A gene encoding superoxide dismutase (SOD) from Neospora caninum, a causative agent of neosporosis, has been cloned and its gene product functionally expressed and characterized. The gene had an open reading frame of 606 bp and deduced 201 amino acids. Sequence analysis showed that the gene had conserved metal-binding residues and conserved amino acid residues that were found in Fe-SODs. Comparison of the deduced amino acid sequence of the enzyme with previously reported Fe-SOD amino acid sequences of the other parasitic protozoans revealed significant high homology. The coding region of the N. caninum Fe-SOD was cloned and functionally expressed in Escherichia coli. Enzyme activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide and potassium cyanide, and the enzyme showed similar biochemical properties with typical Fe-SODs of other parasitic protozoans. Southern blot analysis showed that the SOD gene appears to be present as a single-copy gene in N. caninum genome. Semiquantitative reverse transcription–polymerase chain reaction and immunoblot using antiserum raised against the purified recombinant protein showed that Fe-SOD is expressed in both developmental stages of N. caninum, i.e., in bradyzoites and tachyzoites. In an immunofluorescence assay, the enzyme was localized on the cell surface of N. caninum tachyzoites. These results suggest that Fe-SOD might be essential for the intracellular survival of N. caninum and may play an important role in the pathogenesis of the parasite by protecting the parasite from oxidative killing.
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April 2004
Research Article|
April 01 2004
CLONING, EXPRESSION, AND CHARACTERIZATION OF IRON-CONTAINING SUPEROXIDE DISMUTASE FROM NEOSPORA CANINUM
Min-Hee Cho;
Min-Hee Cho
Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea. nihkim@nih.go.kr
* These authors have contributed equally in this study
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Byoung-Kuk Na;
Byoung-Kuk Na
Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea. nihkim@nih.go.kr
* These authors have contributed equally in this study
† Present address: Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94143-0811
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Kyoung-Ju Song;
Kyoung-Ju Song
Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea. nihkim@nih.go.kr
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Jung-Hwa Cho;
Jung-Hwa Cho
Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea. nihkim@nih.go.kr
‡ Department of Tropical and Endemic Parasitic Diseases, National Institute of Health, Seoul 122-701, Korea
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Seung-Won Kang;
Seung-Won Kang
Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea. nihkim@nih.go.kr
§ National Veterinary Research and Quarantine Service, Anyang 430- 016, Korea
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Kwang-Ho Lee;
Kwang-Ho Lee
Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea. nihkim@nih.go.kr
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Chul-Yong Song;
Chul-Yong Song
Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea. nihkim@nih.go.kr
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Tong-Soo Kim
Tong-Soo Kim
Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea. nihkim@nih.go.kr
‡ Department of Tropical and Endemic Parasitic Diseases, National Institute of Health, Seoul 122-701, Korea
∥ To whom correspondence should be addressed
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J Parasitol (2004) 90 (2): 278–285.
Citation
Min-Hee Cho, Byoung-Kuk Na, Kyoung-Ju Song, Jung-Hwa Cho, Seung-Won Kang, Kwang-Ho Lee, Chul-Yong Song, Tong-Soo Kim; CLONING, EXPRESSION, AND CHARACTERIZATION OF IRON-CONTAINING SUPEROXIDE DISMUTASE FROM NEOSPORA CANINUM. J Parasitol 1 April 2004; 90 (2): 278–285. doi: https://doi.org/10.1645/GE-3222
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