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 (O(2)()). With stimulation of the respiratory burst, cytosolic oxidase components, p47(phox), p67(phox), and Rac, translocate to the phagolysomal and plasma membranes where they form a complex with cytochrome b(558) and express enzyme activity. A 29-kDa neutrophil protein (p29) was identified by co-immunoprecipitation with p67(phox). 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 p67(phox) 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 p47(phox) and p67(phox). In addition, rh-p29 exhibited PLA(2) activity, which was Ca(2+) independent, optimal at low pH, and preferential for phosphatidylcholine substrates. The recombinant protein protected glutathione synthetase and directly inactivated H(2)O(2). By activity and sequence homology, rh-p29 can be classified as a peroxiredoxin. Finally, O(2)() 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 PLA(2) 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 H(2)O(2) or altering signaling pathways affected by H(2)O(2).
Highlights
Production of oxygen metabolites is critical to the function of the neutrophil
A 29-kDa Protein Associated with p67phox Enhances NADPH Oxidase Activity has been demonstrated by a variety of studies with intact neutrophils and cell-free systems activated by anionic detergents (9 –11)
MJ33 (2 M), the inhibitor of the nonclassic PLA2, had a minimal inhibitory effect (% inhibition, 17.5 Ϯ 5.9, mean Ϯ S.E., n ϭ 5). These results suggested that rh-p29 affects the NADPH oxidase through its peroxiredoxin activity
Summary
O2., superoxide anion; DTT, dithiothreitol; NBD PC, 1-o-palmitoyl-2-(N-4-nitrobenzo-2-oxo-1,3-diazole)aminododecanoyl phosphatidylcholine; NBD PE, 1-o-palmitoyl-2-(N-4-nitrobenzo-2-oxo-1,3-diazole)aminododecanoyl phosphatidylethanolamine; MJ33, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol; PLA2, phospholipase A2; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; RT, reverse transcriptase; rh, recombinant human. A 29-kDa Protein Associated with p67phox Enhances NADPH Oxidase Activity has been demonstrated by a variety of studies with intact neutrophils and cell-free systems activated by anionic detergents (9 –11). This 29-kDa protein was first detected in immunoprecipitation studies suggesting an interaction with p67phox in cytosol. In the SDS cell-free system of oxidase activation, rh-p29 enhanced O2 consumption and O2. In the SDS cell-free system of oxidase activation, rh-p29 enhanced O2 consumption and O2. production suggesting that it may play a critical role in regulating toxic oxygen metabolites and, perhaps, protecting the oxidase complex from oxidative damage
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