Characterization of a cDNA of peroxiredoxin II responding to hydrogen peroxide and phagocytosis in Amoeba proteus.
Phagocytic cells have defense systems against reactive oxygen species generated as the first non-specific defense mechanism against invading pathogens or microorganisms. We cloned a cDNA encoding a 21.69-kDa protein in Amoeba proteus homologous to 2-Cys peroxiredoxin (Prx-Ap). In the disk inhibition assay using H2O2 as an oxidizing agent, Escherichia coli overproducing Prx-Ap showed better viability than did E. coli transformed with pBluescript II SK for control. Monoclonal antibodies (mAb) produced against Prx-Ap reacted with a 22.5-kDa protein and several minor proteins. In Western blot analysis, levels of the 22.5-kDa protein in amoebae treated with 2-mM H2O2 for 1 h increased about 2-fold over those in control cells. Immunofluorescence scattered throughout the cytoplasm also increased after H2O2 treatment. In Northern blot analysis using the cDNA as a probe, the level of transcripts also changed with H2O2 treatment. When amoebae were fed with Tetrahymena, the intensity of immunofluorescence increased from 15 min and persisted until 2 h after phagocytosis. These results suggest that the 22.5-kDa protein of A. proteus is a Prx protein and that it has an antioxidant property responding to phagocytosis.
Park
Miey
M
Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Shin
Hae J
HJ
Lee
Soo Y
SY
Ahn
Tae I
TI
D000595:Amino Acid Sequence; D000656:Amoeba / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000911:Antibodies, Monoclonal; D000975:Antioxidants; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D018076:DNA, Complementary; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D054464:Peroxiredoxins; D010587:Phagocytosis; D015800:Protozoan Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis
D000900:Anti-Bacterial Agents; D000911:Antibodies, Monoclonal; D000975:Antioxidants; D018076:DNA, Complementary; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; D010544:Peroxidases; D054464:Peroxiredoxins