Cloning, expression, and characterization of iron-containing superoxide dismutase from Neospora caninum.
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.
Cho
Min-Hee
MH
Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea.
Na
Byoung-Kuk
BK
Song
Kyoung-Ju
KJ
Cho
Jung-Hwa
JH
Kang
Seung-Won
SW
Lee
Kwang-Ho
KH
Song
Chul-Yong
CY
Kim
Tong-Soo
TS
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D018688:Neospora / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D013482:Superoxide Dismutase