Molecular characterization of peroxiredoxin from Entamoeba moshkovskii and a comparison with Entamoeba histolytica.
Peroxiredoxin of the pathogenic parasite, Entamoeba histolytica, is thought to be involved in protection from oxidative attack by host phagocytic cells and endogenously generated hydrogen peroxide. In this study, we cloned peroxiredoxin genes from the nonpathogenic ameba, Entamoeba moshkovskii, and characterized the peroxiredoxin protein. The open reading frame of three cloned cDNAs was demonstrated to encode a polypeptide of 218 or 217 amino acids. Identity of the amino acid sequence of peroxiredoxins between E. moshkovskii and E. histolytica was considerably high (77-81%), but the N-terminus portion of E. moshkovskii peroxiredoxin was shorter than that of E. histolytica. A recombinant peroxiredoxin of E. moshkovskii expressed in Escherichia coli exhibited hydrogen peroxidase activity. Its K(m) and V(max) values of 35 microM and 0.07 micromol/min/mg protein were approximately 1 and 1.5 times greater than E. histolytica peroxiredoxin, respectively. In addition, the protective effect of E. moshkovskii peroxiredoxin against oxidative-nicking of supercoiled plasmid DNA was shown to be greater than that of E. histolytica peroxiredoxin. Confocal laser scanning microscopy, using polyclonal antibody against the recombinant E. moshkovskii peroxiredoxin, demonstrated that this protein was localized in the nucleus and cytoplasm of trophozoites, supporting its function as a protectant against DNA damage. Southern blot and real-time reverse transcription PCR analyses of the E. moshkovskii peroxiredoxin gene demonstrated that it was a multi-copy gene and its expression was comparable to that of E. histolytica. These results suggest that the antioxidant peroxiredoxin is important for protection against endogenously generated hydrogen peroxide in the nonpathogenic ameba.
Cheng
Xun-Jia
XJ
Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.
Yoshihara
Eisaku
E
Takeuchi
Tsutomu
T
Tachibana
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D018076:DNA, Complementary; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D004747:Entamoeba / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D005786:Gene Expression Regulation; D006861:Hydrogen Peroxide / Q000378:metabolism; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D054464:Peroxiredoxins; D012333:RNA, Messenger / Q000032:analysis; D016053:RNA, Protozoan / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases; D054464:Peroxiredoxins