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Glutathione S-transferase from the Icelandic scallop (Chlamys islandica): Isolation and partial characterization
Comparative Biochemistry and Physiology, Part C 144 (2007) 403 – 407 www.elsevier.com/locate/cbpc
Glutathione S-transferase from the Icelandic scallop (Chlamys islandica): Isolation and partial characterization Bjørnar Myrnes ⁎, Inge W. Nilsen Marine Biotechnology and Fish Health, Norwegian Institute of Fisheries and Aquaculture, P. O. Box 6122, N-9291 Tromsø, Norway Received 14 August 2006; received in revised form 25 October 2006; accepted 23 November 2006 Available online 29 November 2006
Abstract Glutathione S-transferase from the digestive gland of the cold-adapted marine bivalve Icelandic scallop was purified to apparent homogeneity by single GSTrap chromatography. The enzyme appeared to be a homodimer with subunit Mr 22,000 having an optimum catalytic activity at pH 6.5–7. Enzymatic analysis of scallop GST using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione resulted in apparent values for KGST and KCDNB of 0.3 mM and 0.4 mM, respectively. The scallop GST lost activity faster than porcine GST when exposed to increased m m temperatures, but both enzymes needed 10 min incubation at 60 °C for complete inactivation. A partial coding sequence was identified in cDNA synthesised from digestive gland mRNA. Comparison to known sequences indicates that the gene product is a glutathione S-transferase, and the predicted Icelandic scallop GST protein scores 40% sequence identity and 60% sequence similarity to mu-class proteins. © 2006 Elsevier Inc. All rights reserved. Keywords: Invertebrate; Icelandic scallop; GST; Protein; Coding sequence; Purification
1. Introduction The glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of enzymes which catalyse the conjugation of a broad range of electrophilic substances to glutathione (Keen and Jakoby, 1978; Hayes and Wolf, 1988). The primary function of the enzyme is considered to be the detoxification of both endogenous and xenobiotic alkylating agents. GSTs have been classified into several classes (Alpha, Mu, Pi, Theta, Sigma, Kappa, Zeta and Omega) based on primary structure, immunological and kinetic properties (Mannevik et al., 1985; Meyer et al., 1991; Meyer and Thomas, 1995; Pemble et al., 1996; Board et al., 1997, 2000). The enzymes have been most intensively studied in mammals, but there has also been considerable interest in the role of GST in non-vertebrate organisms (Mannervik and Danielson, 1989; Clark, 1989; Sheehan et al., 2001). GST is a part of the repertoire of adaptive response mechanisms to chemical stress in eukaryotic cells (Norrpa, 2003) and its activity in aquatic organisms has been exploited as a biomarker of pollution (Goldberg, 1975; ⁎ Corresponding author. Tel.: +47 776 2900; fax: +47 776 29100. E-mail address: [email protected] (B. Myrnes). 1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2006.11.011
Goldberg and Bertine, 2000; Pennec and Pennec, 2003; Moreiro and Guilhmino, 2005). Relative to vertebrate systems, little information is available concerning GSTs from marine invertebrate organisms such as molluscs although GST isoforms were isolated and characterized from gills and digestive gland of blue mussel more than a decade ago (Fitzpatrick and Sheehan, 1993; Fitzpatrick et al., 1995). In recent years several glutathione S-transferase protein and gene isoforms from marine shellfishes have been isolated and characterized (Blanchette and Signh, 2002; Hoarau et al., 2002, 2006; Yang et al., 2002, 2003, 2004). In this study we report the isolation and partial characterization of a GST protein and a GST cDNA gene from the Icelandic scallop. 2. Materials and methods 2.1. Materials and biological samples Wild Icelandic scallops Chlamys islandica were collected at 15 m depth from a site in northern Norway. The digestive glands were collected and used as material for enzyme and gene isolation.
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The GSTrap FF (5 mL) chromatography column was supplied by Amersham Pharmacia Biotech. Porcine glutathione S-transferase (G 6636) was obtained from Sigma-Aldrich and all other chemicals used were of analytical grade. 2.2. Purification of scallop glutathione S-transferase Digestive glands from five scallops were homogenized at 4 °C in 250 mL buffer A (10 mM sodium phosphate buffer pH 7.2 containing 0.15 M KCl, 1 mM EDTA and 1 mM dithiothreitol), using a Waring blender. The extract obtained was centrifuged at 30,000 g for 30 min and the supernatant containing GST activity was collected and filtered through Whatman GF/C filter at 4 °C and finally centrifuged for 60 min at 30,000 g. The supernatant (173 mL) containing GST activity was collected and used as crude extract. All further steps in purification of scallop GST were carried out at 6–8 °C. The crude extract was subjected to GSTrap FF chromatography in buffer A in three separate experiments. The column was further washed with buffer A and bound GST eluted with 50 mM Tris– HCl (pH 8), containing 10 mM reduced glutathione. Fractions containing GST activity from these experiments were combined, dialysed against 10 mM Tris–HCl buffer pH 8, followed by concentration on a PM 10 ultrafilter (cut off 10 kDa) using an Amicon Diaflo stirred cell. The fraction obtained (1.1 mL) produced a single Coomassie-stained protein band when analysed after SDS-PAGE and was used as isolated scallop GST. 2.3. Determination of GST activity GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) was measured as described by Habig and Jakoby (1981) using 1 mM CDNB and 1 mM GSH in 0.1 M potassium phosphate buffer pH 6.5. One unit of GST activity is defined as the amount of enzyme that produce 1 μmol S-(2,4 dinitrophenyl)glutathione at 340 nm (ε = 9.6 mM− 1 cm− 1) per min at 24 °C.
(Clontech). PCR reaction mixtures were as recommended in the kit for 3′-RACE. Employing touch-down PCR (5 s denaturation at 95 °C and 90 s annealing/elongation temperatures 72 °C, 70 °C and 68 °C for 5 cycles, 5 cycles and 30 cycles, respectively) produced a single band of approximately 0.9 kb in agarose gel analysis. The amplified product was subcloned by the use of pGEM-T Easy vector system II (Promega) and E. coli JM109 cells for transformation. Recombinant plasmids were analysed by PCR, and DNA sequencing of both strands using vector-contained Sp6 and T7 primer sequences were performed by a commercial service supplier (MedProbe, Norway). The PCR product contained an open reading frame of 642 bp coding for a protein that by sequence similarity searches was recognised to be a GST and probably belonging to the mu-class. The finalised sequence also revealed that the insert was an amplified product containing the AP1 sites on both ends with no sequence corresponding to the lysozyme primer. Although rare, occasionally a template is created with full adaptor sequences in both ends according to the instruction manual of the kit. 2.6. Other methods Protein concentrations were determined by the Pierce BCA Protein Assay (Pierce, Rockford, IL, USA) using bovine serum albumin (BSA) as standard protein. SDS/PAGE was performed in 10% NuPAGE Bis–Tris gel system (Novex, San Diego, CA, USA) using an MES SDS running buffer, and the GST migration length was compared to the Mark12™ protein standard (Novex). Protein in gel was visualised by Coomassie G-250 staining. The EBI-hosted programs Blast-2 (Altschul et al., 1997), InterProScan (Zdobnov and Apweiler, 2001), and ClustalW (Higgins et al., 1994) were used for search of sequence homology and protein motif and for multiple sequence alignments, respectively. Sequence editing for publication quality was performed in GeneDoc (Nicholas et al., 1997). 3. Results and discussion
2.4. Kinetic studies 3.1. Purification of Icelandic scallop glutathione S-transferase The apparent Km value for CDNB was determined using a CDNB range of 0.2–1 mM and a fixed GSH concentration of 1 mM. The apparent Km value for GSH was determined using a GSH range of 0.2–1 mM and a fixed CDNB concentration of 1 mM. Data were plotted as a double-reciprocal Lineweaver– Burk plot to determine the apparent Km.
GST (52.4 units) from digestive gland of scallop was extracted and subjected to GSTrap chromatography yielding
2.5. Identifying a GST coding sequence Isolation of mRNA and subsequent cDNA synthesis from the crystalline style one digestive gland of Icelandic scallop was performed as previously described (Nilsen et al., 1999). In an unrelated project, the synthesised cDNA was used as template for attempted amplification of a second lysozyme gene from scallops using degenerated and supposedly gene-specific primers in combination with the AP1 primer (5′-CCATCCTAATACGACTCACTATAGGGC-3′) corresponding to the adaptor sequence in the Marathon cDNA Amplification Kit
Fig. 1. SDS-PAGE analysis of purified scallop GST. Lanes: M, molecular markers; A, 1.4 μg scallop GST.
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18.7 μg of GST protein. When this purified scallop GST was analysed by SDS-PAGE a single band corresponding to an Mr of ∼ 22 kDa was detected after Coomassie staining (Fig. 1). By Sephadex S 200 gel filtration of an aliquot of the crude extract, the glutathione transferase activity eluted in a single peak having a molecular mass of approximately 44 kDa (data not shown). These results indicate strongly that the scallop GST is a homodimer of 22 kDa subunits. This is in agreement with results obtained from other bivalve GSTs (Fitzpatrick et al., 1995; Yang et al., 2002, 2003, 2004). Heterodimeric forms of GST have been reported in Northern quahog (Blanchette and Signh, 2002). In general, all non-invertebrate GSTs studied are homodimer proteins having subunits which fall within the range of 20 to 27 kDa (Clark, 1989). 3.2. Characterization of scallop GST The specific activity of the isolated C. islandica GST against CDNB was 300.6 μmol/min/mg. This is one of the highest specific activities reported for bivalve GSTs (Table 1). The GSH CDNB apparent Km and Km for scallop GST are 0.3 mM and CDNB 0.4 mM, respectively. The scallop GST has Km values comparable to the other bivalve GSTs listed in Table 1 except for those from Mytilus edulis and Ruditapes decussatus, indicating that these GSTs have similar affinity for the CDNB substrate. The pH optimum of scallop GST catalysed conjugation of CDNB to GSH was determined in sodium acetate and potassium phosphate buffers having an ionic strength of 0.1 (Fig 2A). The results obtained show that the optimum pH for the Table 1 Comparison of the kinetic constants and specific activities with CDNB and GSH as substrates of isolated marine bivalve GSTs GST
KCDNB m
KGSH m
Specific References activity
(mM− 1s− 1) (mM− 1s− 1) (μmol/ min/mg) Chlamys islandica GST (digestive gland) Mytilus edulis GST1 (gills) Mytilus edulis mGST⁎ (hepatopancreas) Atactodea striata asGST (liver and intestine) Asaphis dichotoma adGST (liver and intestine) Corbicula fluminea GST (liver) Ruditapes decussatus GST 6–6 Ruditapes decussatus GST 5–5 Ruditapes decussatus GST 3–3 Mercenaria mercenaria GST ⁎Recombinant.
0.4
0.3
300.6
3.7
0.5
10.1
0.68
0.06
5.9
0.43
0.19
108
0.68
0.11
283
0.59
0.46
2.8
1.75
361
1.65
0.37
86
0.17
4.82
290
0.4
Not tested
2.1
25
This study Fitzpatrick et al., 1995 Yang et al., 2004 Yang et al., 2003 Yang et al., 2002 Vidal and Norbonne, 2000 Hoarau et al., 2002 Hoarau et al., 2002 Hoarau et al., 2002 Blanchette and Signh, 2002
Fig. 2. Effect of pH and temperature on GST. A) Effect of pH on scallop GST activity. GST activity (-O-) were measured in 0.1 M sodium acetate (pH 4–6), 0.04 M potassium phosphate (pH 6.0–8.0). B) Effect of temperature on scallop GST (-O-) and porcine GST (-◊-) stability after 10 min incubation at 0 to 80 °C. Assays were performed at pH 6.5 and results are expressed as relative activities.
scallop GST is pH 6.5–7 and no catalytic activity was detected below pH 4.5. Non-vertebrate GSTs are reported to have pH optima in the range of pH 7 to pH 10 (Clark, 1989). The recently reported recombinant M. edulis GST has maximum catalytic activity at pH 8.5 (Yang et al., 2004). The effect of temperature on the stability of the purified scallop GST and the porcine GST are shown in Fig. 2B. Pre-incubating the enzymes for 10 min at different temperatures showed that the scallop GST is more susceptible to heat denaturation than the porcine enzyme. For the scallop enzyme, 10 min incubation at 22 °C resulted in 20% reduction in enzyme activity. 3.3. Scallop GST coding sequence cDNA synthesised from mRNA of digestive gland was previously used in an effort to amplify a potential second gene to the already isolated lysozyme gene from Icelandic scallop (Nilsen et al., 1999). This produced a seemingly specific PCR product containing a 760 bp sequence that by analysis revealed an open reading frame for a 214 amino acid residues protein (Fig. 3A). BLAST search demonstrated high sequence resemblance of the predicted protein to GSTs from various sources, all scoring around 40% identity and 60% similarity to the scallop
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Fig. 3. Icelandic scallop GST sequence. A) The cDNA (Accession no. AM279651) and its predicted protein sequence. Lower-letter cases denote the open reading frame. B) Sequence alignment of the scallop protein together with selected mu-class GSTs that represent the top scores of 40% identity and 60% similarity in 191 residues or more overlap in homology search. One-letter symbols signify amino acids and positions varying from the majority in the respective sequences. Major conservations (8 out of 10) are indicated by grey shading and total conservations are shaded black. The following protein sequence entries were used: Q6JVNO (Bush tick), Q8MW50 (pork tapeworm), Q4RV52 (green puffer fish), Q28G37 (Western claw frog), Q6FGJ9 (human), Q9BEA9 (Japanese macaque), P46409 (rabbit), Q9NOV4 (bovine), and Q9MZB4 (goat).
protein. All top-scoring known sequences were GSTs of the muclass. InterProScan recognised several GST-characteristic sequence motifs including the N- and C-terminal domains IPR004045 (for glutathione binding) and IPR004046 (for interaction with hydrophobic ligand). The predicted protein has a calculated molecular mass of 24.8 kDa and an isoelectric point of 6.0 with a net charge of minus 1.5. Alignments revealed that the scallop GST sequence in its presented form lacks 10–16 N-terminal residues compared to other published GST proteins (Fig. 3B). A number of amino acids involved in binding of glutathione to various classes of GSTs have been identified (reviewed by Dirr et al., 1994). The alignment shows that the majority of these residues are
conserved in the deduced scallop enzyme sequence, such as Trp-35, Lys-40, Asn-49, Leu-50, and Asp-96. Other residues like Cys-32, Glu-62 and Thr-63 in the scallop sequence replace the normally conserved amino acids arginine, glutamine and serine, respectively. Exclusive to mu-class GSTs, and hence called mu-loop, is an insertion in their sequence compared to other classes of GST (Dirr et al., 1994). In the predicted scallop GST sequence this loop is extended to 9 amino acids (residues 23–31) instead of the 7-residues loop usually seen in mu-GSTs. Unfortunately, attempts to sequence the N-terminal part of the purified scallop GST protein were not successful, and it is therefore not possible to link the cDNA-derived GST sequence to the purified protein and its enzyme activities. The gel
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analyses in Fig. 1 revealed a single protein band indicating little or no GST protein heterogeneity in scallop glands with respect to molecular mass. The same type of organ was the source of mRNA leading to the cDNA used as template for the amplification of a potential mu-class GST coding sequence. Still it is too speculative to claim that the presented cDNA is encoding the purified protein. The putative translation product has a molecular mass of 24.8 kDa (or even higher with complete N-terminus), in contrast to the measured 22 kDa of the purified protein. Thus, this may be an indication for a translation product other than the isolated protein. References Altschul, S.F., Madden, T.L., Schäffer, A.A., Zang, J., Zang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Blanchette, B.N., Signh, B.R., 2002. Isolation and characterization of the glutathione-S-transferase isozyme Q3 from the Northern Quahog Mercinaria mercinaria. J. Protein Chem. 21, 151–159. Board, P.G., Baker, R.T, Chelvanayagan, G., Jermiin, L.S., 1997. Zeta, a novel class of glutathione transferase in a range of species from plant to humans. Biochem. J. 328, 929–935. Board, P.G., Coggan, M., Chelvanaygam, G., Easteal, S., Jermin, L.S., Schulte, G.K., Danley, D.E., Hoth, L.R., Griffor, M.C., Kamath, A., Rosner, M.H., Chrunyk, B.A., Perregwux, D.E., Gabel, C.A., Geoghegan, K.F., Pandit, J., 2000. Identification, characterization, and crystal structure of the Omega class gluthion transferases. J. Biol. Chem. 275, 24798–24804. Clark, A., 1989. The comparative enzymology of the glutathione S-transferase from non-vertebrate organisms. Comp. Biochem. Physiol. B 92, 419–446. Dirr, H., Reinemer, P., Huber, R., 1994. X-ray crystal structures of cytosolic glutathione S-transferases: implications for protein architecture, substrate recognition and catalytic function. Eur. J. Biochem. 220, 645–661. Fitzpatrick, P.J., Sheehan, D., 1993. Separation of multiple forms of glutathione S-transferase from the Blue Mussel, Mytilus edulis. Xenobiotica 23, 851–861. Fitzpatrick, P., Krag, T.O.B., Højrup, P., Sheehan, D., 1995. Characterization of glutathione S-transferase and a related glutathione-binding protein from gill of the blue mussel, Mytilus edulis. Biochem. J. 305, 145–150. Goldberg, E.D., 1975. The mussel watch — a first step in global marine monitoring. Mar. Pollut. Bull. 6, 111. Goldberg, E.D., Bertine, K.K., 2000. Beyond the mussel watch — new directions for monitoring marine pollution. Sci. Total Environ. 247, 165–174. Habig, W.H., Jakoby, W.B., 1981. Assays for differentiation of glutathione S-transferase. Methods Enzymol. 77, 398–405. Hayes, J.D., Wolf, C.R., 1988. In: Sies, H., Ketterer, B. (Eds.), In Glutathion Conjugation: Mechanism and Biological Significances. Academic Press, London, pp. 315–355. Higgins, D., Thompson, J., Gibson, T., Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Hoarau, P., Garello, G., Gnassia-Barelli, M., Romeo, M., Girard, J.P., 2002. Purification and partial characterization of seven glutathione S-transferase
407
isoforms from the clam Ruditapes decussatus. Eur. J. Biochem. 269, 4359–4366. Hoarau, P., Damiens, G., Roméo, M., Gnassia-Barelli, M., Bebianno, M.J., 2006. Cloning end expression of a GST-pi gene in Mytilus galloprovincialis. Attempt to use GST-pi transcript as a biomarker of pollution. Comp. Biochem. Physiol. C 143, 196–203. Keen, J.H., Jakoby, W.B., 1978. Glutathione transferases. Catalysis of nucleophilic reactions of glutathione. J. Biol. Chem. 253, 5654–5657. Mannevik, B., Alin, P., Guthenberg, C., Jenson, H., Tahir, M.K., Warholm, M., Jornvall, H., 1985. Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural and enzymatic properties. Proc. Natl. Acad. Sci. U. S. A. 82, 7202–7206. Mannervik, B., Danielson, U.H., 1989. Glutathione transferases — structure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283–337. Meyer, D.J., Thomas, M., 1995. Characterization of rat spleen prostaglandin H D-isomerase as a sigma-class GSH transferase. Biochem. J. 311, 739–742. Meyer, D.J., Coles, B., Pemble, S.E., Gilmore, K.S., Fraser, G.M., Ketter, B., 1991. Theta, a new class of glutathione transferase purified from rat and man. Biochem. J. 274, 409–414. Moreiro, S.M., Guilhmino, L., 2005. The use of Mytilus galloprovincialis acethylesterase and glutathoon S-transferase activities as biomarkers of environmental contamination along the northwest Portuguese coast. Environ. Monit. Asses. 105, 309–325. Nicholas, K.B., Nicholas Jr., H.B., Deerfield Jr., D.W., 1997. GeneDoc: analysis and visualization of genetic variation. EMBNET NEWS 4, 14. Nilsen, I.W., Øverbø, K., Sandsdalen, E., Sandaker, E., Sletten, K., Myrnes, B., 1999. Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity. FEBS Lett. 464, 153–158. Norrpa, H., 2003. Genetic susceptibility, biomarker response, and cancer. Mutat. Res. 544, 339–348. Pemble, S.E., Wardle, A.F., Taylor, J.B., 1996. Glutathion S-transferase class Kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homolog. Biochem. J. 319, 749–754. Pennec, G.L., Pennec, M.L., 2003. Induction of glutathione-S-transferases in primary cultured digestive gland acini from the mollusc bivalve Pecten maximus (L.): application of a new cellular model in biomonitoring studies. Aquat. Toxicol. 64, 131–142. Sheehan, D., Mead, G., Foley, V.M., Dowd, C.A., 2001. Structure, function and evolution of glutathione transferases: implication for classification of nonmammalian members of an ancient enzyme superfamily. Biochem. J. 360, 1–16. Vidal, M.L., Norbonne, J.F., 2000. Characterization of glutathione S-transferase activity in the Asiatic clam Corbicula fluminea. Bull. Environ. Contam. Toxicol. 64, 455–462. Yang, H.-L., Nie, L., Zhu, S., Zhou, X.-W., 2002. Purification and characterization of a novel glutatione S-transferase from Asaphis dichotoma. Arch. Biochem. Biophys. 403, 202–208. Yang, H.-L., Zeng, Q., Nie, L., Zhu, S., Zhou, X.-W., 2003. Purification and characterization of a novel glutathione S-transferase from Atacodea striata. Biochem. Biophys. Res. Commun. 307, 626–631. Yang, H.-L., Zeng, Q., Nie, L., Zhu, S., Zhou, X.-W., 2004. Molecular cloning, expression and characterization of glutathione S-transferase from Mytilus edulis. Comp. Biochem. Physiol. B 139, 175–182. Zdobnov, E.M., Apweiler, R., 2001. InterProScan — an integration platform for the signature–recognition methods in InterPro. Bioinformatics 17, 847–848.
Glutathione S-transferase from the Icelandic scallop (Chlamys islandica): Isolation and partial characterization Bjørnar Myrnes ⁎, Inge W. Nilsen Marine Biotechnology and Fish Health, Norwegian Institute of Fisheries and Aquaculture, P. O. Box 6122, N-9291 Tromsø, Norway Received 14 August 2006; received in revised form 25 October 2006; accepted 23 November 2006 Available online 29 November 2006
Abstract Glutathione S-transferase from the digestive gland of the cold-adapted marine bivalve Icelandic scallop was purified to apparent homogeneity by single GSTrap chromatography. The enzyme appeared to be a homodimer with subunit Mr 22,000 having an optimum catalytic activity at pH 6.5–7. Enzymatic analysis of scallop GST using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione resulted in apparent values for KGST and KCDNB of 0.3 mM and 0.4 mM, respectively. The scallop GST lost activity faster than porcine GST when exposed to increased m m temperatures, but both enzymes needed 10 min incubation at 60 °C for complete inactivation. A partial coding sequence was identified in cDNA synthesised from digestive gland mRNA. Comparison to known sequences indicates that the gene product is a glutathione S-transferase, and the predicted Icelandic scallop GST protein scores 40% sequence identity and 60% sequence similarity to mu-class proteins. © 2006 Elsevier Inc. All rights reserved. Keywords: Invertebrate; Icelandic scallop; GST; Protein; Coding sequence; Purification
1. Introduction The glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of enzymes which catalyse the conjugation of a broad range of electrophilic substances to glutathione (Keen and Jakoby, 1978; Hayes and Wolf, 1988). The primary function of the enzyme is considered to be the detoxification of both endogenous and xenobiotic alkylating agents. GSTs have been classified into several classes (Alpha, Mu, Pi, Theta, Sigma, Kappa, Zeta and Omega) based on primary structure, immunological and kinetic properties (Mannevik et al., 1985; Meyer et al., 1991; Meyer and Thomas, 1995; Pemble et al., 1996; Board et al., 1997, 2000). The enzymes have been most intensively studied in mammals, but there has also been considerable interest in the role of GST in non-vertebrate organisms (Mannervik and Danielson, 1989; Clark, 1989; Sheehan et al., 2001). GST is a part of the repertoire of adaptive response mechanisms to chemical stress in eukaryotic cells (Norrpa, 2003) and its activity in aquatic organisms has been exploited as a biomarker of pollution (Goldberg, 1975; ⁎ Corresponding author. Tel.: +47 776 2900; fax: +47 776 29100. E-mail address: [email protected] (B. Myrnes). 1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2006.11.011
Goldberg and Bertine, 2000; Pennec and Pennec, 2003; Moreiro and Guilhmino, 2005). Relative to vertebrate systems, little information is available concerning GSTs from marine invertebrate organisms such as molluscs although GST isoforms were isolated and characterized from gills and digestive gland of blue mussel more than a decade ago (Fitzpatrick and Sheehan, 1993; Fitzpatrick et al., 1995). In recent years several glutathione S-transferase protein and gene isoforms from marine shellfishes have been isolated and characterized (Blanchette and Signh, 2002; Hoarau et al., 2002, 2006; Yang et al., 2002, 2003, 2004). In this study we report the isolation and partial characterization of a GST protein and a GST cDNA gene from the Icelandic scallop. 2. Materials and methods 2.1. Materials and biological samples Wild Icelandic scallops Chlamys islandica were collected at 15 m depth from a site in northern Norway. The digestive glands were collected and used as material for enzyme and gene isolation.
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The GSTrap FF (5 mL) chromatography column was supplied by Amersham Pharmacia Biotech. Porcine glutathione S-transferase (G 6636) was obtained from Sigma-Aldrich and all other chemicals used were of analytical grade. 2.2. Purification of scallop glutathione S-transferase Digestive glands from five scallops were homogenized at 4 °C in 250 mL buffer A (10 mM sodium phosphate buffer pH 7.2 containing 0.15 M KCl, 1 mM EDTA and 1 mM dithiothreitol), using a Waring blender. The extract obtained was centrifuged at 30,000 g for 30 min and the supernatant containing GST activity was collected and filtered through Whatman GF/C filter at 4 °C and finally centrifuged for 60 min at 30,000 g. The supernatant (173 mL) containing GST activity was collected and used as crude extract. All further steps in purification of scallop GST were carried out at 6–8 °C. The crude extract was subjected to GSTrap FF chromatography in buffer A in three separate experiments. The column was further washed with buffer A and bound GST eluted with 50 mM Tris– HCl (pH 8), containing 10 mM reduced glutathione. Fractions containing GST activity from these experiments were combined, dialysed against 10 mM Tris–HCl buffer pH 8, followed by concentration on a PM 10 ultrafilter (cut off 10 kDa) using an Amicon Diaflo stirred cell. The fraction obtained (1.1 mL) produced a single Coomassie-stained protein band when analysed after SDS-PAGE and was used as isolated scallop GST. 2.3. Determination of GST activity GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) was measured as described by Habig and Jakoby (1981) using 1 mM CDNB and 1 mM GSH in 0.1 M potassium phosphate buffer pH 6.5. One unit of GST activity is defined as the amount of enzyme that produce 1 μmol S-(2,4 dinitrophenyl)glutathione at 340 nm (ε = 9.6 mM− 1 cm− 1) per min at 24 °C.
(Clontech). PCR reaction mixtures were as recommended in the kit for 3′-RACE. Employing touch-down PCR (5 s denaturation at 95 °C and 90 s annealing/elongation temperatures 72 °C, 70 °C and 68 °C for 5 cycles, 5 cycles and 30 cycles, respectively) produced a single band of approximately 0.9 kb in agarose gel analysis. The amplified product was subcloned by the use of pGEM-T Easy vector system II (Promega) and E. coli JM109 cells for transformation. Recombinant plasmids were analysed by PCR, and DNA sequencing of both strands using vector-contained Sp6 and T7 primer sequences were performed by a commercial service supplier (MedProbe, Norway). The PCR product contained an open reading frame of 642 bp coding for a protein that by sequence similarity searches was recognised to be a GST and probably belonging to the mu-class. The finalised sequence also revealed that the insert was an amplified product containing the AP1 sites on both ends with no sequence corresponding to the lysozyme primer. Although rare, occasionally a template is created with full adaptor sequences in both ends according to the instruction manual of the kit. 2.6. Other methods Protein concentrations were determined by the Pierce BCA Protein Assay (Pierce, Rockford, IL, USA) using bovine serum albumin (BSA) as standard protein. SDS/PAGE was performed in 10% NuPAGE Bis–Tris gel system (Novex, San Diego, CA, USA) using an MES SDS running buffer, and the GST migration length was compared to the Mark12™ protein standard (Novex). Protein in gel was visualised by Coomassie G-250 staining. The EBI-hosted programs Blast-2 (Altschul et al., 1997), InterProScan (Zdobnov and Apweiler, 2001), and ClustalW (Higgins et al., 1994) were used for search of sequence homology and protein motif and for multiple sequence alignments, respectively. Sequence editing for publication quality was performed in GeneDoc (Nicholas et al., 1997). 3. Results and discussion
2.4. Kinetic studies 3.1. Purification of Icelandic scallop glutathione S-transferase The apparent Km value for CDNB was determined using a CDNB range of 0.2–1 mM and a fixed GSH concentration of 1 mM. The apparent Km value for GSH was determined using a GSH range of 0.2–1 mM and a fixed CDNB concentration of 1 mM. Data were plotted as a double-reciprocal Lineweaver– Burk plot to determine the apparent Km.
GST (52.4 units) from digestive gland of scallop was extracted and subjected to GSTrap chromatography yielding
2.5. Identifying a GST coding sequence Isolation of mRNA and subsequent cDNA synthesis from the crystalline style one digestive gland of Icelandic scallop was performed as previously described (Nilsen et al., 1999). In an unrelated project, the synthesised cDNA was used as template for attempted amplification of a second lysozyme gene from scallops using degenerated and supposedly gene-specific primers in combination with the AP1 primer (5′-CCATCCTAATACGACTCACTATAGGGC-3′) corresponding to the adaptor sequence in the Marathon cDNA Amplification Kit
Fig. 1. SDS-PAGE analysis of purified scallop GST. Lanes: M, molecular markers; A, 1.4 μg scallop GST.
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18.7 μg of GST protein. When this purified scallop GST was analysed by SDS-PAGE a single band corresponding to an Mr of ∼ 22 kDa was detected after Coomassie staining (Fig. 1). By Sephadex S 200 gel filtration of an aliquot of the crude extract, the glutathione transferase activity eluted in a single peak having a molecular mass of approximately 44 kDa (data not shown). These results indicate strongly that the scallop GST is a homodimer of 22 kDa subunits. This is in agreement with results obtained from other bivalve GSTs (Fitzpatrick et al., 1995; Yang et al., 2002, 2003, 2004). Heterodimeric forms of GST have been reported in Northern quahog (Blanchette and Signh, 2002). In general, all non-invertebrate GSTs studied are homodimer proteins having subunits which fall within the range of 20 to 27 kDa (Clark, 1989). 3.2. Characterization of scallop GST The specific activity of the isolated C. islandica GST against CDNB was 300.6 μmol/min/mg. This is one of the highest specific activities reported for bivalve GSTs (Table 1). The GSH CDNB apparent Km and Km for scallop GST are 0.3 mM and CDNB 0.4 mM, respectively. The scallop GST has Km values comparable to the other bivalve GSTs listed in Table 1 except for those from Mytilus edulis and Ruditapes decussatus, indicating that these GSTs have similar affinity for the CDNB substrate. The pH optimum of scallop GST catalysed conjugation of CDNB to GSH was determined in sodium acetate and potassium phosphate buffers having an ionic strength of 0.1 (Fig 2A). The results obtained show that the optimum pH for the Table 1 Comparison of the kinetic constants and specific activities with CDNB and GSH as substrates of isolated marine bivalve GSTs GST
KCDNB m
KGSH m
Specific References activity
(mM− 1s− 1) (mM− 1s− 1) (μmol/ min/mg) Chlamys islandica GST (digestive gland) Mytilus edulis GST1 (gills) Mytilus edulis mGST⁎ (hepatopancreas) Atactodea striata asGST (liver and intestine) Asaphis dichotoma adGST (liver and intestine) Corbicula fluminea GST (liver) Ruditapes decussatus GST 6–6 Ruditapes decussatus GST 5–5 Ruditapes decussatus GST 3–3 Mercenaria mercenaria GST ⁎Recombinant.
0.4
0.3
300.6
3.7
0.5
10.1
0.68
0.06
5.9
0.43
0.19
108
0.68
0.11
283
0.59
0.46
2.8
1.75
361
1.65
0.37
86
0.17
4.82
290
0.4
Not tested
2.1
25
This study Fitzpatrick et al., 1995 Yang et al., 2004 Yang et al., 2003 Yang et al., 2002 Vidal and Norbonne, 2000 Hoarau et al., 2002 Hoarau et al., 2002 Hoarau et al., 2002 Blanchette and Signh, 2002
Fig. 2. Effect of pH and temperature on GST. A) Effect of pH on scallop GST activity. GST activity (-O-) were measured in 0.1 M sodium acetate (pH 4–6), 0.04 M potassium phosphate (pH 6.0–8.0). B) Effect of temperature on scallop GST (-O-) and porcine GST (-◊-) stability after 10 min incubation at 0 to 80 °C. Assays were performed at pH 6.5 and results are expressed as relative activities.
scallop GST is pH 6.5–7 and no catalytic activity was detected below pH 4.5. Non-vertebrate GSTs are reported to have pH optima in the range of pH 7 to pH 10 (Clark, 1989). The recently reported recombinant M. edulis GST has maximum catalytic activity at pH 8.5 (Yang et al., 2004). The effect of temperature on the stability of the purified scallop GST and the porcine GST are shown in Fig. 2B. Pre-incubating the enzymes for 10 min at different temperatures showed that the scallop GST is more susceptible to heat denaturation than the porcine enzyme. For the scallop enzyme, 10 min incubation at 22 °C resulted in 20% reduction in enzyme activity. 3.3. Scallop GST coding sequence cDNA synthesised from mRNA of digestive gland was previously used in an effort to amplify a potential second gene to the already isolated lysozyme gene from Icelandic scallop (Nilsen et al., 1999). This produced a seemingly specific PCR product containing a 760 bp sequence that by analysis revealed an open reading frame for a 214 amino acid residues protein (Fig. 3A). BLAST search demonstrated high sequence resemblance of the predicted protein to GSTs from various sources, all scoring around 40% identity and 60% similarity to the scallop
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Fig. 3. Icelandic scallop GST sequence. A) The cDNA (Accession no. AM279651) and its predicted protein sequence. Lower-letter cases denote the open reading frame. B) Sequence alignment of the scallop protein together with selected mu-class GSTs that represent the top scores of 40% identity and 60% similarity in 191 residues or more overlap in homology search. One-letter symbols signify amino acids and positions varying from the majority in the respective sequences. Major conservations (8 out of 10) are indicated by grey shading and total conservations are shaded black. The following protein sequence entries were used: Q6JVNO (Bush tick), Q8MW50 (pork tapeworm), Q4RV52 (green puffer fish), Q28G37 (Western claw frog), Q6FGJ9 (human), Q9BEA9 (Japanese macaque), P46409 (rabbit), Q9NOV4 (bovine), and Q9MZB4 (goat).
protein. All top-scoring known sequences were GSTs of the muclass. InterProScan recognised several GST-characteristic sequence motifs including the N- and C-terminal domains IPR004045 (for glutathione binding) and IPR004046 (for interaction with hydrophobic ligand). The predicted protein has a calculated molecular mass of 24.8 kDa and an isoelectric point of 6.0 with a net charge of minus 1.5. Alignments revealed that the scallop GST sequence in its presented form lacks 10–16 N-terminal residues compared to other published GST proteins (Fig. 3B). A number of amino acids involved in binding of glutathione to various classes of GSTs have been identified (reviewed by Dirr et al., 1994). The alignment shows that the majority of these residues are
conserved in the deduced scallop enzyme sequence, such as Trp-35, Lys-40, Asn-49, Leu-50, and Asp-96. Other residues like Cys-32, Glu-62 and Thr-63 in the scallop sequence replace the normally conserved amino acids arginine, glutamine and serine, respectively. Exclusive to mu-class GSTs, and hence called mu-loop, is an insertion in their sequence compared to other classes of GST (Dirr et al., 1994). In the predicted scallop GST sequence this loop is extended to 9 amino acids (residues 23–31) instead of the 7-residues loop usually seen in mu-GSTs. Unfortunately, attempts to sequence the N-terminal part of the purified scallop GST protein were not successful, and it is therefore not possible to link the cDNA-derived GST sequence to the purified protein and its enzyme activities. The gel
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analyses in Fig. 1 revealed a single protein band indicating little or no GST protein heterogeneity in scallop glands with respect to molecular mass. The same type of organ was the source of mRNA leading to the cDNA used as template for the amplification of a potential mu-class GST coding sequence. Still it is too speculative to claim that the presented cDNA is encoding the purified protein. The putative translation product has a molecular mass of 24.8 kDa (or even higher with complete N-terminus), in contrast to the measured 22 kDa of the purified protein. Thus, this may be an indication for a translation product other than the isolated protein. References Altschul, S.F., Madden, T.L., Schäffer, A.A., Zang, J., Zang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Blanchette, B.N., Signh, B.R., 2002. Isolation and characterization of the glutathione-S-transferase isozyme Q3 from the Northern Quahog Mercinaria mercinaria. J. Protein Chem. 21, 151–159. Board, P.G., Baker, R.T, Chelvanayagan, G., Jermiin, L.S., 1997. Zeta, a novel class of glutathione transferase in a range of species from plant to humans. Biochem. J. 328, 929–935. Board, P.G., Coggan, M., Chelvanaygam, G., Easteal, S., Jermin, L.S., Schulte, G.K., Danley, D.E., Hoth, L.R., Griffor, M.C., Kamath, A., Rosner, M.H., Chrunyk, B.A., Perregwux, D.E., Gabel, C.A., Geoghegan, K.F., Pandit, J., 2000. Identification, characterization, and crystal structure of the Omega class gluthion transferases. J. Biol. Chem. 275, 24798–24804. Clark, A., 1989. The comparative enzymology of the glutathione S-transferase from non-vertebrate organisms. Comp. Biochem. Physiol. B 92, 419–446. Dirr, H., Reinemer, P., Huber, R., 1994. X-ray crystal structures of cytosolic glutathione S-transferases: implications for protein architecture, substrate recognition and catalytic function. Eur. J. Biochem. 220, 645–661. Fitzpatrick, P.J., Sheehan, D., 1993. Separation of multiple forms of glutathione S-transferase from the Blue Mussel, Mytilus edulis. Xenobiotica 23, 851–861. Fitzpatrick, P., Krag, T.O.B., Højrup, P., Sheehan, D., 1995. Characterization of glutathione S-transferase and a related glutathione-binding protein from gill of the blue mussel, Mytilus edulis. Biochem. J. 305, 145–150. Goldberg, E.D., 1975. The mussel watch — a first step in global marine monitoring. Mar. Pollut. Bull. 6, 111. Goldberg, E.D., Bertine, K.K., 2000. Beyond the mussel watch — new directions for monitoring marine pollution. Sci. Total Environ. 247, 165–174. Habig, W.H., Jakoby, W.B., 1981. Assays for differentiation of glutathione S-transferase. Methods Enzymol. 77, 398–405. Hayes, J.D., Wolf, C.R., 1988. In: Sies, H., Ketterer, B. (Eds.), In Glutathion Conjugation: Mechanism and Biological Significances. Academic Press, London, pp. 315–355. Higgins, D., Thompson, J., Gibson, T., Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Hoarau, P., Garello, G., Gnassia-Barelli, M., Romeo, M., Girard, J.P., 2002. Purification and partial characterization of seven glutathione S-transferase
407
isoforms from the clam Ruditapes decussatus. Eur. J. Biochem. 269, 4359–4366. Hoarau, P., Damiens, G., Roméo, M., Gnassia-Barelli, M., Bebianno, M.J., 2006. Cloning end expression of a GST-pi gene in Mytilus galloprovincialis. Attempt to use GST-pi transcript as a biomarker of pollution. Comp. Biochem. Physiol. C 143, 196–203. Keen, J.H., Jakoby, W.B., 1978. Glutathione transferases. Catalysis of nucleophilic reactions of glutathione. J. Biol. Chem. 253, 5654–5657. Mannevik, B., Alin, P., Guthenberg, C., Jenson, H., Tahir, M.K., Warholm, M., Jornvall, H., 1985. Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural and enzymatic properties. Proc. Natl. Acad. Sci. U. S. A. 82, 7202–7206. Mannervik, B., Danielson, U.H., 1989. Glutathione transferases — structure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283–337. Meyer, D.J., Thomas, M., 1995. Characterization of rat spleen prostaglandin H D-isomerase as a sigma-class GSH transferase. Biochem. J. 311, 739–742. Meyer, D.J., Coles, B., Pemble, S.E., Gilmore, K.S., Fraser, G.M., Ketter, B., 1991. Theta, a new class of glutathione transferase purified from rat and man. Biochem. J. 274, 409–414. Moreiro, S.M., Guilhmino, L., 2005. The use of Mytilus galloprovincialis acethylesterase and glutathoon S-transferase activities as biomarkers of environmental contamination along the northwest Portuguese coast. Environ. Monit. Asses. 105, 309–325. Nicholas, K.B., Nicholas Jr., H.B., Deerfield Jr., D.W., 1997. GeneDoc: analysis and visualization of genetic variation. EMBNET NEWS 4, 14. Nilsen, I.W., Øverbø, K., Sandsdalen, E., Sandaker, E., Sletten, K., Myrnes, B., 1999. Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity. FEBS Lett. 464, 153–158. Norrpa, H., 2003. Genetic susceptibility, biomarker response, and cancer. Mutat. Res. 544, 339–348. Pemble, S.E., Wardle, A.F., Taylor, J.B., 1996. Glutathion S-transferase class Kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homolog. Biochem. J. 319, 749–754. Pennec, G.L., Pennec, M.L., 2003. Induction of glutathione-S-transferases in primary cultured digestive gland acini from the mollusc bivalve Pecten maximus (L.): application of a new cellular model in biomonitoring studies. Aquat. Toxicol. 64, 131–142. Sheehan, D., Mead, G., Foley, V.M., Dowd, C.A., 2001. Structure, function and evolution of glutathione transferases: implication for classification of nonmammalian members of an ancient enzyme superfamily. Biochem. J. 360, 1–16. Vidal, M.L., Norbonne, J.F., 2000. Characterization of glutathione S-transferase activity in the Asiatic clam Corbicula fluminea. Bull. Environ. Contam. Toxicol. 64, 455–462. Yang, H.-L., Nie, L., Zhu, S., Zhou, X.-W., 2002. Purification and characterization of a novel glutatione S-transferase from Asaphis dichotoma. Arch. Biochem. Biophys. 403, 202–208. Yang, H.-L., Zeng, Q., Nie, L., Zhu, S., Zhou, X.-W., 2003. Purification and characterization of a novel glutathione S-transferase from Atacodea striata. Biochem. Biophys. Res. Commun. 307, 626–631. Yang, H.-L., Zeng, Q., Nie, L., Zhu, S., Zhou, X.-W., 2004. Molecular cloning, expression and characterization of glutathione S-transferase from Mytilus edulis. Comp. Biochem. Physiol. B 139, 175–182. Zdobnov, E.M., Apweiler, R., 2001. InterProScan — an integration platform for the signature–recognition methods in InterPro. Bioinformatics 17, 847–848.