Molecular cloning, characterization, and expression in Escherichia coli of iron superoxide dismutase cDNA from Leishmania donovani chagasi.
A cDNA corresponding to superoxide dismutase (SOD; EC 1.15.1.1.) was isolated from a Leishmania donovani chagasi (L. d. chagasi) promastigote cDNA library, using PCR with a set of primers derived from conserved amino acids of manganese SODs (MnSODs) and iron SODs (FeSODs). Comparison of the deduced amino acid sequences with previously reported SOD amino acid sequences revealed that the L. d. chagasi 585-bp open reading frame had considerable homology with FeSODs and MnSODs. The highest homology was shared with prokaryotic FeSODs. The coding region of L. d. chagasi SOD cDNA has been expressed in fusion with glutathione-S-transferase, using an Escherichia coli mutant, QC779, lacking both MnSOD and FeSOD genes (sodA and sodB). Staining of native polyacrylamide gels for SOD activity of Leishmania crude lysate and the recombinant SOD revealed that both had SOD activity that was inactivated by 5 mM hydrogen peroxide but not by 2 mM potassium cyanide, which is indicative of FeSOD. The recombinant enzyme also protected E. coli mutant QC779 from paraquat toxicity. This indicated that the glutathione-S-transferase peptide does not interfere with the in vivo and in vitro activities of the recombinant SOD. Cross-species hybridization showed that FeSOD is highly conserved in the Leishmania genus. Interestingly, the hybridization pattern of the FeSOD gene(s) coincided with other classification schemes that divide Leishmania species into complexes. The cloning of FeSOD cDNA may contribute to the understanding of the role of SODs in Leishmania pathogenesis.
Ismail
S O
SO
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Skeiky
Y A
YA
Bhatia
A
A
Omara-Opyene
L A
LA
Gedamu
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D018314:Leishmania infantum / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010269:Paraquat / Q000633:toxicity; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D013482:Superoxide Dismutase; D010269:Paraquat