EXPERIMENTAL PARASITOLOGY 88, 43–50 (1998) ARTICLE NO. PR974217 Dirofilaria immitis: Molecular Cloning and Expression of a cDNA Encoding a Selenium-Independent Secreted Glutathione Peroxidase 1 Cindy Tripp,* ,2 Rexann S. Frank,* Murray E. Selkirk,² Liang Tang,* Marcia M. Grieve,* Glenn R. Frank,* and Robert B. Grieve* *Heska Corporation, 1825 Sharp Point Drive, Fort Collins, Colorado 80525, U.S.A.; ² Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, U.K. Tripp, C., Frank, R. S., Selkirk, M. E., Tang, L., Grieve, M. M., glutathione peroxidse (gp29); Dirofilaria immitis glutathione peroxi- dase cDNA clone (Di29); third stage larvae (L3); fourth stage larvae Frank, G. R., and Grieve, R. B. 1998. Dirofilaria immitis: Molecular cloning and expression of a cDNA encoding a selenium-independent (L4). secreted glutathione peroxidase. Experimental Parasitology 88, 43–50. A cDNA clone, Di29, encoding a homolog of glutathione peroxidase, was isolated from a Dirofilaria immitis adult female cDNA expression library by a combination of polymerase chain reaction amplification with primers designed from the Brugia pahangi glutathione peroxidase INTRODUCTION gene sequence and hybridization screening of D. immitis cDNA librar- ies. The Di29 nucleotide and deduced amino acid sequences were very similar to those described for lymphatic filariae and predicted a secreted form of glutathione peroxidase with a cysteine residue substituted for Most organisms counter oxidative stress via a combination selenocysteine in the active site. The cDNA clone was expressed in of nonenzymatic scavengers and the synthesis of antioxidant Escherichia coli and Spodoptera frugiperda Sf9 insect cells, and the enzymes (Halliwell and Gutteridge 1989). Infectious agents resulting recombinant proteins were purified for antibody production may also be exposed to oxidants derived from host leuko- and assessment of enzymatic properties, respectively. An antiserum generated against the E. coli-expressed protein detected a protein of cytes and appear to have adapted to this challenge by the 29 kDa in D. immitis via immunoblotting. This protein is expressed production of secreted variants of these enzymes. Direct in adult worms (both sexes) and fourth stage larvae generated via 6 evidence for the protective role of antioxidants in infectious days of in vitro culture, but was undetectable in microfilariae, and organisms has been obtained via construction of superoxide third stage larvae obtained either directly from mosquitoes or following dismutase deletion mutants in Brucella abortus, which are 2 days of culture. The Di29-encoded recombinant protein was secreted from Sf9 insect cells and displayed low-level glutathione peroxidase cleared more rapidly from infected mice than wild-type bac- activity against a range of hydroperoxide substrates, including hydrogen teria (Tatum et al. 1992). peroxide.  1998 Academic Press Recently, the major soluble cuticular protein of adult Index Descriptors and Abbreviations: Dirofilaria immitis; nematode; Brugia pahangi was identified as a secreted variant of gluta- heartworm; glutathione peroxidase; antioxidant enzyme; cDNA clone; thione peroxidase (GPx) (Cookson et al. 1992). GPxs cata- deoxyribonucleic acid (DNA); ribonucleic acid (RNA); complementary deoxyribonucleic acid (cDNA); glutathione peroxidase (GPx); glutathi- lyze the reduction of hydroperoxides by glutathione and play one S-transferase (GST); glutathione (GSH); hydrogen peroxide an important role in the metabolism of hydrogen peroxide (H 2 O 2 ); polymerase chain reaction (PCR); spliced leader (SL); 29-kDa (H 2 O 2 ) and protection against oxidative stress. Classical GPx is a tetrameric cytosolic enzyme which contains a selenocys- teine residue in the active site which is involved in redox 1 The sequence data reported herein have been submitted to GenBank catalysis, but this residue is substituted by cysteine in the and assigned the accession numbers U87457 and U87458. 2 To whom correspondence should be addressed. GPx homolog from B. pahangi, termed gp29. Analysis of 43 0014-4894/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.