A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity

Abstract

Production of toxic oxygen metabolites provides a mechanism for microbicidal activity of the neutrophil. The NADPH oxidase enzyme system initiates the production of oxygen metabolites by reducing oxygen to form superoxide anion (O2.-). With stimulation of the respiratory burst, cytosolic oxidase components, p47phox, p67phox, and Rac, translocate to the phagolysomal and plasma membranes where they form a complex with cytochrome b558 and express enzyme activity. A 29-kDa neutrophil protein (p29) was identified by co-immunoprecipitation with p67phox. N-terminal sequence analysis of p29 revealed homology to an open reading frame gene described in a myeloid leukemia cell line. A cDNA for p29 identical to the open reading frame protein was amplified from RNA of neutrophils. Significant interaction between p29 and p67phox was demonstrated using a yeast two-hybrid system. A recombinant (rh) p29 was expressed in Sf9 cells resulting in a protein with an apparent molecular weight of 34,000. The rh-p29 showed immunoreactivity with the original rabbit antiserum that detected p47phox and p67phox. In addition, rh-p29 exhibited PLA2 activity, which was Ca2+ independent, optimal at low pH, and preferential for phosphatidylcholine substrates. The recombinant protein protected glutathione synthetase and directly inactivated H2O2. By activity and sequence homology, rh-p29 can be classified as a peroxiredoxin. Finally, O2.- production by plasma membrane and recombinant cytosolic oxidase components in the SDS-activated, cell-free NADPH oxidase system were enhanced by rh-p29. This effect was not inhibited by PLA2 inhibitors. Thus, p29 is a novel protein that associates with p67 and has peroxiredoxin activity. This protein has a potential role in protecting the NADPH oxidase by inactivating H2O2 or altering signaling pathways affected by H2O2.