Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase

Figures & data

Figure 1. Effect of ROS on PAD activity. (A) Microtiter wells were coated with human fibrinogen and incubated for 3 h at room temperature with rhPAD2 (300 ng/ml) or rhPAD4 (3000 ng/ml) in 100 mM Tris-HCl including 15 mM reduced glutathione (GSH) and 5 mM CaCl₂. (B) Supernatants from unstimulated leukocytes or leukocytes stimulated with PMA for 30 min. were added 1:1 to wells containing rhPAD2 (300 ng/ml) in 100 mM Tris-HCl including various concentrations of glutathione (GSH) and 5 mM CaCl₂. In both experiments, mAb 20B2 recognizing a citrullinated epitope on fibrinogen was used as probe. PAD activity is shown as per cent of maximal activity for each isoform, expressed as mean and range of duplicate measurements.

Figure 2. Influence of oxidation on PAD activity and amount of PAD2 released from activated leukocytes. (A) Bars show the median fluorescence intensity (MFI) of rhodamine-123 by CD15-positive, live (NIR-negative) granulocytes from two independent experiments. Leukocytes from two donors resuspended in RPMI medium containing 5% normal human serum were left unstimulated or were stimulated with PMA in the absence or presence of the NADPH oxidase inhibitor DPI. (B) Microtiter wells were coated with human fibrinogen and incubated for 3 h with PMA-stimulated leukocytes in the absence or presence of DPI, and mAb 20B2 was used as probe for citrullination. The average of duplicate measurements for each of eight donors is shown. (C) The corresponding concentration of PAD2 in the supernatants is shown.

Figure 3. PAD activity and PAD2 release in stimulated leukocytes in the presence of H₂O₂. (A) Leukocytes were exposed to various concentration of H₂O₂ in the absence and presence of DPI. Histograms show the median fluorescence intensity (MFI) of rhodamine-123 in CD15-positive, live (NIR-negative) granulocytes. (B) Microtiter wells coated with human fibrinogen were incubated for 3 h at room temperature with unstimulated or PMA-stimulated leukocytes resuspended in RPMI medium containing 5% normal human serum with or without DPI and various concentrations of H₂O₂. Citrullination of fibrinogen was measured using mAb 20B2 as detecting antibody. Bars and error bars show mean and SEM of experiments involving four healthy donors. (C) PAD2 concentrations in the corresponding cell supernatants are shown.