- Email: [email protected]
Molecular cloning and characterization of a novel glutathione S-transferase gene induced by light stimulation in the protozoan Blepharisma japonicum
FEMS Microbiology Letters 231 (2004) 185^189
www.fems-microbiology.org
Molecular cloning and characterization of a novel glutathione S-transferase gene induced by light stimulation in the protozoan Blepharisma japonicum Yuichi Takada a
a;
, Kouji Uda b , Kazuo Kawamura b , Tatsuomi Matsuoka
a
Department of Natural Environmental Science, Faculty of Science, Kochi University, Kochi 780-8520, Japan b Department of Material Science, Faculty of Science, Kochi University, Kochi 780-8520, Japan Received 22 October 2003; received in revised form 4 December 2003; accepted 5 December 2003 First published online 9 January 2004
Abstract A cDNA clone that is inducible by light stimulation was cloned by a differential screening method from a cDNA library of the protozoan Blepharisma japonicum, and the light-dependent expression was checked by semi-quantitative reverse transcription polymerase chain reaction analysis. Sequence analysis showed that the cDNA encodes a glutathione S-transferase (GST) that has not been characterized in the protozoa. Multiple alignment of B. japonicum GST (BjGST1), known protozoan, and mammalian K-, W-, Z-, c-, a-, j-, U-, and g-class GSTs suggested that the BjGST1 may be a novel class GST. Furthermore, highly conserved amino acid residues among the GSTs and the substrate specificity of recombinant BjGST1 showed that BjGST1 is related to K-, W-, Z-, and c-class GSTs rather than the other class of GSTs. 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Light stimulation; Di¡erential screening ; Glutathione S-transferase ; Substrate speci¢city ; Blepharisma japonicum
1. Introduction Glutathione S-transferases (GSTs; EC 2.5.1.18) are phase II detoxi¢cation enzymes that catalyze the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds. GSTs also have peroxidase and isomerase activities, playing multifunctional roles such as removing reactive oxygen species (ROS), transporting hydrophobic compounds, and regulating cell signaling pathways [1^3]. In mammals the soluble GSTs are well characterized, and divided into eight classes, termed K, W, Z, c, a, j, U, and g. The criteria of classi¢cation are based on the comparison of primary and tertiary structure, the substrate speci¢city, and the immunological cross-reactivity [3^5]. However, microbial, and especially protozoan, GSTs have been much less well char-
* Corresponding author. Tel. : +81 (88) 844-8315; Fax : +81 (88) 844-8356. E-mail address : [email protected] (Y. Takada).
acterized, with only a few reports appearing in the literature [6,7]. The ciliated protozoan Blepharisma japonicum exhibits a noticeable step-up photophobic response when suddenly exposed to strong light. The initiation of this reaction to light is triggered by a pink-colored photoreceptor pigment called blepharismin [8,9]. The chemical structure of blepharismin resembles that of hypericin, which is a lightsensitive antibiotic chemical isolated from the higher plant [10^12]. It has also been reported that the blepharismin functions as a toxic product against protozoa and bacteria, with the toxicity depending on the light intensity [8,13,14]. It has recently been suggested that the photoactivated toxicity is caused by the generation of ROS such as singlet oxygen and hydroxyl radicals [15,16]. ROS are involved in various kinds of cytotoxic reactions such as lipid peroxidation, DNA damage, and protein oxidation [17]. As such, B. japonicum must be accustomed to hazardous conditions because of its numerous ROS-generating pigments. In addition, B. japonicum must have acquired some antioxidant systems. In the present study, we attempted to characterize a novel gene encoding a detoxi¢cation enzyme isolated by di¡erential screening.
0378-1097 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00927-3
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CSPD, and exposed onto X-ray ¢lm (Amersham). Finally, when comparing the two ¢lms, positive signals were picked up.
2. Materials and methods 2.1. Cell culture B. japonicum was cultured at 23‡C in the dark in a 0.1% cereal leaf infusion containing bacteria (Enterobacter aerogenes) as food. The bacteria, which were supplied by the Institute for Fermentation, Osaka, Japan, were cultured on 1.5% agar plates containing 0.5% polypeptone, 1% meat extract, and 0.5% NaCl. For the RNA extraction, cells were collected by gentle centrifugation (150Ug, 1 min), resuspended in a saline solution containing 5 mM Tris^HCl (pH 7.4), 1 mM CaCl2 , and 1 mM KCl, and then kept at 23‡C in the dark overnight. 2.2. Construction of a cDNA library from light-stimulated B. japonicum The cultured B. japonicum were stimulated with white light (5 W m32 ) for 24 h, and total RNA was isolated by the acid guanidinium thiocyanate^phenol^chloroform method [18]. Then poly(A)þ RNA was puri¢ed with Oligotex-dT30 Gsuperf (Takara). The EcoRI/NotI-ended double-stranded cDNA was synthesized from poly(A)þ RNA according to the protocols of TimeSaver cDNA Synthesis Kit and Directional Cloning Toolbox (Amersham). The cDNA was inserted into the dephosphorylated EcoRI/NotI site of the Lambda ExCell cloning vector (Amersham). The lambda phage DNA was introduced into the phage capsid with a Gigapack III Gold Packaging Extract (Stratagene) and transfected into Escherichia coli XL1-Blue. 2.3. Di¡erential screening Two types of ¢rst-strand cDNAs for di¡erential plaque hybridization were prepared with the Ready-To-Go T-Primed First Strand Kit (Amersham) from poly(A)þ RNA isolated from light-stimulated or dark-conditioned cells, as described above. Digoxigenin (DIG)-labeled cDNA probes were synthesized from the cDNAs with DIG-High Prime (Roche). Plaques from the cDNA library were continuously transferred to Biodyne B nylon membrane (Pall). Hybridization and detection of DIG-labeled probes were performed with the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). Pre-hybridization was performed for 1 h at 42‡C in DIG Easy Hyb. The probes were added to the exchanged hybridization solution, and hybridization was performed for 3 h at 42‡C. The ¢nal washes were performed at 68‡C in 0.5Usodium saline citrate and 0.1% sodium dodecyl sulfate (SDS) for 30 min. The hybridized probes were detected immunologically with anti-DIG Fab fragments conjugated to alkaline phosphatase, visualized with the chemiluminescence substrate
2.4. DNA sequence analysis The insert cDNA was ampli¢ed by polymerase chain reaction (PCR) with a speci¢c primer pair of the phage DNA (sense primer 5P-CCAAGCTATTTAGGTGACAC3P and antisense primer 5P-TAATACGACTCACTATAGGG-3P), and subcloned into the pGEM-T Easy vector (Promega). The nucleotide sequences were determined with an automated DNA sequencer (ABI Prism 3100, Applied Biosystems). The 5P-upstream region of the cDNA was ampli¢ed by 5P-rapid ampli¢cation of cDNA ends. The poly-C tail was added to the 3P-end of the cDNAs using terminal deoxynucleotidyl transferase, and PCR was performed with the primer pair (oligo-dG adapter 5PGAATTCG15 -3P and the reverse primer). The nucleotide sequences were determined as described above. A database search using cDNA sequences was performed with the BLAST program for nucleotide sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/blast/). The sequence data have been deposited in the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession number AB122092. 2.5. Semi-quantitative reverse transcription (RT)-PCR analysis Two types of cDNAs were prepared as PCR templates. Each of the cDNAs was concurrently ampli¢ed with two pairs of primers, BjGST1-speci¢c primers (sense primer 5PTTTTGACGCCTATGGGAGAG-3P and antisense primer 5P-CGTCTTGTGAAAGCAGTCCA-3P), and actinspeci¢c primers (cDNA sequences for actin were obtained from GenBank, AB056698; sense primer 5P-CCAGTCCTCCTCACTGAAGC-3P and antisense primer 5P-GCCGTCAATATCAAGCCCTA-3P) as an internal standard. Di¡erent ampli¢cation cycles were carried out, each at 94‡C for 30 s, 58‡C for 30 s, and 72‡C for 30 s. 2.6. Construction of the BjGST1 expression vector The open reading frame (including the stop codon) of the cDNA encoding BjGST1 was ampli¢ed by PCR with the upstream primer 5P-GGGAATTCCATATGGCTATCAGGTTCCACTA-3P (the NdeI site is underlined), and the downstream primer 5P-CCGCTCGAGTTAAAACTGAGACTCCGCTC-3P (the XhoI site is underlined). The PCR product was digested with NdeI and XhoI, and subcloned into the plasmid expression vector pET30b (Novagen) previously cut with the same restriction enzymes. The identity of the insert was veri¢ed by sequencing, and the plasmid was designated pET30b/BjGST1.
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2.7. Expression and puri¢cation of recombinant BjGST1 E. coli BL21 was transformed with plasmid pET30b/ BjGST1 and cultured overnight in LB broth containing kanamycin (20 Wg ml31 ). The culture was diluted 1:20 with LB broth and subjected to further incubation at 37‡C for 2 h. Isopropyl L-D-thiogalactoside (IPTG) was added to the culture at a ¢nal concentration of 0.1 mM. After further incubation at 37‡C for 5 h, bacteria were harvested and resuspended in phosphate-bu¡ered saline (PBS). Bacteria were sonicated, and the cell debris was removed by centrifugation (15 000Ug, 10 min). The supernatant was loaded onto a GSTrap FF column (Amersham), and the column was washed with PBS. The enzyme was eluted with 50 mM Tris^HCl bu¡er (pH 8.0) containing 10 mM GSH. The purity of the enzyme was ascertained by SDS^polyacrylamide gel electrophoresis (PAGE). 2.8. Enzyme assay GST activity with 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-nitrobenzyl chloride, and 4-nitrophenyl acetate was measured as described by Habig and Jakoby [19]. Activity with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was measured as described previously [20]. The GSH peroxidase activity of GST was measured with cumene hydroperoxide as described previously [21], and the conjugation of GSH to trans-2-nonenal was measured as reported previously [22]. Protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard [23].
3. Results and discussion A number of positive clones were isolated from the light-stimulated B. japonicum cDNA library by di¡erential screening, and their DNA sequences were determined. The sequences were analyzed with the BLAST program in the GenBank database, and it was found that one of the isolated clones showed signi¢cant local homology (E value was less than 0.001) with already known GSTs. It is widely recognized that GSTs play multifunctional roles in cellular detoxi¢cation systems, whereas GSTs have not been well characterized in the protozoa [3,6,7]. The cDNA (BjGST1) encoding B. japonicum GST contained a 627-bp open reading frame (including the stop codon) and encoded a protein of 208 residues with a calculated molecular mass of 23 945 Da. The stop codon (TAA) was followed by a 3P-untranslated region containing a putative polyadenylation signal (AAATAA) at 14 bp upstream of the poly(A)þ tail. To determine whether BjGST1 is strongly expressed in the light-stimulated B. japonicum, semi-quantitative RTPCR analysis was performed with actin mRNA as an in-
Fig. 1. Con¢rmation of up-regulation of BjGST1 expression after light stimulation by semi-quantitative RT-PCR. A and B pro¢les were obtained from dark-conditioned and light-stimulated B. japonicum, respectively, and actin mRNA was used as an internal standard.
ternal standard. As shown in Fig. 1, BjGST1 expression in light-stimulated B. japonicum was greater than that in dark-conditioned B. japonicum, suggesting that B. japonicum has a transcription mechanism that is induced by light irradiation, and that the possible second messenger could be a ROS such as singlet oxygen or hydroxyl radicals generated from the blepharismin pigments [15,16]. Using the ClustalW alignment program (http://clustalw.genome.ad.jp/), the deduced amino acid sequences of BjGST1 were compared with protozoan GST (Plasmodium falciparum), and the mammalian K-, W-, Z-, c-, a-, j-, U-, and g-class GSTs, resulting in BjGST1 exhibiting 9.6^ 23.1% pairwise identity towards selected typical GSTs (Fig. 2). It is generally recognized that the same class GSTs show greater than 40% identity, whereas the identity between separate GST classes is less than 30%, suggesting that BjGST1 may be a novel class GST [2,24]. In taking note of highly conserved amino acid residues, it was also found that BjGST1 possesses important residues in K-, W-, Z-, and c-class GSTs. Tyrosine in the N-terminus (Tyr7) is essential to interacting with the thiol group of GSH, and Phe49 (tyrosine in the Z class) acts as a key residue in the hydrophobic lock-and-key motif that is mediated by intersubunit stability and catalytic function [3,25,26]. To investigate the GST activity, an expression vector including the entire open reading frame of BjGST1 was constructed and transformed into E. coli. The expression was induced by IPTG, and the sonicated cell lysate was subjected to GSH-a⁄nity column chromatography. The expression and puri¢cation procedures were monitored by SDS^PAGE (Fig. 3), which showed that the recombinant BjGST1 had a molecular mass of approx. 25 000 Da, corresponding to the calculated molecular mass (23 945 Da). The puri¢ed BjGST1 showed relatively high GST activities towards 1-chloro-2,4-dinitrobenzene, 7-chloro-4nitrobenzo-2-oxa-1,3-diazole, and ethacrynic acid but not towards 1,2-dichloro-4-nitrobenzene and 4-nitrobenzyl chloride (Table 1), indicating that the GST activities of BjGST1 are similar to those of K- and Z-class GSTs [20,27]. Furthermore, BjGST1 also showed GSH peroxidase activity with the substrate cumene hydroperoxide and slight conjugation activity with trans-2-nonenal as the secondary product of lipid peroxidation. These ¢ndings sug-
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Fig. 2. Multiple alignment of amino acid sequences of BjGST1 with protozoan and mammalian GSTs. The shaded box represents highly conserved residues (more than ¢ve residues) among the 10 aligned residues. Residues forming the GSH-binding site in human prostaglandin D synthase are marked by an asterisk at the top of the alignment. Amino acid sequences were obtained from SwissProt (sw) and GenBank (gb) as follows : Protozoa (P. falciparum GST1, gb AF426836), K (human GSTA1-1, sw P08263), W (human GSTM4-4, sw Q03013), Z (human GSTP1-1, sw P09211), c (human prostaglandin D synthase, sw O60760), a (human GSTT1-1, sw P30711), j (human GSTZ1-1, sw O43708), U (rat GSTK1, sw P24473), g (human GSTO 1-1, sw P78417).
gest that BjGST1 can act as the antioxidant protein, which reduces endogenous toxic products generated from cellular oxidation. Previously reported phylogenic trees of GST classes showed that K-, W-, Z-, and c-class GSTs were able to
cluster into one group [28,29]. The BjGST1 showed higher homology with these GST classes (15.9^23.1%) than with the other GST classes (9.6^17.3), and the features of the BjGST1 are also similar to the former GST classes. Thus, the possible novel classed GST, BjGST1, seems to be closely related to K-, W-, Z-, and c-class GSTs. To more closely study BjGST1, which is one of the little known protozoan GSTs, it is useful to understand the function and evolution of GSTs.
Table 1 Speci¢c activities of recombinant BjGST1
Fig. 3. The expression and puri¢cation of recombinant BjGST1. Lane 1, total cellular extracts from uninduced E. coli BL21 containing pET30b/ BjGST1; lane 2, as lane 1, but after induction by IPTG; lane 3, a⁄nity-puri¢ed recombinant BjGST1.
Substrate
Speci¢c activity (Wmol min31 mg31 )
1-Chloro-2,4-dinitrobenzene 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole Ethacrynic acid 4-Nitrophenyl acetate 1,2-Dichloro-4-nitrobenzene 4-Nitrobenzyl chloride Cumene hydroperoxide trans-2-Nonenal
6.46 T 0.65 3.92 T 0.46 1.68 T 0.16 0.102 T 0.015 Not detected Not detected 0.472 T 0.018 0.0747 T 0.0056
Results are given as means T S.D. for three determinations.
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Acknowledgements We thank Dr. H.J. Yuasa, Dr. S. Fujiwara, and Ms. A. Kida for important suggestions and technical help throughout this work. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (No. 13640684).
[14]
[15]
[16]
References [1] Mannervik, B. (1985) The isoenzymes of glutathione transferase. Adv. Enzymol. Relat. Areas Mol. Biol. 57, 357^417. [2] Hayes, J.D. and Pulford, D.J. (1995) The glutathione S-transferase supergene family : regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30, 445^600. [3] Sheehan, D., Meade, G., Foley, V.M. and Dowd, C.A. (2001) Structure, function and evolution of glutathione transferases : implications for classi¢cation of non-mammalian members of an ancient enzyme superfamily. Biochem. J. 360, 1^16. [4] Board, P.G., Baker, R.T., Chelvanayagam, G. and Jermiin, L.S. (1997) Zeta, a novel class of glutathione transferases in a range of species from plants to humans. Biochem. J. 328, 929^935. [5] Board, P.G., Coggan, M., Chelvanayagam, G., Easteal, S., Jermiin, L.S., Schulte, G.K., Danley, D.E., Hoth, L.R., Gri¡or, M.C., Kamath, A.V., Rosner, M.H., Chrunyk, B.A., Perregaux, D.E., Gabel, C.A., Geoghegan, K.F. and Pandit, J. (2000) Identi¢cation, characterization, and crystal structure of the Omega class glutathione transferases. J. Biol. Chem. 275, 24798^24806. [6] Overbaugh, J.M., Lau, E.P., Marino, V.A. and Fall, R. (1988) Puri¢cation and preliminary characterization of a monomeric glutathione S-transferase from Tetrahymena thermophila. Arch. Biochem. Biophys. 261, 227^234. [7] Liebau, E., Bergmann, B., Campbell, A.M., Teesdale-Spittle, P., Brophy, P.M., Luersen, K. and Walter, R.D. (2002) The glutathione Stransferase from Plasmodium falciparum. Mol. Biochem. Parasitol. 124, 85^90. [8] Giese, A.C. (1973) Blepharisma : The Biology of a Light-Sensitive Protozoa, pp. 266^303. Stanford University Press, Stanford, CA. [9] Matsuoka, T. (1983) Negative phototaxis in Blepharisma japonicum. J. Protozool. 30, 409^414. [10] Brockmann (1957) Zur Kenntnis des Hypericin und Pseudohypericins. Chem. Ber. 90, 2480^2491. [11] Checcucci, G., Shoemaker, R.S., Bini, E., Cerny, R., Tao, N., Hyon, J.-S., Gio¡re, D., Ghetti, F., Lenci, F. and Song, P.-S. (1997) The chemical structure of blepharismin, the photosensor pigment for Blepharisma japonicum. J. Am. Chem. Soc. 119, 5762^5763. [12] Maeda, M., Naoki, H., Matsuoka, T., Kato, Y., Kotsuki, H., Utsumi, K. and Tanaka, T. (1997) Blepharismin 1^5, novel photoreceptor from the unicellular organism Blepharisma japonicum. Tetrahedron Lett. 38, 7411^7414. [13] Pant, B., Kato, Y., Kumagai, T., Matsuoka, T. and Sugiyama, M. (1997) Blepharismin produced by a protozoan Blepharisma functions
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as an antibiotic e¡ective against methicillin-resistant Staphylococcus aureus. FEMS Microbiol. Lett. 155, 67^71. Noda Terazima, M., Iio, H. and Harumoto, T. (1999) Toxic and phototoxic properties of the protozoan pigments blepharismin and oxyblepharismin. Photochem. Photobiol. 69, 47^54. Checcucci, G., Lenci, F., Ghetti, F. and Song, P.-S. (1991) A videomicroscopic study of the e¡ect of a singlet oxygen quencher on Blepharisma japonicum photobehavior. J. Photochem. Photobiol. 11, 49^ 55. Kato, Y., Watanabe, Y., Sagara, Y., Murakami, Y., Sugiyama, M. and Matsuoka, T. (1996) The photoreceptor pigment of unicellular organism Blepharisma generates hydroxyl radicals. J. Photochem. Photobiol. 34, 29^33. Cabiscol, E., Tamarit, J. and Ros, J. (2000) Oxidative stress in bacteria and protein damage by reactive oxygen species. Int. Microbiol. 3, 3^8. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156^159. Habig, W.H. and Jakoby, W.B. (1981) Assays for di¡erentiation of glutathione S-transferases. Methods Enzymol. 77, 398^405. Ricci, G., Caccuri, A.M., Lo Bello, M., Pastore, A., Piemonte, F. and Federici, G. (1994) Colorimetric and £uorometric assays of glutathione transferase based on 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Anal. Biochem. 218, 463^465. Lawrence, R.A. and Burk, R.F. (1976) Glutathione peroxidase activity in selenium-de¢cient rat liver. Biochem. Biophys. Res. Commun. 71, 952^958. Brophy, P.M., Southan, C. and Barrett, J. (1989) Glutathione transferases in the tapeworm Moniezia expansa. Biochem. J. 262, 939^946. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. Cha, C.J., Kim, S.J., Kim, Y.H., Stingley, R. and Cerniglia, C.E. (2002) Molecular cloning, expression and characterization of a novel class glutathione S-transferase from the fungus Cunninghamella elegans. Biochem. J. 368, 589^595. Stenberg, G., Board, P.G. and Mannervik, B. (1991) Mutation of an evolutionarily conserved tyrosine residue in the active site of a human class Alpha glutathione transferase. FEBS Lett. 293, 153^155. Sayed, Y., Wallace, L.A. and Dirr, H.W. (2000) The hydrophobic lock-and-key intersubunit motif of glutathione transferase A1-1: implications for catalysis, ligandin function and stability. FEBS Lett. 465, 169^172. Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M.K., Warholm, M. and Jornvall, H. (1985) Identi¢cation of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties. Proc. Natl. Acad. Sci. USA 82, 7202^7206. Snyder, M.J. and Maddison, D.R. (1997) Molecular phylogeny of glutathione-S-transferases. DNA Cell Biol. 16, 1373^1384. Agianian, B., Tucker, P.A., Schouten, A., Leonard, K., Bullard, B. and Gros, P. (2003) Structure of a Drosophila sigma class glutathione S-transferase reveals a novel active site topography suited for lipid peroxidation products. J. Mol. Biol. 326, 151^165.
FEMSLE 11365 11-2-04
www.fems-microbiology.org
Molecular cloning and characterization of a novel glutathione S-transferase gene induced by light stimulation in the protozoan Blepharisma japonicum Yuichi Takada a
a;
, Kouji Uda b , Kazuo Kawamura b , Tatsuomi Matsuoka
a
Department of Natural Environmental Science, Faculty of Science, Kochi University, Kochi 780-8520, Japan b Department of Material Science, Faculty of Science, Kochi University, Kochi 780-8520, Japan Received 22 October 2003; received in revised form 4 December 2003; accepted 5 December 2003 First published online 9 January 2004
Abstract A cDNA clone that is inducible by light stimulation was cloned by a differential screening method from a cDNA library of the protozoan Blepharisma japonicum, and the light-dependent expression was checked by semi-quantitative reverse transcription polymerase chain reaction analysis. Sequence analysis showed that the cDNA encodes a glutathione S-transferase (GST) that has not been characterized in the protozoa. Multiple alignment of B. japonicum GST (BjGST1), known protozoan, and mammalian K-, W-, Z-, c-, a-, j-, U-, and g-class GSTs suggested that the BjGST1 may be a novel class GST. Furthermore, highly conserved amino acid residues among the GSTs and the substrate specificity of recombinant BjGST1 showed that BjGST1 is related to K-, W-, Z-, and c-class GSTs rather than the other class of GSTs. 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Light stimulation; Di¡erential screening ; Glutathione S-transferase ; Substrate speci¢city ; Blepharisma japonicum
1. Introduction Glutathione S-transferases (GSTs; EC 2.5.1.18) are phase II detoxi¢cation enzymes that catalyze the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds. GSTs also have peroxidase and isomerase activities, playing multifunctional roles such as removing reactive oxygen species (ROS), transporting hydrophobic compounds, and regulating cell signaling pathways [1^3]. In mammals the soluble GSTs are well characterized, and divided into eight classes, termed K, W, Z, c, a, j, U, and g. The criteria of classi¢cation are based on the comparison of primary and tertiary structure, the substrate speci¢city, and the immunological cross-reactivity [3^5]. However, microbial, and especially protozoan, GSTs have been much less well char-
* Corresponding author. Tel. : +81 (88) 844-8315; Fax : +81 (88) 844-8356. E-mail address : [email protected] (Y. Takada).
acterized, with only a few reports appearing in the literature [6,7]. The ciliated protozoan Blepharisma japonicum exhibits a noticeable step-up photophobic response when suddenly exposed to strong light. The initiation of this reaction to light is triggered by a pink-colored photoreceptor pigment called blepharismin [8,9]. The chemical structure of blepharismin resembles that of hypericin, which is a lightsensitive antibiotic chemical isolated from the higher plant [10^12]. It has also been reported that the blepharismin functions as a toxic product against protozoa and bacteria, with the toxicity depending on the light intensity [8,13,14]. It has recently been suggested that the photoactivated toxicity is caused by the generation of ROS such as singlet oxygen and hydroxyl radicals [15,16]. ROS are involved in various kinds of cytotoxic reactions such as lipid peroxidation, DNA damage, and protein oxidation [17]. As such, B. japonicum must be accustomed to hazardous conditions because of its numerous ROS-generating pigments. In addition, B. japonicum must have acquired some antioxidant systems. In the present study, we attempted to characterize a novel gene encoding a detoxi¢cation enzyme isolated by di¡erential screening.
0378-1097 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00927-3
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CSPD, and exposed onto X-ray ¢lm (Amersham). Finally, when comparing the two ¢lms, positive signals were picked up.
2. Materials and methods 2.1. Cell culture B. japonicum was cultured at 23‡C in the dark in a 0.1% cereal leaf infusion containing bacteria (Enterobacter aerogenes) as food. The bacteria, which were supplied by the Institute for Fermentation, Osaka, Japan, were cultured on 1.5% agar plates containing 0.5% polypeptone, 1% meat extract, and 0.5% NaCl. For the RNA extraction, cells were collected by gentle centrifugation (150Ug, 1 min), resuspended in a saline solution containing 5 mM Tris^HCl (pH 7.4), 1 mM CaCl2 , and 1 mM KCl, and then kept at 23‡C in the dark overnight. 2.2. Construction of a cDNA library from light-stimulated B. japonicum The cultured B. japonicum were stimulated with white light (5 W m32 ) for 24 h, and total RNA was isolated by the acid guanidinium thiocyanate^phenol^chloroform method [18]. Then poly(A)þ RNA was puri¢ed with Oligotex-dT30 Gsuperf (Takara). The EcoRI/NotI-ended double-stranded cDNA was synthesized from poly(A)þ RNA according to the protocols of TimeSaver cDNA Synthesis Kit and Directional Cloning Toolbox (Amersham). The cDNA was inserted into the dephosphorylated EcoRI/NotI site of the Lambda ExCell cloning vector (Amersham). The lambda phage DNA was introduced into the phage capsid with a Gigapack III Gold Packaging Extract (Stratagene) and transfected into Escherichia coli XL1-Blue. 2.3. Di¡erential screening Two types of ¢rst-strand cDNAs for di¡erential plaque hybridization were prepared with the Ready-To-Go T-Primed First Strand Kit (Amersham) from poly(A)þ RNA isolated from light-stimulated or dark-conditioned cells, as described above. Digoxigenin (DIG)-labeled cDNA probes were synthesized from the cDNAs with DIG-High Prime (Roche). Plaques from the cDNA library were continuously transferred to Biodyne B nylon membrane (Pall). Hybridization and detection of DIG-labeled probes were performed with the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). Pre-hybridization was performed for 1 h at 42‡C in DIG Easy Hyb. The probes were added to the exchanged hybridization solution, and hybridization was performed for 3 h at 42‡C. The ¢nal washes were performed at 68‡C in 0.5Usodium saline citrate and 0.1% sodium dodecyl sulfate (SDS) for 30 min. The hybridized probes were detected immunologically with anti-DIG Fab fragments conjugated to alkaline phosphatase, visualized with the chemiluminescence substrate
2.4. DNA sequence analysis The insert cDNA was ampli¢ed by polymerase chain reaction (PCR) with a speci¢c primer pair of the phage DNA (sense primer 5P-CCAAGCTATTTAGGTGACAC3P and antisense primer 5P-TAATACGACTCACTATAGGG-3P), and subcloned into the pGEM-T Easy vector (Promega). The nucleotide sequences were determined with an automated DNA sequencer (ABI Prism 3100, Applied Biosystems). The 5P-upstream region of the cDNA was ampli¢ed by 5P-rapid ampli¢cation of cDNA ends. The poly-C tail was added to the 3P-end of the cDNAs using terminal deoxynucleotidyl transferase, and PCR was performed with the primer pair (oligo-dG adapter 5PGAATTCG15 -3P and the reverse primer). The nucleotide sequences were determined as described above. A database search using cDNA sequences was performed with the BLAST program for nucleotide sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/blast/). The sequence data have been deposited in the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession number AB122092. 2.5. Semi-quantitative reverse transcription (RT)-PCR analysis Two types of cDNAs were prepared as PCR templates. Each of the cDNAs was concurrently ampli¢ed with two pairs of primers, BjGST1-speci¢c primers (sense primer 5PTTTTGACGCCTATGGGAGAG-3P and antisense primer 5P-CGTCTTGTGAAAGCAGTCCA-3P), and actinspeci¢c primers (cDNA sequences for actin were obtained from GenBank, AB056698; sense primer 5P-CCAGTCCTCCTCACTGAAGC-3P and antisense primer 5P-GCCGTCAATATCAAGCCCTA-3P) as an internal standard. Di¡erent ampli¢cation cycles were carried out, each at 94‡C for 30 s, 58‡C for 30 s, and 72‡C for 30 s. 2.6. Construction of the BjGST1 expression vector The open reading frame (including the stop codon) of the cDNA encoding BjGST1 was ampli¢ed by PCR with the upstream primer 5P-GGGAATTCCATATGGCTATCAGGTTCCACTA-3P (the NdeI site is underlined), and the downstream primer 5P-CCGCTCGAGTTAAAACTGAGACTCCGCTC-3P (the XhoI site is underlined). The PCR product was digested with NdeI and XhoI, and subcloned into the plasmid expression vector pET30b (Novagen) previously cut with the same restriction enzymes. The identity of the insert was veri¢ed by sequencing, and the plasmid was designated pET30b/BjGST1.
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2.7. Expression and puri¢cation of recombinant BjGST1 E. coli BL21 was transformed with plasmid pET30b/ BjGST1 and cultured overnight in LB broth containing kanamycin (20 Wg ml31 ). The culture was diluted 1:20 with LB broth and subjected to further incubation at 37‡C for 2 h. Isopropyl L-D-thiogalactoside (IPTG) was added to the culture at a ¢nal concentration of 0.1 mM. After further incubation at 37‡C for 5 h, bacteria were harvested and resuspended in phosphate-bu¡ered saline (PBS). Bacteria were sonicated, and the cell debris was removed by centrifugation (15 000Ug, 10 min). The supernatant was loaded onto a GSTrap FF column (Amersham), and the column was washed with PBS. The enzyme was eluted with 50 mM Tris^HCl bu¡er (pH 8.0) containing 10 mM GSH. The purity of the enzyme was ascertained by SDS^polyacrylamide gel electrophoresis (PAGE). 2.8. Enzyme assay GST activity with 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-nitrobenzyl chloride, and 4-nitrophenyl acetate was measured as described by Habig and Jakoby [19]. Activity with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was measured as described previously [20]. The GSH peroxidase activity of GST was measured with cumene hydroperoxide as described previously [21], and the conjugation of GSH to trans-2-nonenal was measured as reported previously [22]. Protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard [23].
3. Results and discussion A number of positive clones were isolated from the light-stimulated B. japonicum cDNA library by di¡erential screening, and their DNA sequences were determined. The sequences were analyzed with the BLAST program in the GenBank database, and it was found that one of the isolated clones showed signi¢cant local homology (E value was less than 0.001) with already known GSTs. It is widely recognized that GSTs play multifunctional roles in cellular detoxi¢cation systems, whereas GSTs have not been well characterized in the protozoa [3,6,7]. The cDNA (BjGST1) encoding B. japonicum GST contained a 627-bp open reading frame (including the stop codon) and encoded a protein of 208 residues with a calculated molecular mass of 23 945 Da. The stop codon (TAA) was followed by a 3P-untranslated region containing a putative polyadenylation signal (AAATAA) at 14 bp upstream of the poly(A)þ tail. To determine whether BjGST1 is strongly expressed in the light-stimulated B. japonicum, semi-quantitative RTPCR analysis was performed with actin mRNA as an in-
Fig. 1. Con¢rmation of up-regulation of BjGST1 expression after light stimulation by semi-quantitative RT-PCR. A and B pro¢les were obtained from dark-conditioned and light-stimulated B. japonicum, respectively, and actin mRNA was used as an internal standard.
ternal standard. As shown in Fig. 1, BjGST1 expression in light-stimulated B. japonicum was greater than that in dark-conditioned B. japonicum, suggesting that B. japonicum has a transcription mechanism that is induced by light irradiation, and that the possible second messenger could be a ROS such as singlet oxygen or hydroxyl radicals generated from the blepharismin pigments [15,16]. Using the ClustalW alignment program (http://clustalw.genome.ad.jp/), the deduced amino acid sequences of BjGST1 were compared with protozoan GST (Plasmodium falciparum), and the mammalian K-, W-, Z-, c-, a-, j-, U-, and g-class GSTs, resulting in BjGST1 exhibiting 9.6^ 23.1% pairwise identity towards selected typical GSTs (Fig. 2). It is generally recognized that the same class GSTs show greater than 40% identity, whereas the identity between separate GST classes is less than 30%, suggesting that BjGST1 may be a novel class GST [2,24]. In taking note of highly conserved amino acid residues, it was also found that BjGST1 possesses important residues in K-, W-, Z-, and c-class GSTs. Tyrosine in the N-terminus (Tyr7) is essential to interacting with the thiol group of GSH, and Phe49 (tyrosine in the Z class) acts as a key residue in the hydrophobic lock-and-key motif that is mediated by intersubunit stability and catalytic function [3,25,26]. To investigate the GST activity, an expression vector including the entire open reading frame of BjGST1 was constructed and transformed into E. coli. The expression was induced by IPTG, and the sonicated cell lysate was subjected to GSH-a⁄nity column chromatography. The expression and puri¢cation procedures were monitored by SDS^PAGE (Fig. 3), which showed that the recombinant BjGST1 had a molecular mass of approx. 25 000 Da, corresponding to the calculated molecular mass (23 945 Da). The puri¢ed BjGST1 showed relatively high GST activities towards 1-chloro-2,4-dinitrobenzene, 7-chloro-4nitrobenzo-2-oxa-1,3-diazole, and ethacrynic acid but not towards 1,2-dichloro-4-nitrobenzene and 4-nitrobenzyl chloride (Table 1), indicating that the GST activities of BjGST1 are similar to those of K- and Z-class GSTs [20,27]. Furthermore, BjGST1 also showed GSH peroxidase activity with the substrate cumene hydroperoxide and slight conjugation activity with trans-2-nonenal as the secondary product of lipid peroxidation. These ¢ndings sug-
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Fig. 2. Multiple alignment of amino acid sequences of BjGST1 with protozoan and mammalian GSTs. The shaded box represents highly conserved residues (more than ¢ve residues) among the 10 aligned residues. Residues forming the GSH-binding site in human prostaglandin D synthase are marked by an asterisk at the top of the alignment. Amino acid sequences were obtained from SwissProt (sw) and GenBank (gb) as follows : Protozoa (P. falciparum GST1, gb AF426836), K (human GSTA1-1, sw P08263), W (human GSTM4-4, sw Q03013), Z (human GSTP1-1, sw P09211), c (human prostaglandin D synthase, sw O60760), a (human GSTT1-1, sw P30711), j (human GSTZ1-1, sw O43708), U (rat GSTK1, sw P24473), g (human GSTO 1-1, sw P78417).
gest that BjGST1 can act as the antioxidant protein, which reduces endogenous toxic products generated from cellular oxidation. Previously reported phylogenic trees of GST classes showed that K-, W-, Z-, and c-class GSTs were able to
cluster into one group [28,29]. The BjGST1 showed higher homology with these GST classes (15.9^23.1%) than with the other GST classes (9.6^17.3), and the features of the BjGST1 are also similar to the former GST classes. Thus, the possible novel classed GST, BjGST1, seems to be closely related to K-, W-, Z-, and c-class GSTs. To more closely study BjGST1, which is one of the little known protozoan GSTs, it is useful to understand the function and evolution of GSTs.
Table 1 Speci¢c activities of recombinant BjGST1
Fig. 3. The expression and puri¢cation of recombinant BjGST1. Lane 1, total cellular extracts from uninduced E. coli BL21 containing pET30b/ BjGST1; lane 2, as lane 1, but after induction by IPTG; lane 3, a⁄nity-puri¢ed recombinant BjGST1.
Substrate
Speci¢c activity (Wmol min31 mg31 )
1-Chloro-2,4-dinitrobenzene 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole Ethacrynic acid 4-Nitrophenyl acetate 1,2-Dichloro-4-nitrobenzene 4-Nitrobenzyl chloride Cumene hydroperoxide trans-2-Nonenal
6.46 T 0.65 3.92 T 0.46 1.68 T 0.16 0.102 T 0.015 Not detected Not detected 0.472 T 0.018 0.0747 T 0.0056
Results are given as means T S.D. for three determinations.
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Acknowledgements We thank Dr. H.J. Yuasa, Dr. S. Fujiwara, and Ms. A. Kida for important suggestions and technical help throughout this work. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (No. 13640684).
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