Purification and biochemical characterization of cytosolic glutathione-S-transferase from filarial worms Setaria cervi

Comparative Biochemistry and Physiology, Part B 151 (2008) 237–245

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Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b

Purification and biochemical characterization of cytosolic glutathione-S-transferase from filarial worms Setaria cervi Rumana Ahmad a, Arvind K. Srivastava a,⁎, Rolf D. Walter b a b

Division of Biochemistry, P.O. Box No. 173, Central Drug Research Institute, Chattar Manzil Palace, Lucknow-226001, India Abteilung fur Biochemie, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

A R T I C L E

I N F O

Article history: Received 13 November 2007 Received in revised form 23 March 2008 Accepted 31 March 2008 Available online 8 April 2008 Keywords: Glutathione-S-transferase Purification Characterization Drug target Enzyme inhibition Glutathione metabolism Glutathione peroxidase Filariasis Helminth parasites Setaria cervi

A B S T R A C T The present study reports the purification and characterization of GST from cytosolic fraction of Setaria cervi. GST activity was determined in various subcellular fractions of bovine filarial worms S. cervi (Bubalus bubalis Linn.) and was found to be localized mainly in the cytosolic and microsomal fractions. The soluble enzyme from S. cervi was purified to homogeneity using a combination of salt precipitation, centrifugation, cation exchange and GSH-Sepharose affinity chromatography followed by ultrafiltration. SDS-PAGE analysis revealed a single band and activity staining was also detected on PAGE gels. Gel filtration and MALDI-TOF studies revealed that the native enzyme is a homodimer with a subunit molecular mass of 24.6 kDa. Comparison of kinetic properties of the parasitic and mammalian enzymes revealed significant differences between them. The substrate specificity and inhibitor profile of cytosolic GST from S. cervi appeared to be different from GST from mammalian sources. © 2008 Published by Elsevier Inc.

1. Introduction Lymphatic filariasis (LF) is a debilitating disease caused by nematode worms of the genera Wuchereria and Brugia. Approximately 120 million people in the world have the disease and infection rates are increasing with the continued expansion of urbanization that is underway in the tropics. Control of filariasis remains disappointing due to lack of appropriate one-shot chemotherapeutic agents capable of eliminating adult parasites (Ottesen, 1992; Ottesen et al., 1997). Therefore, there is an urgent need to develop an antifilarial drug that has both macrofilaricidal and microfilaricidal activity or at least disrupting transmission by killing the developing forms in the female worms. The components of the glutathione (GSH) system are major chemotherapeutic targets in filarial species, as it has been proposed to constitute the antioxidant system responsible for the long-term existence of filarial worms in mammalian host (Callahan et al., 1988; Brophy and Pritchard, 1992a). Many inhibitors of enzymes involved in GSH metabolism are known to be associated with antiparasitic activities because they inhibit the parasitic enzymes to a greater

⁎ Corresponding author. Tel.: +91 522 2612411x4346; fax: +91 522 2623938. E-mail address: [email protected] (A.K. Srivastava). 1096-4959/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.cbpb.2008.03.019

extent than the mammalian counterparts (Srivastava et al., 1995; Arora et al., 2004; Gupta et al., 2004). One of those vulnerable targets is the enzyme glutathione-S-transferase (Srivastava et al., 1994; Tiwari et al., 2003; Ahmad et al., 2004). Glutathione-S-transferase(s); GST(s) (E.C.2.5.1.18) are multifunctional enzymes that play an important role in the detoxification of xenobiotics and function as intracellular binding proteins (Mannervik et al., 1985; Mannervik and Danielson, 1988; Precious and Barrett, 1989a,b). GST(s) are abundant in mammals, plants and invertebrates. GST, an important enzyme of GSH cycle, is considered to be an essential detoxification enzyme in parasitic species. GST(s) of protozoan and metazoan parasites have gained increasing attention in the past few years due to their involvement in drug resistance. A number of GST(s) from parasitic nematodes, trematodes, ticks and malarial parasites have been studied so far and some of these proteins might be exploited as a drug target or might be good candidates for vaccine development (Mitchell, 1989; McTigue et al., 1995; Morrison et al., 1996; Rossjohn et al., 1997; Ouaissi et al., 2002; Fritz-Wolf et al., 2003). GST from Schistosoma japonicum has been identified as a potential vaccine candidate (Smith et al., 1986; Smith and Johnson, 1988; Riveau et al., 1998). The interest in helminth GST(s) stems from the fact that they represent the major detoxification enzymes in these organisms and that their role in detoxification protect helminth parasites from host

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immune attack (Brophy et al., 1990a). As helminths contain very low levels of other detoxification enzymes such as catalase, superoxide dismutase (SOD) and cytochrome P450, GST may provide the worm's primary defense against electrophilic and oxidative damage (Precious and Barrett, 1989a,b; Brophy and Barrett, 1990). A vaccine capable of inducing the antibody-mediated neutralization of this function has the potential for rendering parasites vulnerable to the toxic products generated by immune attack (Tiu et al., 1988; Porchet et al., 1994). The GST enzymatic activity has been described in the adult and larval stages of helminths. Glutathione affinity chromatography and two-dimensional electrophoresis (2-DE) have been used to purify glutathione binding proteins from Caenorhabditis elegans. Most of the GST(s) identified were of the nematode specific class, however, three alpha class GST(s), a pi and a sigma class GST have also been isolated (van Rossum et al., 2001). GST(s) from helminths have been reported from every developmental stage (Vibanco-Perez and Landa-Piedra, 1998; Wildenburg et al., 1998; Rao et al., 2000). The detection of Ancylostoma caninum (Ac-GST-1) in the hypodermis, intestine, and adult excretory-secretory (ES) products suggests that it not only functions as an intracellular cytosolic housekeeping enzyme, but is also acting at the host–parasite interface and could function in blood feeding and other parasitic roles (Zhan et al., 2005). Nematodes secrete antioxidant enzymes that might protect exposed cuticular surfaces by neutralizing host free radicals generated by the oxidative burst of leukocytes. Genes encoding superoxide dismutases (SODs) have been cloned from a range of parasitic nematodes (James et al., 1994; Tang et al., 1994). Another family of antioxidants secreted by parasitic helminths is glutathione-S-transferases (GSTs) (Brophy and Barrett, 1990), which are thought to neutralize lipid hydroperoxides (Brophy and Pritchard, 1992b). GST(s) have been purified from extracts and ES products of Necator americanus and A. ceylanicum (Brophy et al., 1995), but their gene sequences are not available. Secretory GST has also been localized in parts of the cuticle and in the outer lamellae of the hypodermis in O. volvulus (Sommer et al., 2001). Although their biological roles in parasite infections are poorly understood, GST(s) are of interest in helminth vaccine research, given the high levels of protection they induce against trematode infections (Sexton et al., 1994). Parasite GST(s) play a major role by detoxifying the secondary products of lipid peroxidation produced via immune initiated free-radical attack on host or parasite membranes (Ketterer et al., 1988; Brophy and Pritchard, 1992b; Cervi et al., 1999; Kampkotter et al., 2003). GST activity has also been detected in adult female Setaria cervi, a bovine filarial parasite. This parasite resembles the human parasite in its nocturnal periodicity and antigenic pattern (Kaushal et al., 1987). The role of S. cervi GST antigen in inducing immunity in the host against Brugia malayi microfilariae and infective larvae has been studied in vitro and results suggest that native GST from S. cervi is effective in inducing protection against heterologous B. malayi filarial parasite and thus has potential in immunoprophylaxis (Gupta et al., 2005; Gupta and Rathaur, 2005). The present work describes the isolation of native GST from adult females S. cervi and subsequent biochemical and kinetic studies on the native enzyme that reveal significant differences between the parasitic and mammalian enzyme. 2. Materials and methods 2.1. Chemicals Reduced glutathione (GSH), trizma, trisodium citrate, ethylene diamine tetra acetic acid (EDTA), ammonium sulfate, sodium chloride (NaCl), epoxy activated sepharose 6B, ethanolamine, sodium borate, potassium chloride (KCl), sodium azide, NADPH, glutathione reductase (GR), bromosulphathalein, t-butyl hydroperoxide, cumene

hydroperoxide, linoleic hydroperoxide, dithiothreitol (DTT), CHAPS, urea, thiourea, sodium sulfate, hemin, S-hexylglutathione and BSA were purchased from Sigma-Aldrich Chemical Co., USA. All reagents used in electrophoresis and activity staining were purchased from Sigma. 1-chloro-2, 4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and sodium acetate were from Spectrochem Pvt. Ltd. Mumbai, India. Folin & Ciocalteu's phenol reagent was purchased from Sisco Research Laboratories, Mumbai, India. Carboxymethyl Sepharose (CM-Sepharose) was purchased from Amersham Pharmacia and Hanks balanced salt solution (HBSS) was from Himedia laboratories Pvt. Ltd. Mumbai, India. All other chemicals used were of analytical grade. 2.2. Biological materials 2.2.1. Collection of filarial worms S. cervi Adult bovine filarial worms S. cervi females of average body mass 35 ± 5 mg and length 6.0 ± 1.0 cm were collected from the peritoneal cavity of freshly slaughtered naturally infected water buffaloes Bubalus bubalis (Linn.) at a local abattoir. They were thoroughly washed with saline and kept in Hanks balanced salt solution with sodium bicarbonate and glucose (5.55 mM) for 60 min at 37 °C in Dubnoff metabolic shaker for complete revival before being used in enzymatic studies. 2.2.2. Preparation of GST from filarial worms S. cervi A 10% homogenate of actively motile bovine filarial worms S. cervi was prepared in 50 mM Tris–HCl buffer, pH 7.4 containing 250 mM sucrose and 0.2 mM EDTA using a Potter Elvehjem glass homogenizer fitted with a Teflon pestle. The homogenate was centrifuged at 1000 g for 15 min, 10,000 g for 30 min and subsequently at 100,000 g for 60 min to obtain mitochondrial, post mitochondrial, cytosolic and microsomal fractions respectively. Each fraction was dialyzed against 100 volumes of 20 mM potassium phosphate, pH 7.4 containing 0.2 mM EDTA and dialyzed preparation was used as enzyme source. 2.3. GST activity determination GST(s) activity in various dialyzed fractions obtained as a result of subcellular fractionation was determined spectrophotometrically at 340 nm according to the method of Habig et al. (1974). All reactions were corrected for non-enzymatic conjugation, with reaction mixtures without enzyme serving as controls. Under standard assay conditions, the reaction mixture contained 100 mM phosphate, pH 6.5, 1.0 mM CDNB in 20 μL ethanol, 1.0 mM GSH and enzyme protein unless stated otherwise. A unit of enzyme activity was expressed as the amount that catalyzes the formation of 1 μmol S-2, 4-dinitrophenyl-GSH adduct per minute, using a molar extinction coefficient of 9.6 mM− 1 cm− 1 for CDNB. Protein was estimated by the method of Lowry et al. (1951) using BSA as standard. 2.4. Purification of GST from S. cervi 2.4.1. Ammonium sulfate precipitation The cytosolic fraction of S. cervi was subjected to ammonium sulfate fractionation (0–80%). After each fractionation, the sample was stirred in cold for 30 min and then centrifuged at 13,000 g for 15 min. All the fractions were analyzed for enrichment in GST activity. The fraction having highest enrichment in GST activity was used as the source of GST. 2.4.2. Ion-exchange chromatography Solvents A (0.05 M citrate Buffer, pH 5.5) and B (0–3.0 M NaCl in 0.05 M citrate buffer, pH 5.5) were degassed for 5 min. Carboxymethyl-Sepharose (CM-Sepharose) preswollen in 20% ethanol was

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100 mL of water and the remaining active groups were blocked by allowing the gel to stand in 1.0 M ethanolamine for 4 h.

Table 1 Subcellular distribution of GST activity in filarial worms S. cervi Fraction

Total activity (μmol/min)

Total protein (mg)

Specific Activity⁎

Crude homogenate Mitochondrial fraction Post mitochondrial fraction Cytosolic fraction Microsomal fraction

1.40 ± 0.028 0 1.20 ± 0.042 2.024 ± 0.018 0.026 ± 0.02

20.1 ± 0.152 1.25 ± 0.080 18.2 ± 0.056 13.9 ± 0.033 2.54 ± 0.025

0.070 ± 0. 0185 Not detectable 0.066 ± 0.017 0.122 ± 0.024 0.010 ± 0.0052

1

2.5.2. Chromatographic procedures The protein sample was applied to the GSH-Sepharose affinity column (15 mL volume), washed with 0.5 M KCl and equilibrated with buffer A (10 mM Tris–HCl, 1.0 mM EDTA and 3.0 mM DTT, pH 8.0) at a flow rate of 30 mL/h. After sample application, the column was washed with buffer B (10 mM Tris–HCl, 1.0 mM EDTA, 3.0 mM DTT and 0.7 M NaCl, pH 8.0). GST was eluted from the column with buffer C (10 mM Tris–HCl, 1.0 mM EDTA, 3.0 mM DTT and 10 mM GSH, pH 8.0). The peak of activity determined by conjugation activity towards CDNB and absorbance at 280 nm was collected. The fractions containing at least 25% of enzyme activity of the peak fraction were pooled and concentrated about 10 times using centricon centrifugal filter devices (Millipore) fitted with a 10 kDa molecular mass cut-off filter (YM-10) at 4000 g for 60 min at 4 °C.

50.6

2.8

2.6. Electrophoresis

62.3 67.9

1.4 2

11.1

43.2

Specific Activity⁎ was expressed as μmol S-2, 4-dinitrophenyl-GSH adduct formed/min/ mg protein ± S.D. based on experiments done in quadruplicates.

Table 2 Summary of steps employed in the purification of GST from S. cervi S. Purification no. step 1 2 3 4 5

Total protein (mg)

Cytosolic 399.0 fraction 80% ammo. 71.3 sulfate cut Ultrafiltration 65.1 Cation 36.0 exchange 0.135 GSHSepharose affinity

Total activity (μmoles/min)

Specific activity Yield (μmoles/min/mg) (%)

16.2

0.041

8.2

0.115

10.1 11.0

0.155 0.31

1.8

13.4

239

100

Fold purification

prepared by decanting 20% ethanol solution and replacing it with starting buffer (0.05 M citrate buffer pH 5.5). The CM-Sepharose slurry was degassed and packed in a glass column up to a vertical height of 12 cm. About 2 bed-volumes of citrate buffer were passed through the column for equilibration. 3.0 mL of 55–80% ammonium sulfate precipitate from S. cervi was loaded on exchanger bed. The flow-rate was adjusted between 10–15 mL/h (0.3 mL/min) and 1.0 mL fractions were collected in different tubes. The column was subsequently washed with citrate buffer and 1.0 mL fractions were collected in different tubes. Finally, the bounded proteins were eluted by applying a linear gradient of NaCl from 0–0.3 M in starting buffer. 2.5. GSH-Sepharose affinity chromatography 2.5.1. Coupling of ligand to sepharose The GSH affinity column was prepared according to the method of Simons and Vander Jagt (1977). The coupled gel was washed with

2.6.1. SDS-PAGE SDS-PAGE was performed on a 12% gel according to the method of Laemmli (1970). All reagents for SDS-PAGE were prepared and stored under specified conditions. The following molecular mass standards were used: aprotinin (6.5 kDa), α-lactalbumin (14.2 kDa), trypsin inhibitor (20 kDa), trypsinogen (24 kDa), carbonic anhydrase (29 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), ovalbumin (45 kDa), and albumin (66 kDa). 2.6.2. Activity staining Native PAGE was performed on a 7.5% gel according to the method of Laemmli (1970). After electrophoresis, gels were stained for GST activity using the method of Ricci et al. (1984). Blue insoluble formazan appeared on the gel surface in about 3–5 min, except in the GST area (data not shown). 2.7. Biochemical and functional characterization of GST from S. cervi 2.7.1. Two-Dimensional Electrophoresis (2D-E) The native protein from S. cervi was subjected to 2D-E for detection of isoenzymes, if any. The precipitated protein was centrifuged at 10,000 g for 5 min. The resulting pellet was washed 2–3 times with acetone and vortexed each time. The pellet was resuspended in 0.1 mL TDW and the sample was frozen for 30 min at −20 °C. The frozen sample was lyophilized in a Maxi Dry Plus apparatus for 1 h and the

Fig. 1. Elution profile for total protein and total activity of S. cervi GST from CM-Sepharose column (a) and GSH-Sepharose column (b). W1–W3): 1.0 mL fractions were collected after washing the column with citrate buffer; 1–9): 1.0 mL fractions (proteins) eluted by applying a linear gradient of NaCl from 0–3.0 M in starting buffer. E1–E25): 1.0 mL fractions collected by applying 10 mM Tris–HCl buffer containing 10 mM GSH, 1.0 mM EDTA and 3.0 mM DTT, pH 8.0. Activity was expressed as μmol S-2, 4-dinitrophenyl-GSH adduct formed/ min ± S.D. based on experiments done in quadruplicates.

1.93 1.14

W1–W3): 1.0 mL fractions collected after washing the column with citrate buffer; 1–9): 1.0 mL fractions (proteins) eluted by applying a linear gradient of NaCl from 0–3.0 M in starting buffer. E1–E25): 1.0 mL fractions collected by applying 10 mM Tris–HCl buffer containing 10 mM GSH, 1.0 mM EDTA and 3.0 mM DTT, pH 8.0. Specific Activity was expressed as μmol S-2, 4-dinitrophenyl-GSH adduct formed/min/mg ± S.D. based on experiments done in quadruplicates. The native enzyme from S. cervi was found to be fairly stable at 0 to 4 °C and could be frozen at −20 °C without any appreciable change in its catalytic activity. GST activity also remained unchanged at room temperature and under all conditions of storage. For all practical purposes and to avoid protein degradation, the enzyme protein had to be first lyophilized and then stored frozen at −20 °C.

0.85 0.09 1.22 1.67 2.81 1.55 3.51 8.33 7.19 5.07 7.11 7.33 12.62 13.83 4.17 7.45 25 37.07 13.89 6.25 2.98 2.92

0.069 0.140

1.94

0.050 0.050 0.050 0.080 0.14 0.095 0.080 0.080 0.12 0.082 0.1 0.072 0.094 0.12 0.34 0.4 0.29 0.12 0.08 0.07

E13 0.91 E12 1.3 E11 0.5 E10 2.54 E9 10 E8 10.8 E7 1.67 E6 0.5 E5 0.21 E4 0.18 E3 0.12 E2 0.13 E1 0.16

Fraction Total activity (μmol/min) Total protein (mg) Sp. activity (μmol/min/mg)

GSH-Sepharose affinity chromatography

(b)

0.06

E21 0.23 E20 0.22 E19 0.33 E17 0.58 E16 0.61 E14 0.73

E15 0.58

4 0.186 0.429 0.31 3 0.148 1.22 0.12 2 0.098 0.918 0.11 1 0 0.149 0 W3 0 0 0 W2 0.0025 0.315 0.01 W1 0.018 0.096 0.19 Fraction Total activity (μmol/min) Total protein (mg) Specific activity (μmol/min/mg)

Cation exchange chromatography

2.7.4. Kinetic and inhibition studies All kinetic and inhibition studies were carried out using dialyzed preparations of enzyme to avoid interference due to ammonium sulfate and Tris–HCl. The Km value of native GST for substrate GSH was determined using varying GSH concentrations and a fixed CDNB concentration of 1.0 mM. The Km value for substrate CDNB was determined using varying substrate concentrations and a fixed GSH concentration of 1.0 mM. Data were plotted as double reciprocal Lineweaver–Burk plots to determine the apparent Km values. In order to determine dose-dependent effect of inhibitor hemin and its respective inhibitor constant (Ki), enzyme was incubated with varying concentrations of inhibitor for 10 min at room temperature in the presence of 100 mM potassium phosphate buffer and 1.0 mM GSH. Reaction was initiated by the addition of 1.0 mM CDNB and the absorbance at 340 nm was monitored for 5 min at 30 s intervals. The percentage inhibition of the enzyme activity by hemin was

(a)

2.7.3. Matrix Assisted Laser Desorption Ionization (MALDI) Determination of subunit molecular mass of native GST from S. cervi was done by MALDI-TOF MS in a micro mass MALDI-TOF analyzer. For this, the isolated native protein was washed several times with TDW to remove ammonium sulfate and Tris buffer followed by centrifugation at 8000 g for 15 min. Subsequently, the protein was resuspended in 100 μL TDW for MALDI-TOF analysis.

Table 3 Elution profiles for total protein and total activity of S. cervi GST from CM-Sepharose column (a) and GSH-Sepharose column (b)

2.7.2. Determination of the native mass of GST by gel filtration chromatography The molecular mass of native GST from S. cervi was estimated by gel filtration. The retention time of purified protein and standard proteins were determined on Sephacryl-400 HR (dextran crosslinked with N,N'-methylene bisacrylamide). The column used was Waters Protein-Pak™ 300 SW. Column length was 7.5 × 300 mm. The mobile phase was 50 mM Tris–Acetate buffer containing 100 mM sodium sulfate (pH 8.0). The flow rate was 1.0 mL/min and the wavelength of detection was 280 nm. The following molecular mass standards were used: α-chymotrypsinogen (25 kDa), albumin (67 kDa), glutathione reductase (118 kDa) and γ-globulin (160 kDa).

E18 0.67

5 0.028 0.612 0.05

pellet was dissolved in 160 μL rehydration buffer (5.0 mL) containing 2.1 g urea, 0.76 g thiourea, 0.0242 g Tris, 0.015 g DTT and 0.2 g CHAPS with 1:2 carrier ampholytes (pH 3–10). The pellet was vortexed for about 45 min and then centrifuged at 13,000 g for 20 min. Immobilized pH gradient (IPG) strip (pH 3–10) was hydrated with the rehydration buffer containing the dissolved protein. Isoelectric focusing (IEF) was done in first dimension for about 10 h followed by SDS PAGE on a 12% gel in the second dimension.

0.06

E25 0.017 E24 0.043 E22 0.083

E23 0.061

8 0.0042 0.122 0.03 7 0.012 0.14 0.09 6 0.021 0.219 0.1

Fig. 2. Elution profiles for total protein and total activity of S. cervi GST from GSHSepharose column. E1–E25): 1.0 mL fractions collected by applying 10 mM Tris–HCl buffer containing 10 mM GSH, 1.0 mM EDTA and 3.0 mM DTT, pH 8.0. Activity was expressed as μmol S-2, 4-dinitrophenyl-GSH adduct formed/min ± S.D. based on experiments done in quadruplicates.

0.020

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Fig. 3. (a) SDS PAGE analysis of purified GST from S. cervi on 12% gel. Lanes: 1) low range marker, Sigma (6.5 kDa–66 kDa); 2) α-chymotrypsinogen, Sigma (25 kDa); 3, 4) purified GST from S. cervi; 5) unfractionated cytosolic fraction; 6) fraction from 0–55% ammonium sulfate cut precipitate of cytosolic fraction; 7) fraction from 55–80% ammonium sulfate cut precipitate of cytosolic fraction; 8) active fraction from CM-Sepharose column. (b) Elution profile of standard proteins and purified GST of S. cervi from gel filtration column.

2.7.5. GST peroxidase activity assay GST activity in dialyzed preparations was measured by the glutathione reductase-coupled assay using lipid hydroperoxides (tbutyl hydroperoxide/cumene hydroperoxide/linoleic hydroperoxide) as substrates. The selenium independent glutathione peroxidase (GPx) activity was assayed by a modification of the method of Paglia and Valentine (1967) and Ahmad and Pardini (1988). NADPH oxidation was recorded at 340 nm for 1 min at 30 s intervals. Next, NADPH oxidation was recorded for an additional 1 min following the addition of 0.1 mL sample (cytosolic fraction). GST activity was obtained by subtracting the former rate from the latter. One unit of GST activity was defined as the oxidation of 1 μmol NADPH/min at 25 °C and pH 7.0. Enzyme activity was calculated using a molar extinction coefficient of 6.22 mM− 1 cm− 1 for NADPH.

purification of GST from S. cervi. The enzyme from S. cervi was purified to around 43.2 fold with a total recovery of 11.1% from affinity chromatography. Figs. 1 and 2 and Table 3 show the elution profiles of S. cervi GST from CM-Sepharose and GSH-Sepharose affinity columns, respectively. Fig. 3 depicts the SDS PAGE profile, showing single band purification of GST from S. cervi after GSH-Sepharose affinity chromatography and ultrafiltration respectively. Purified GST from mouse liver was run along with purified GST from S. cervi on a 10% gel revealing that the mammalian enzyme had a subunit molecular mass of approximately 25 kDa (data not shown). The enzyme was found to retain its biological activity when PAGE was done under non-denaturing conditions on a 7.5% gel (data not shown). Fig. 3 also depicts the elution profile of GST from gel filtration column and determination of native molecular mass of GST. Determination of subunit molecular mass of GST from S. cervi was done by MALDI-TOF MS. Native GST from S. cervi was found to be homodimeric having a subunit molecular mass of 24.6 kDa. Gel filtration studies indicated that the protein has a native molecular mass of about 49.2 kDa. One prominent feature of GST is the presence of a number of isoenzymes in a given species (Mannervik, 1985). Twodimensional electrophoresis of GST from S. cervi yielded a single spot thereby suggesting that adult S. cervi has only a single GST (Fig. 4). The trematode parasite Paragonimus westermani has also been found to possess a single sigma class GST in adult stage (Hong et al., 2000).

2.8. Statistical analysis.

3.3. Effect of pH

Results were expressed in terms of mean ± standard deviation (S.D.) based on all experiments performed in either triplicates or quadruplicates.

The optimum pH of S. cervi GST was determined within the pH range of 4.5 to 8.0 of 0.1 M potassium phosphate buffer. Cytosolic GST

calculated by comparing with the enzyme activity in the absence of inhibitor. For determining the type of inhibition, enzyme was incubated with varying concentrations of inhibitor at different substrate concentrations for 10 min at room temperature in the presence of 100 mM potassium phosphate buffer. Percent inhibition OD=min in the absence of inhibitor  OD=min of experimental tube ¼  100 OD=min in the absence of inhibitor

3. Results 3.1. GST localization GST activity profile in various dialyzed subcellular fractions of S. cervi homogenate is depicted in Table 1. It is evident from the results that GST activity in these worms was mainly associated with cytosolic and microsomal fractions. 3.2. Characterization of GST from S. cervi The specific activity profile of S. cervi GST after salt fractionation of cytosolic fraction showed enrichment in 55–80% ammonium sulfate fraction. Table 2 summarizes the results of steps employed in the

Fig. 4. Two-dimensional electrophoresis (2D-E) of purified GST from S. cervi.

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R. Ahmad et al. / Comparative Biochemistry and Physiology, Part B 151 (2008) 237–245 Table 4 Substrate specificities of GST from cytosolic fraction of filarial worms S. cervi Substrate

[Substrate] (mM)

[GSH] (mM)

Specific Activity⁎ (μmol/min/mg protein)

1-chloro-2,4-dinitobenzene (CDNB)

1.0

1.0

13.4 ± 0.032

5.0 5.0

NDa NDa

1.0 1.0 1.0

NDa 0.012 ± 0.002 NDa

Model substrate Bromosulphathalein 0.03 1,2-dichloro-4-nitrobenzene 1.0 (DCNB) Lipid hydroperoxides t-Butyl hydroperoxide Cumene hydroperoxide Linoleic hydroperoxide Fig. 5. Effect of pH of the assay buffer on S. cervi GST activity. Specific Activity was expressed as μmol S-2, 4-dinitrophenyl-GSH adduct formed/min/mg protein ± S.D. based on experiments done in triplicates.

from S. cervi females showed an optimum pH centered at about 6.5. The enzyme was found to be maximally active between pH 6.5 to 7.5 (Fig. 5). Any substitution of catalytically active amino acids of the active site, is expected to affect the pH dependence of the enzyme to display its optimal activity due to a change in the pKa values of the substituted. 3.4. Kinetic studies The reaction catalyzed by cytosolic GST from S. cervi was found to be fairly linear with respect to time (30 s to 5 min) and amount of enzyme protein (4.4 to 88 μg). GST from S. cervi displayed Michaelis– Menten behavior with regards to the standard substrate CDNB and co-substrate GSH. Reciprocal plots of 1/v versus 1/[S] gave the kinetic parameters relating to one substrate in presence of fixed concentration of the second substrate. It was observed that when GSH concentration was progressively increased beyond 0.5 mM keeping the concentration of the purified enzyme fixed, a very slight increase in the activity of the enzyme was observed till GSH concentration reached 1.0 mM. At this substrate concentration, the activity increased no further but remained constant. This indicated that the enzyme had become saturated with GSH at this concentration. A decrease in the activity of the enzyme was observed when GSH concentration was increased beyond 1.0 mM. This implied inhibition of the enzyme by high substrate concentrations. CDNB exhibited similar behavior at high concentrations. Therefore, for determination

0.25 1.0 0.25

⁎ All values are the means ± SD of at least triplicate determinations (ND = not detectable). a Detection limit 0.005 μmol/min/mg protein.

of Km values, an appropriate range of concentrations were chosen for GSH (0.05–0.4 mM) and CDNB (0.5–5.0 mM). The Km values of native S. cervi GST with respect to GSH and CDNB were found to be 0.11 ± 0.010 and 2.5 ± 0.018 mM respectively (Fig. 6). A relatively low level of activity of GST was detected with model lipid hydroperoxide substrate cumene hydroperoxide and no activity with t-butyl hydroperoxide and linoleic hydroperoxide thereby indicating that selenium independent GSH peroxidase activity of S. cervi GST is very limited. With bromosulphathalein and 1, 2dichloro-4-nitrobenzene (DCNB), no significant activity was detected (Table 4). 3.5. Inhibition studies Hemin, a known inhibitor of mammalian GST inhibited S. cervi GST in a concentration dependent fashion when studied for its effect in the 10 to 50 μM range. It maximally inhibited GST to around 92.4% at 50 μM, in a non-competitive manner with respect to substrates GSH and CDNB. The IC50 value was calculated to be around 25 μM. The enzyme had an apparent Ki of around 4.0 μM with respect to hemin. The GSH analog S-hexylglutathione inhibited GST activity from S. cervi in a concentration dependent manner when studied for its effect in the concentration range 0.025 to 0.2 mM. It maximally inhibited GST to around 90.2% in a competitive manner with respect to substrate GSH. The IC50 value was found to be around 0.1 mM. The enzyme had an apparent Ki of around 0.04 mM with respect to

Fig. 6. Lineweaver Burk double reciprocal plots for Km of purified GST from S. cervi with respect to GSH (a) and CDNB (b). Activity was expressed as μmol S-2, 4-dinitrophenyl-GSH adduct formed/min ± S.D. based on experiments done in triplicates.

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Fig. 7. Type of inhibition of Hemin (a) and Hexylglutathione (b) on the activity of purified S. cervi GST with respect to GSH. Specific Activity was expressed as μmol S-2, 4dinitrophenyl-GSH adduct formed/min/mg ± S.D. based on experiments done in triplicates.

hexylglutathione. Fig. 7 depicts the pattern of inhibition of S. cervi GST by hemin and S-hexylglutathione. 4. Discussion The present study describes the characterization of a cytosolic SvGST25 from adult filarial worms S. cervi. Although microsomal forms have been detected (Morgenstern and DePierre, 1983), an appreciable amount of GST(s) activity has also been detected in the cytosol. It has been found that the cytosolic glutathione transferase from S. cervi is clearly distinct from the microsomal glutathione transferase catalyzing the same reaction (Ahmad and Srivastava, 2007). As is evident from the single band in Fig. 3, native GST preparation from S. cervi was homogeneous and devoid of any contaminating protein. A study of kinetic properties of the isolated enzyme from S. cervi revealed significant differences between the parasitic enzymes and their mammalian counterparts. The Km value obtained with respect to GSH for S. cervi GST was 0.11 mM, as compared to a value of 0.1– 2.0 mM reported for rat liver GST(s) (Habig et al., 1974) and was comparable to those reported for cloned GST(s) from Schistosoma species i.e. 0.37–0.43 mM (Walker et al., 1993). The Km value obtained with respect to CDNB was 2.5 mM for S. cervi GST, as compared to a value of 0.06–0.8 mM reported for rat liver GST(s) (Habig et al., 1974) and was comparable to that reported for cloned O. volvulus GST2 i.e. 2.71 mM (Liebau et al., 1996b). Thus, the purification of native GST by GSH affinity chromatography and the activity with CDNB confirmed the functional integrity of the protein. The activity obtained was found to be in the range of those of other nematode GST(s) (Liebau et al., 1996a,b, 1997). Several parasitic worms possess more than one GST (Liebau et al., 1996a,b), while for example Plasmodium falciparum seems to have only one classical GST (Fritz-Wolf et al., 2003). GST has been found to be localized near the host–parasite interface. Tissue distribution and localization of these nematode GST(s) differ, depending on the protein and organism. Thus, some of the GST(s) (but not all) act at the host– parasite interface and are indeed accessible to inhibitors (Liebau et al., 1996b, 1997; Wildenburg et al., 1998). In the present study, 2D-E was done to ascertain the number of isoenzymes of GST present in S. cervi. A single spot was obtained, which confirmed the presence of a single enzyme in affinity purified S. cervi GST (Fig. 4). Since the cytosolic enzyme has the ability to reduce cumene hydroperoxide, this demonstrated to some extent, that cytosolic GST can act as a Se-independent GPx. Se-independent GPx activity has also been observed in Dirofilaria immitis (Jaffe and Lambert, 1986) and O. volvulus (Liebau et al., 1996b).

GST(s) have been characterized from a number of parasitic helminths including cestodes, digeneans and nematodes (Brophy et al., 1990b, 1994a,b; Brophy and Pritchard, 1994). Significantly higher activity has been found in intestinal cestodes and digeneas compared with parasitic nematodes. To date, several glutathione dependent components of the helminth parasite defense system have been characterized, including GST, glyoxylase and GR. GST(s) have been characterized in a number of helminth parasites including Ascaris suum (Liebau et al., 1994c, 1997), O. volvulus (Liebau et al., 1994a,b,d), Heligmosomoides polygyrus (Brophy et al., 1994a), N. americanus (Brophy et al., 1995), D. immitis and Brugia pahangi (Jaffe and Lambert, 1986), Echinococcus species (Fernandez and Hormaeche, 1994; Liebau et al., 1996a), Moniezia expansa (Brophy and Barrett, 1987; Brophy et al., 1989), Fasciola hepatica (Howell et al., 1988; Brophy et al., 1990a; Hillyer et al., 1992; Panaccio et al., 1992; Salvatore et al., 1995) and the Schistosome species (Smith et al., 1986; Balloul et al., 1987; Sexton et al., 1990; Trottein et al., 1990; Henkle et al., 1990; Liu et al., 1993). Recently, mass spectrometric studies were performed on isolated filarial GST (Gupta et al., 2007). The purified GST from S. cervi was also subjected to a number of inhibition studies. A variation in the inhibitor profile between human and parasitic GST was found to be more common than selective inhibition. GST(s) from cestodes, S. mansoni and S. cervi were found to be relatively more sensitive to inhibition by the dye cibacron blue and triphenyltin chloride as compared to their mammalian counterparts (results not shown). Since the studied kinetic and inhibition parameters of ScGST are sufficiently different from that of mammals, ScGST can be considered as a potential drug target. The potential of GST as a drug target has previously been studied by analyzing the in vitro effects of some known GST inhibitors on the motility and viability of S. cervi adults and microfilariae (mf). Of those tested, ethacrynic acid (EA) at micromolar concentrations reduced the viability and motility of mf, third stage larvae (L3) and adult worms (Rao et al., 2000). These results suggest that helminth GST(s) contain sufficient regions of structural difference compared to host enzymes to hold out the prospect of development of parasite-specific vaccines and antihelminthic drugs. The hematin binding range for nematode GST(s) has been found to be similar to a number of mammalian liver tissue alpha class GST(s) which have proposed roles in heme detoxification and/or transport (Mannervik et al., 1985). Binding studies have revealed that hematin binds to the parasite enzyme at a site different from the active site (van Rossum et al., 2004).A number of mammalian liver GST(s) also have proposed secondary binding sites, distinct from the substrate site, for hydrophobic ligands (Salinas and Wong, 1999).

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GST from human filarial parasites W. bancrofti (Wb), B. malayi (By) and O. volvulus (Ov) is significantly different from human GST in sequence and structure (Bhargavi et al., 2005; Perbandt et al., 2005). Thus, Wb, By and Ov GST(s) are potential chemotherapeutic targets for antifilarial drug treatment. Comparison of modelled Wb, By and Ov GST(s) with human GST has revealed structural differences between them. Analysis of active site residues for the binding of electrophilic substrates has provided insights towards the design of parasite specific GST inhibitors. GST inhibitors are emerging as promising therapeutic agents not only for the chemotherapy of filariasis but also in the control of resistance amongst anticancer agents. In diagnostic medicine, as well as in antiparasitic drug development, GST inhibitors are important lead molecules. An exhaustive review on antifilarial drug development has been presented by Mathew et al. (2006, 2007) in which important molecules known for their GST inhibition along with their potential therapeutic uses have been summarized. A three dimensional structural model of glutathione-S-transferase (GST) of the lymphatic filarial parasite Wb has been constructed by homology modeling as a target for drug development against adult worm (Nathan et al., 2005). New drugs that affect new molecular targets are essential to improve treatment and control by killing the adult filarial worms. In conclusion, the comparison of the parasitic GST and the isofunctional host enzyme might open a new approach to studies on the redox metabolism, growth and differentiation of the parasites and as a drug target for chemotherapeutic intervention against filariasis. Acknowledgements Financial assistance from Council for Scientific and Industrial Research (CSIR), New Delhi (India) in the form of Senior Research Fellowship to RA is gratefully acknowledged. References Ahmad, S., Pardini, R.S., 1988. Evidence for the presence of glutathione peroxidase activity towards an organic hydroperoxide in larvae of the cabbage looper moth, Trichoplusia ni. Insect Biochem. 18, 861–866. Ahmad, R., Mishra, R.C., Tewari, N., Tripathi, R.P., Srivastava, A.K., Walter, R.D., 2004. Modulation of filarial glutathione-S-transferase(s) activity: a possibility towards the synthesis of new classes of antifilarial agents. Med. Chem. Res. 13, 724–745. Ahmad, R., Srivastava, A.K., 2007. Cytosolic and microsomal Glutathione-S-transferase (s) from bovine filarial worms Setaria cervi. J. Parasitol. 93, 1285–1290. Arora, K., Mishra, R.C., Tripathi, R.P., Srivastava, A.K., Walter, R.D., 2004. Glutathione synthesis in filarial worms: an attractive target for the design and synthesis of new antifilarials. Med. Chem. Res. 13, 687–706. Balloul, J.M., Grzych, J.M., Pierce, R.J., Capron, A., 1987. A purified 28,000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis. J. Immunol. 138, 3448–3453. Bhargavi, R., Vishwakarma, S., Murty, U.S., 2005. Modelling analysis of GST from Wuchereria bancrofti and Brugia malayi. Bioinformation 25–27. Brophy, P.M., Barrett, J., 1987. Glutathione-S-transferase in the cestode Moniezia expansa. Biochem. Soc. Trans. 15, 1105. Brophy, P.M., Barrett, J., 1990. Glutathione-S-transferase in helminths. Parasitology 100, 345–349. Brophy, P.M., Pritchard, D.I., 1992a. Immunity to helminths: ready to tip the biochemical balance? Parasitol. Today 8, 419–422. Brophy, P.M., Pritchard, D.I., 1992b. Metabolism of lipid peroxidation products by the gastro-intestinal nematodes Necator americanus, Ancylostoma ceylanicum and Heligmosomoides polygyrus. Int. J. Parasitol. 22, 1009–1012. Brophy, P.M., Pritchard, D.I., 1994. Parasitic helminth glutathione-S-transferase(s): an update on their potential as targets for immuno- and chemotherapy. Exp. Parasitol. 79, 89–96. Brophy, P.M., Southan, C., Barrett, J., 1989. Glutathione-S-transferase(s) in the tapeworm Moniezia expansa. Biochem. J. 262, 939–946. Brophy, P.M., Crowley, P., Barrett, J., 1990a. Detoxification reactions of Fasciola hepatica cytosolic glutathione-S-transferase(s). Mol. Biochem. Parasitol. 39, 155–162. Brophy, P.M., Crowley, P., Barrett, J., 1990b. Relative distribution of glutathione transferase, glyoxalase I and glyoxalase II in helminths. Int. J. Parasitol. 20, 259–261. Brophy, P.M., Ben-Smith, A., Brown, A., Behnke, J.M., Pritchard, D.I., 1994a. GlutathioneS-transferase(s) from gastrointestinal tract nematode Heligmosomoides polygrus and mammalian liver compared. Comp. Biochem. Physiol. 109B, 585–592. Brophy, P.M., Brown, A., Pritchard, D.I., 1994b. A PCR strategy for the isolation of GST(s) from the gastro intestinal nematodes. Int. J. Parasitol. 24, 1059–1061.

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