Characterization of superoxide overproduction by the D-LoopNox4-Nox2 cytochrome b558 in phagocytes—Differential sensitivity to calcium and phosphorylation events

NADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound cytochrome b558, the redox core, is assembled with cytosolic p47phox, p67phox, p40phox, and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from FAD to O2. Moreover, replacement of this loop by the Nox4-D-loop (D-loopNox4-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca2+ influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated cytochrome b558 was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loopNox4-Nox2 mutant may come from a change of responsiveness to intracellular Ca2+ level and to phosphorylation events during oxidase activation. Finally the D-loopNox4-Nox2-PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step.