Characterization of a cDNA of Peroxiredoxin II Responding to Hydrogen Peroxide and Phagocytosis in Amoeba proteus

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Abstract
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.