Antioxidant Effects of Neutrophil p29 Peroxiredoxin.

Production of reactive oxygen species through a respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of the components of the NADPH oxidase enzyme system (p47phox, p67phox, p40phox, gp91phox, p22phox and Rac) and expression of the activity of the system to produce superoxide anion (O^{2 −}). We recently identified a neutrophil protein with an approximate MW of 29 kDa which binds to the p67phox, is classified as a peroxiredoxin (Prx) and translocates to the plasma membrane during stimulation of the neutrophil. Additional studies demonstrate that this protein (p29 Prx) increases production of O^{2 −} in a cell-free system of oxidase activity in a specific, stoichiometric manner and that the cysteine residues of p29 Prx, at amino acid positions 47 and 91, are required for this activity. The current studies demonstrate the antioxidant activity of p29 Prx using the technique of small interfering RNA (siRNA) to degrade specific mRNA and decrease the expression of the protein. siRNA probes for p29 Prx were constructed based on standard constraints for unique 19 nucleotide binding sites along with other sequence selection criteria. Nine probes were constructed based on the cDNA sequence of p29 Prx; one resulted in significant knockdown of p29 Prx. A vector for green fluorescent protein was obtained commercially. U293 cell line was cultured under standard conditions and expressed p29 Prx message by RT-PCR and protein by Western blot. U293 cells were transfected with siRNA or GFP with calcium chloride. Cell counts and viability were also determined by standard techniques. For Western blots, proteins from cell lysates were separated on 10% SDS-PAGE and blotted onto nitrocellulose, and specific proteins were detected with polyclonal antibodies to p29 Prx, lamin or actin by chemiluminescent technique. U293 cells were transfected with the siRNA for p29 Prx and the vector for GFPb and the cells were harvested at 24, 48, and 72 hours. A knockdown by one of the 6 siRNAs resulted in decreased levels of p29 Prx by Western blot. Optimum knockdown was achieved by transfection of 1 μg siRNA and the decrease in p29 Prx observed at 24 hours but was maximum at 48 and 72 hours. Greater than 90% transfection was achieved documented by green fluorescence of the cells. Under these conditions, a decrease in p29 Prx >90% detected by Western blot was achieved with no differences in levels of actin or lamin in the cell lysates. In separate experiments, U293 cells were transfected with siRNA for p29 Prx and the vector for GFP and exposed to an oxidant stress (paraquot, 100 μM). The level of p29 Prx, actin, and lamin; cell counts; and viability were determined. Cells transfected with siRNA had lower p29 Prx (<10%) but not actin or lamin. The viability of control cells and siRNA transfected cells after 48 hours was no different (91% and 94%, respectively). Paraquot decreased the viability of nontransfected cells (68%), and knockdown of p29 Prx in the presence of paraquot resulted in poorer viability (49%). The number of cells harvested was slightly decreased in the paraquot treated groups but not in the untreated control or siRNA groups. These results suggest an antioxidant effect of p29 Prx in the cell and support its role in protecting the oxidase enzyme system in the neutrophil.