Abstract
The assembly of cytosolic subunits p47(phox), p67(phox), and p40(phox) with flavocytochrome b(558) at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47(phox) subunit plays a critical role in oxidase assembly. Recent studies showed that the p47(phox) Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47(phox) PX domain in neutrophils is unclear, we investigated its role using p47(phox) knock-out (KO) mouse neutrophils to express human p47(phox) and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47(phox) proteins were expressed at levels similar to endogenous murine p47(phox), with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47(phox) rescued p47(phox) KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47(phox) with Arg(90) substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47(phox) Arg(90) mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47(phox) PX domain for neutrophil NADPH oxidase activation.
Highlights
The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytic leukocytes plays a key role in innate host defense against bacterial and fungal infections [1,2,3]
These results indicate that Arg90 in p47phox, a residue that plays a critical role in phosphoinositide binding to its Phox homology (PX) domain, is a positive regulator of both phorbol ester- and chemoattractant-induced NADPH oxidase activity on neutrophil plasma membranes
Unlike the p40phox PX domain, which is specific for PI3P [23, 33], the p47phox PX domain binds preferentially to PI[3,4]P2 and can bind with lower affinity to other phosphoinositides [23, 29, 30, 32, 47]
Summary
Reagents and Antibodies—Chemicals were purchased from Sigma-Aldrich unless otherwise stated. Amaxa kit V (Amaxa Biosystems) was used to transiently transfect 2 ϫ 106 K562-gp91/p67Cherry cells with 2 g of pRK5-p40phox and 2 g of pEYFP-N1-p47phox (p47YFP) or mutants [40]. PMA (300 ng/ml) or hIgG-latex beads was used to activate 2 ϫ 105 K562-gp91/p67Cherry cells, co-transfected with p40phox and p47YFP or mutants in the presence of 20 M isoluminol and 20 units/ml horseradish peroxidase (HRP). Live Images by Confocal Video Microscopy—SOZ-induced phagocytosis in p47phox KO neutrophils expressing p47YFP or mutants was filmed using a spinning disk (CSU10) confocal system mounted on a Nikon TE-2000U inverted microscope with an Ixon air-cooled EMCCD camera (Andor Technology, South Windsor, CT) and a Nikon Plan Apo ϫ100 1.4 numerical aperture objective as described previously [28, 40]. Phagosomes were monitored and analyzed at each stage (cup, closure (time of sealing), and postinternalization (200 –300 s)) with this method, including determination of the mean Ϯ S.E
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