Molecular and Biochemical Parasitology, 52 (1991) 127-130

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(0 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 01749

Short Communication

Molecular cloning and sequencing of glutathione peroxidase from Schistosoma mansoni D a v i d L. W i l l i a m s 1, R a y m o n d J. Pierce l, E d i t h C o o k s o n 2 a n d A n d r 6 C a p r o n 1 1Centre d'Immunologie et de Biologie Parasitaire, Unit~ Mixte INSERM U167, CNRS URA624, Institut Pasteur, Lille, France; and 2Wolfson Laboratories, Department of Biochemistry. Imperial College of Science, Technology and Medicine, London, UK (Received 21 January 1992; accepted 21 January 1992)

Key words: Schistosoma mansoni; Glutathione peroxidase; cDNA clone; Selenocysteine; Vaccine development; Polymerase chain reaction

The immune system utilises reactive oxygen intermediates in response to many parasitic infections and the ability of parasites to escape oxidative damage has been extensively documented. Bacterial pathogen defence against reactive oxygen species includes the overproduction of antioxidant enzymes such as superoxide dismutase and catalase, the induction of D N A repair systems, scavenging substrates, and competition for molecular oxygen [1]. Protozoan and helminth parasites use a variety of mechanisms to escape the host immune response including attenuation of the respiratory burst of phagocytes [2] and the production of protective anti-oxidant enzymes [3]. We have recently characterised a Schistosoma mansoni glutathione (GSH) S-transferase [4,5], Sm28GST, which is thought to be involved in parasite survival strategies [6]. Significant protection is observed after vaccination with Sm28GST in a number of experimental models [4,7]. In order to increase the efficacy of a Sm28GST vaccine we have Correspondence address: D.L. Williams, Centre d'Immunologie et de Biologie Parasitaire, INSERM U167, CNRS U624, Institut Pasteur, 1 rue Calmette, 59019 Lille Cedex, France. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M86510.

attempted to characterise other GSH-dependent antioxidant enzymes. We report here the cloning and sequencing of S. mansoni glutathione peroxidase (GPx). Mammalian GPx is a selenocysteine-conraining homotetrameric enzyme, which protects essential cellular components against oxidative damage [8]. It catalyses the reduction of hydroperoxides, thereby maintaining the integrity of membrane lipids and DNA, and it reduces the cellular level of hydrogen peroxide. Mammalian GPxs have m o n o m e r subunits of approximately 21 kDa, each of which contains one atom of selinium in the form of a selenocysteine residue. The selenocysteine is cotranslationally incorporated at an inframe T G A codon by a selenocysteylcharged opal suppressor tRNA, t R N A (Ser)Sec [9,10]. GPx activity in S. mansoni increases significantly as worms mature in their host and is positively correlated to the resistance to antioxidants [11-12,13]. S. mansoni also contains a gene for the selenocysteine t R N A (ser)sec [14], suggesting that the S. mansoni GPx may be structurally similar to mammalian GPxs. We therefore sought to clone the S. mansoni GPx using a PCR-based strategy with oligonucleotide primers designed from conserved regions of mammalian [15-17] and Brugia

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Fig. 1. Sequence of S. mansoni GPx. Numbering begins with the first A T G as the translation initiation site. The coding region is in capitals and the noncoding regions in lower case letters. The regions corresponding to the synthetic oligonucleotides used in the PCR amplification are doubly underlined. The 2 oligonucleotides were; WI4, a t g a a g c t t G G N T T Y C C N T G ~ A A Y CARTT, corresponding to amino acids 73 79 of the human sequence [16,17] and W4, ccggacttaARRAAYTTYTC R A A R T T C C A , corresponding to the inverse complement of amino acids 160-167 of the human sequence [16,17]; the bases in capitals represent sequences derived from the mammalian and B. pahangi GPxs. Oligonucleotides were synthesised on a Cyclone Plus D N A Synthesizer (Milligen/Biosearch, France). The T G A in the open reading frame is shown in bold type. The EcoRI at the Y-end of the 2gtl 1 clones is boxed. Nucleotide differences between clones GPx9 and GPxl are starred. The consensus (ALF D N A sequencer) polyadenylation site is underlined.

pahangi (E. Cookson, M.L. Blaxter, and M.E. Selkirk, submitted for publication) GPxs. First-strand c D N A synthesis primed with oligo-dT on 8/tg RNA from adult S. mansoni worms and second strand synthesis and subsequent PCR amplification were performed according to the method of Doherty et al. [18]. After the initial PCR amplification or a secondary amplification using 0.5% of the products, bands at 250 bp and 300 bp and a smear at 400~550 bp were present on agarose gels. These bands were cut out and the D N A was purified with Geneclean (BIO101, La Jolla, CA). The D N A fragments were bluntended with T4 D N A polymerase, cloned into pUC18 and then subcloned into MI3 using standard protocols [19]. The M13 clones were sequenced using the Amersham dideoxy sequencing kit (Amersham, UK). Sequences of the 250-bp band and a clone from the 40(~550bp smear had approximately 40% identity to the published GPx sequences when aligned using the P R O S C A N program on D N A S T A R (DNASTAR, Inc., Madison WI), the size difference being due to amplification artefacts in the longer PCR products. The only proteins in the N B R F / P I R database displaying significant homology to these PCR products were GPxs or GPx-related proteins. The 300-bp product was 100% identical to the Escherichia coli btuE gene product [20], a GPx-like protein, assumed to have resulted from spurious

contamination of the PCR reacUons with E. coli DNA. Clone GPx-1, containing the 250-bp product, was used to screen at high stringency a S. mansoni adult worm 2gtl 1 c D N A library [21]. Forty-three positive clones in 100000 recombinant plaques were initially identified, twelve of which were purified to homogeneity. The 2gtll inserts were amplified using PCR and 2gtll PCR primers (Clontech Laboratories, Palo Alto, CA) isolated on agarose gels, purified with Geneclean and subcloned into M13 for sequencing (A.L.F. D N A sequencer, Pharmacia-LKB, Uppsala). The 650-bp inserts of GPx9 and GPxl 1 were entirely sequenced. Except for two base changes in the 3'noncoding region and one silent change in the coding region these clones contained identical inserts. All 12 clones terminated at the same EcoR! site which was 16 amino acids after the initiating A T G relative to the published sequences of the human GPx. Partial sequencing of a number of other 2gtll GPx clones identified other sequence changes, some resulting in potential variations of GPx amino acid sequences (not shown). Whether these differences are a result of errors introduced during the PCR amplification or from there being several copies of GPx in the S. mansoni genome is currently being investigated. In order to obtain a full-length clone of S.

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region contains one in-frame termination codon T G A at position 142 144. This aligns well with the selenocysteine encoding T G A codon of mammalian GPxs. The protein coding region contains two potential Nglycosylation sites (N-X-S/T) at amino acids 107-109 and 133-135. The S. mansoni GPx protein contains 169 amino acids, somewhat fewer than the defined GPxs, with a predicted molecular weight of 19 600. A comparison with human GPx is shown in Fig. 2. The amino acid sequence of S. mansoni GPx is 38% identical to mammalian and B. pahangi GPxs and the E. coli btuE gene product and 34% identical to the human plasma GPx [22].

mansoni GPx, a S. mansoni adult worm c D N A

library in 2ZAP-II, generated according to manufacturer's protocol (Stratagene, La Jolla, CA), was screened with GPx-1. High-stringency screening identified 200 positives out of 100000 plaques. Six clones were plaque purified and rescued according to the manufacturer's protocol (Stratagene). All 6 clones contained 2 EcoRI fragments of about 50 and 650 bp. Two clones were partially sequenced at their 5'-termini. Both clones contained sequences identical to GPx9 and GPx 11 and extended further 5' of the EcoRI site in the 2gtl 1 clones. Both clones contained an inframe A T G 25 bp upstream of this EcoRI site and either 8 bp (GPxZ42) or 15 bp (GPxZ45) of 5'-noncoding sequence. The sequence of the full-length S. mansoni GPx is shown in Fig. 1. The S. mansoni GPx sequence has a short 5'-noncoding region of 15 bases. An ATG, A = position 1, is followed by an open reading frame of 507 bases which terminates at T G A at position 508-510. This is followed by a 175-base 3'-noncoding region which contains a consensus polyadenylation site 14 bases from the poly(A) tail. The coding

Acknowledgements We thank F. Trottein for the 2gtl 1 library, Dr. J. Khalife and A. Cordeiro da Silva for the 2ZAP-II library, and J.P. Kusnierz for synthesising the oligonucleotides. D.L.W. is supported by a fellowship from the Fondation pour la Recherche Medicale. This work was supported by I N S E R M U167-CNRS URA624, the Edna v20

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McConnell Clark Foundation (Grant No. 11389) and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. References 1 Hassett, D.J. and Cohen, M.S. (1989) Bacterial adaption to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. FASEB J. 3, 2574-2582. 2 Dessaint, J.P. and Capron, A. (1989) Immunodeficiencies in parasitic diseases. Immunodeficiency Rev. 1, 311 324. 3 Callahan, H.L., Crouch, R.K. and James, E.R. (1988) Helminth anti-oxidant enzymes: a protective mechanism against host oxidants? Parasitol. Today 4, 218 225. 4 Balloul, J.M., Sondermeyer, P., Dreyer, D., Capron, M., Grzych, J.M., Pierce, R.J., Carvallo, D., Lecocq, J.P. and Capron A. (1987) Molecular cloning of a protective antigen of schistosomes. Nature 326, 149 153. 5 Taylor, J.B., Vidal, A., Torpier, G., Meyer, D.J., Roitsch, C., Balloul, J.M., Southan, C., Sondermeyer, P., Pemble, S. Lecocq, J.P., Capron, A. and Ketterer, B. (1988) The glutatione transferase activity and tissue distribution of a cloned Mr 28K protective antigen of Schistosoma mansoni. EMBO J. 7, 465472. 6 Mitchell, G.F. (1989) Glutathione S-transferase: potential components of anti-schistosome vaccines? Parasitol. Today 5, 34-37. 7 Boulanger, D., Reid, G.D.F., Sturrock, R.F., Wolowczuk, I., Balloul, J.M., Grezel, D., Pierce, R.J., Otieno, M.F., Guerret, S., Grimaud, J.A. and Butterworth, A.E. (1991) Immunization of mice and baboons with recombinant Sm28GST affects both worm viability and fecundity after experimental infection with Schistosoma mansoni. Parasite Immunol. 13, 473490. 8 Meister, A. (1988) Glutathione metabolism and its selective modification. J. Biol. Chem. 263, 17205 17208. 9 Stadtman, T.C. (1991) Biosynthesis and function of selenocysteine-containing enzymes. J. Biol. Chem. 266, 16257 16260. 10 B6ck, A., Forchhammer, K., Heider, J. and Baron, C. (199l) Selenoprotein synthesis: an expansion of the genetic code. Trends Biochem. Sci. 16, 463-467. 11 Nare, B., Smith, J.M. and Prichard, R.K. (1990) Schistosoma mansoni: levels of antioxidants and resistance to oxidants increase during development. Exp. Parasitol. 70, 389 397.

12 Mkoji, G.M., Smith, J.M. and Prichard, R.K. (1988) Antioxidant systems in Schistosoma mansoni: correlation between susceptibility to oxidant killing and the levels of scavengers of hydrogen peroxide and oxygen free radicals. Int. J. Parasitol. 18, 661 665. 13 Mkoji, G.M., Smith, J.M. and Prichard, R.K. (1988) Antioxidant systems in Schistosoma mansoni: evidence for their role in protection of the adult worms against oxidant killing. Int. J. Parasitol. 18, 667 673. 14 Lee, B.J., Rajagopalan, M., Kim, Y.S., You, K.H., Jacobson, K.B. and Hatfield, D. (1990) Selenocysteine tRNA (sER)sEc gene is ubiquitous within the animal kingdom. Mol. Cell. Biol. 10, 1940 1949. 15 Chambers, I., Frampton, J., Goldfarb, P., Affara, N., McBain, W. and Harrison, P.R. (1986) The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the Xermination' codon, TGA. EMBO J. 5, 1221 1227. 16 Mullenbach, G.T., Tabrizi, A., Irvine, B.D., Bell, G.I. and Hallewell, R.A. (1987) Sequence o f a cDNA coding for human glutathione peroxidase confirms TGA encodes active site selenocysteine. Nucleic Acids Res. 15, 5484. 17 Sukenaga, Y., Ishida, K., Takeda, T. and Takagi, K. (1987) cDNA sequence coding for human glutathione peroxidase. Nucleic Acids Res. 15, 7178. 18 Doherty, P.J., Huesca-Contreras, M., Dosch, H.M. and Pan, S. (1989) Rapid amplification of complementary DNA from small amounts of unfractionated RNA. Anal. Biochem. 177, 7 10. 19 Sambrook, J. Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual (second edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 20 Friedrich, M.J., DeVeaux, L.C. and Kadner, R.J. (1986) Nucleotide sequence of the btuCED genes involved in vitamin B~2 transport in Escherichia coli and homology with components of periplasmic binding proteindependent transport systems. J. Bacteriol. 167, 928 934. 21 Trottein, F., Kieny, M.P., Verwaerde, C., Torpier, G., Pierce, R.J., Balloul, J.-M., Schmitt, D., Lecocq, J.-P. and Capron, A. (1990) Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase. Mol. Biochem. Parasitol. 41, 35 44. 22 Takahashi, K., Akasaks, M., Yamamoto, Y., Kobayshai, C., Mizoguchi, J. and Koyama, J. (1990) Primary structure of human plasma glutathione peroxidase deduced from cDNA sequences. J. Biochem. 108, 145 148.