexpressed TST was the missing trypanothione-dependent factor in the resistance phenotype. Indeed, the mechanism of resistance in Leishmania may be similar to that of mammalian cells, where increased GSH levels and transporter expression are coupled to the overexpression of GSTs (23, 24). Interestingly, L. tarentolae is hypersensitive to metals in comparison with pathogenic species (50, 51), and this correlates with a low level of extractable TST activity, compared with the pathogens L. major and L. infantum (Table I). However, many other factors such as thiol levels differ between these species, and more work is required to investigate if the TST activity in Leishmania is involved in metal resistance.

The trypanosomatids appear to be unusual among eukaryotes in lacking a classical low molecular mass S-transferase activity (52). However, as Moutiez et al. (53) suggested, at physiological pH the increased reactivity of T[SH]₂ over GSH may make the non-enzymatic conjugation of xenobiotics far more efficient in the trypanosomatids than other organisms. Thus, high cytosolic T[SH]₂ concentrations may efficiently substitute for a high enzymatic TST activity, and at cytosolic pH the eEF1B TST activity may represent only a small fraction of the non-enzymatic S-transferase activity in the cell. A minor contribution of eEF1B to total cellular S-transferase activity is also consistent with the low specific activity of the rice eEF1B (16) and the failure to detect significant S-transferase activity in T. cruzi, without partial purification of the eEF1B complex (46). This suggests either that the appropriate xenobiotic substrate of eEF1B has not yet been identified or that this activity has a distinct physiological role unrelated to the conjugation and detoxification of xenobiotics. Importantly, the evolution of thiol substrate specificity of the trypanosomatid eEF1B proteins toward trypanothione appears to have paralleled the evolution of trypanothione as the major thiol metabolite of these organisms. This demonstrates that a specific interaction with cellular thiols is required for the function of eEF1B complex.

An alternative activity of GSTs is the binding of ligands. These ligands include hydrophobic compounds such as hemein and glutathione S-conjugates (54). In addition, glutathione can form a mixed disulfide with a cystine residue in the active site of the omega class of GSTs (55). It is therefore possible that the eEF1B complex may be involved in regulating protein synthesis in response to oxidative or toxic stress. Tight control of protein synthesis by the redox state of the GSH/GSSG couple has been observed by Kosower et al. (56), and in vivo studies indicate that it is the elongation step of protein synthesis that is most sensitive to oxidative stress (57). Significantly, translational control may be especially important in trypanosomes, because they lack RNA polymerase II promoters and are thus unusually dependent on post-transcriptional regulation (for review, see Ref. 58).

One mechanism for this response is suggested by the finding that the valyl-tRNA synthetase (ValRS) activity in yeast is controlled by an uncharacterized high molecular weight oxidoreductive regulatory apparatus, which is activated by reduced glutathione (59–61). ValRS has since been found to be present in mammalian cells exclusively as a complex with eEF1B (62) and to associate with the elongation factor complex through an N-terminal extension similar in sequence to eEF1B (63). Furthermore, several other aminoacyl-tRNA synthetases (ARSs) have recently been shown to associate with eEF1B in vivo (64). Our finding that the C. fasciculata eEF1B co-purifies with a probable ARS indicates that this interaction is conserved in the trypanosomatids. The binding of eEF1B to ARS is a functional interaction, because the ValRS activity of the ValRS·eEF1B complex, but not the free ValRS, is activated by eEF1A and GTP (62). This activation could be reversed by the addition of anti-eEF1A and eEF1B antibodies (64). Translational control by eEF1B could therefore be due to either an effect upon tRNA synthetases or by ligands directly modulating the GDP/GTP exchange activity of the complex.

The identification of this unique activity within the trypanosomatid protein synthesis machinery has revealed a potential drug target in what was thought to be a highly conserved part of the eukaryotic cell. Furthermore, the investigation of the role of the eEF1B complex in drug resistance, thiol metabolism, and translational control promises to yield important new insights into the biochemistry of these parasites.

	FOOTNOTES

 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY545587 [GenBank] and AY545588 [GenBank] .

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Funded by a research studentship from the Medical Research Council.

Funded by the Wellcome Trust. To whom correspondence should be addressed. Tel.: 44-1382-345-155; Fax: 44-1382-345-542; E-mail: a.h.fairlamb{at}dundee.ac.uk.

¹ The abbreviations used are: T[SH]₂ and T[S]₂, trypanothione and trypanothione disulfide, respectively; GSH and GSSG, glutathione and glutathione disulfide, respectively; GspdSH, glutathionylspermidine; CDNB, 1-chloro-2,4-dinitrobenzene; T[SDNB]₂, trypanothione bis-dinitrobenzene; BS³, bis(sulfosuccinimidyl)suberate; GST, glutathione S-transferase; TST, trypanothione S-transferase; eEF1A, eukaryotic elongation factor 1A (formerly eEF-1); eEF1B, the eukaryotic elongation factor 1B complex (formerly eEF-1) (eEF1B was formerly eEF-1, eEF1B was formerly eEF-1, and eEF1B was formerly eEF-1); ARS, aminoacyl-tRNA synthetase; RT, reverse transcription; DTT, dithiothreitol; BSA, bovine serum albumin; PEG, polyethylene glycol; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; NEM, N-ethylmaleimide; MALDI-TOF, matrix-assisted laser desorption time of flight; ValRS, valyl-tRNA synthetase.

	ACKNOWLEDGMENTS

 
We thank Dr. Nick Morrice and Dr. Douglas Lamont for protein sequencing and Dr. Angela Mehlert for mass spectrometry.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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3. Zilberstein, D., and Ephros, M. (2002) in World Class Parasites (Vol. 4): Leishmania (Black, S. J., and Seed, J. R., eds) pp. 115-136, Kluwer Academic Press, London
4. Fairlamb, A. H., Blackburn, P., Ulrich, P., Chait, B. T., and Cerami, A. (1985) Science 227, 1485-1487[Abstract/Free Full Text]
5. Fair