Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin [GT], and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically inhibits phorbol myristate acetate (PMA)-stimulated O 2 ؊ generation, while the other two toxins are ineffective. The inhibition is dependent on the disulfide bridge of GT, which interferes with oxidase activation but not catalysis of the activated oxidase. Specifically, GT inhibits PMA-stimulated events: p47 phox phosphorylation, its incorporation into the cytoskeleton, and the membrane translocation of p67 phox , p47 phox , and p40 phox , which are crucial steps in the assembly of the active NADPH oxidase. Thus, damage to p47 phox phosphorylation is likely a key to inhibiting NADPH oxidase activation. GT does not inhibit the membrane translocation of Rac2. The inhibition of p47 phox phosphorylation is due to the defective membrane translocation of protein kinase C (PKC) ␤II rather than an effect of GT on PKC ␤II activity, suggesting a failure of PKC ␤II to associate with the substrate, p47 phox , on the membrane. These results suggest that A. fumigatus may confront PMN by inhibiting the assembly of the NADPH oxidase with its hyphal product, GT.The best-known NADPH oxidase occurs in phagocytes and provides large quantities of reactive oxygen species (ROS) to oxidatively modify microbes as a microbicidal mechanism. The phagocyte NADPH oxidase is a highly regulated multisubunit enzyme composed of p67 phox , p47 phox , p40 phox , and Rac2 in the cytosol, as well as flavocytochrome b 558 , which consists of gp91 phox and p22 phox , in the membrane (4,9,15,29,36,39,57). The enzyme is dormant in resting cells, with these phox (for phagocyte oxidase) components being distributed to their cellular compartments. However, once phagocytes encounter invading microbes or soluble stimulants, such as phorbol myristate acetate (PMA), cytosolic phox components and Rac2 migrate to the membrane to assemble with flavocytochrome b 558 , forming the active enzyme. The activated flavocytochrome b 558 works as the catalytic redox center, where electrons are transferred from NADPH to molecular oxygen to generate O 2 Ϫ , a precursor of ROS. Recently, increasing attention has been directed toward the Nox/Duox family in nonphagocytes (33), which is composed of homologues of gp91 phox . Although the rates of ROS production are quite low in nonphagocytes, their ROS are believed to play important roles in cell signaling, hypoxic response, the immune system, etc.Over the past decade, the biochemistry of phagocyte NADPH oxidase has been intensively studied, particularly concerning the roles of Src homology 3 (SH3) domains and proline-rich regions (PRR), as well as those of other domains recently identified in...

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.

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