NADPH binding component of neutrophil superoxide-generating oxidase

The 2',3'-dialdehyde derivative of NADPH was used as an affinity labeling reagent of a solubilized NADPH-dependent superoxide-generating oxidase preparation of pig neutrophils. The analogue served as both an electron donor and a competitive inhibitor of the NADPH oxidase against NADPH. The apparent Michaelis constant (Km) for the derivative (31 microM) was essentially the same as that for NADPH (33 microM). The activity of the superoxide formation in the presence of 2',3'-dialdehyde NADPH was about a half of that in the presence of NADPH. Incubation of the enzyme with the derivative inactivated the superoxide-generating activity and the inactivation was prevented by the addition of NADPH. We performed the labeling of the oxidase preparation with 2',3'-dialdehyde NADPH and sodium cyanoboro[3H]hydride, based on the above results. A protein of 66,000 daltons was selectively labeled among more than 20 bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were visualized with Coomassie Brilliant Blue. The protein was not labeled when the oxidase preparation was pretreated with p-chloromercuribenzoate or it was labeled in the presence of excess NADPH. The protein is suggested to be the NADPH binding component of the neutrophil superoxide-generating oxidase system.     

NOX3, a superoxide-generating NADPH oxidase of the inner ear

Reactive oxygen species (ROS) play a major role in drug-, noise-, and age-dependent hearing loss, but the source of ROS in the inner ear remains largely unknown. Herein, we demonstrate that NADPH oxidase (NOX) 3, a member of the NOX/dual domain oxidase family of NADPH oxidases, is highly expressed in specific portions of the inner ear. As assessed by real-time PCR, NOX3 mRNA expression in the inner ear is at least 50-fold higher than in any other tissues where its expression has been observed (e.g. fetal kidney, brain, skull). Microdissection and in situ hybridization studies demonstrated that NOX3 is localized to the vestibular and cochlear sensory epithelia and to the spiral ganglions. Transfection of human embryonic kidney 293 cells with NOX3 revealed that it generates low levels of ROS on its own but produces high levels of ROS upon co-expression with cytoplasmic NOX subunits. NOX3-dependent superoxide production required a stimulus in the absence of subunits and upon co-expression with phagocyte NADPH oxidase subunits p47(phox) and p67(phox), but it was stimulus-independent upon co-expression with colon NADPH oxidase subunits NOX organizer 1 and NOX activator 1. Pre-incubation of NOX3-transfected human embryonic kidney 293 cells with the ototoxic drug cisplatin markedly enhanced superoxide production, in both the presence and the absence of subunits. Our data suggest that NOX3 is a relevant source of ROS generation in the cochlear and vestibular systems and that NOX3-dependent ROS generation might contribute to hearing loss and balance problems in response to ototoxic drugs.     

NADPH oxidase involvement in cellular integrity

NADPH oxidase activity is involved in plant adaptation and development. The reactive oxygen species sourced by NADPH oxidase activity may contribute to wall strength and protoplast volume adjustment. Root hair bulge apices of the NADPH oxidase mutant rhd2/Atrbohc were more robust than the kjk cellulose synthase mutant, but burst more readily than the wild type (WT). Root epidermal wall appeared impaired in rhd2/Atrbohc, as revealed by the number of protoplasts released by wall-degrading enzymes. Root hair bulges of rhd2/Atrbohc burst more than the WT when challenged in situ with hypo-osmotic low ionic strength medium. Inhibition of NADPH oxidase activity with diphenylene iodonium caused WT to phenocopy the rhd2/Atrbohc bursting in response to hypo-osmotic shock. This implicates RHD2/AtRBOHC in softening the cell wall to permit protoplast expansion. Overall, the results point to a role for RHD2/AtRBOHC in contributing to wall strength.     

Fungal gliotoxin targets the onset of superoxide-generating NADPH oxidase of human neutrophils

Gliotoxin from Aspergillus, bearing a S&bond;S bond in its structure, prevented the onset of O(-)(2) generation by the human neutrophil NADPH oxidase in response to phorbol myristate acetate (PMA). Gliotoxin affected the activation process harder than the activated oxidase, as shown by its stronger inhibition when added to neutrophils prior to, than post-PMA at maximum enzyme turnover. Decreased O(-)(2) generation persisted even if cells treated with gliotoxin were subsequently washed, with half-inhibition concentrations (IC(50)) of 5.3, and 3.5 microM for treatments of 15 and 30 min, respectively. In addition, gliotoxin made neutrophils reduce cytochrome c regardless of absence of PMA, through its reaction with intracellular reductants in an oxygen-dependent process, named redox cycling. Thus, we next tested whether preincubation of neutrophils with gliotoxin under hypoxic conditions would relieve the inhibition of NADPH oxidase. Instead, this prevention of redox cycling significantly favored damage to the NADPH oxidase with an IC(50) of 0.009 microM. Moreover, conversion of gliotoxin to its dithiol derivative by addition of reduced dithiothreitol during incubation protected cells from losing oxidase activity. These findings support that the disulfide form of gliotoxin targets NADPH oxidase activation.     

Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1

Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22(phox) at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22(phox): the Noxa1-Noxo1 and Noxo1-p22(phox) interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.     

NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system

The superoxide-forming NADPH oxidase of human neutrophils was studied in subcellular fractions of unstimulated cells. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions: alpha, azurophil granules; beta, mostly specific granules; gamma, plasma membrane, and cytosol. NADPH-dependent O2-. formation by these fractions was quantitated as the rate of superoxide dismutase-inhibitable reduction of ferricytochrome c. In the presence of cytosol, NADPH, and either arachidonic acid (optimum 90 microM) or sodium dodecyl sulfate (optimum 160 microM), 70-75% of the oxidase was in the beta fraction and about 25% was in the gamma fraction. A similar distribution was found for cytochrome b559 and FAD, two putative components of the oxidase. The reaction rates observed with arachidonic acid activation were sufficient to account for 25-75% of the O2-. generated by intact neutrophils. The properties of the beta and gamma enzymes were similar and closely resembled those of the oxidase in intact neutrophils or disrupted prestimulated cells. These included resistance to azide and cyanide, a pH optimum of 7.4, and a preference for NADPH (Km approximately 40-45 microM) rather than NADH (Km approximately 2.5 mM) as the electron donor. The combination of beta and gamma fractions displayed additive activity. The activatable oxidase required Mg2+ but not Ca2+. ATP was required for maximum reaction rates. When beta and gamma membranes were preincubated with cytosol and arachidonic acid in the presence of millimolar Mg2+ and then ultracentrifuged membrane-bound O2-. -forming activity was recovered in the pellet and the enzyme required only NADPH (i.e. no cytosol, arachidonic acid, or Mg2+) for expression of activity. These data suggest that cytosol contains a Mg2+-dependent oxidase-activating factor. Molecular sieve chromatography of cytosol indicated a single peak of activity (i.e. ability to activate O2-. generation by beta and/or gamma fraction) eluting with molecules of about 10,000 daltons.     

NADPH oxidase activation and assembly during phagocytosis

Generation of superoxide (O2-) by the NADPH-dependent oxidase of polymorphonuclear leukocytes is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB), and compared it with that elicited by serum-opsonized zymosan (OPZ). Opsonized N. meningitidis- and OPZ-dependent generation of reactive oxygen species by polymorphonuclear leukocytes peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of reactive oxygen species by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal colocalization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). Termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane- and/or phagosome-associated p47phox; in contrast, the specific activity of phosphorylated p47phox at the phagosomal membrane increased. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and OPZ phagosomes despite the continued presence of flavocytochrome b. These data suggest that 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome; and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes and additional phosphorylation of membrane-associated p47phox.     

NADPH oxidase involvement in the pathology of Helicobacter pylori infection

Neutrophil oxidants are hypothesized to damage the gastric mucosa and promote carcinogenesis in people infected with Helicobacter pylori. To investigate this process we used wild-type and chronic granulomatous disease (CGD) mice with a targeted disruption of the gp91(phox) subunit of the NADPH oxidase. The mice were inoculated with a mouse-adapted strain of H. pylori and changes in gastric pathology were examined 12 and 30 weeks after infection. Glandular atrophy, a precursor lesion in the development of intestinal-type gastric carcinoma, and epithelial cell proliferation were both dramatically increased in the gastric body of CGD animals within 12 weeks. This correlated strongly with increased numbers of neutrophils in the mucosa (Pearson coefficient 0.97, P < 0.001). H. pylori is a noninvasive bacterium, and there was no increase in bacterial numbers in the CGD animals. Closer examination of the gastric tissue indicated the presence of degenerate neutrophils associated with atrophy. We hypothesize that the release of granule constituents from these neutrophils contributes to tissue damage. This is exacerbated by the absence of a functional NADPH oxidase, and is consistent with this enzyme complex having an important role in dampening the inflammatory response.     

Glucocerebroside inhibits NADPH oxidase activation in cell-free system

We reported earlier that monocytes and macrophages from patients with type I Gaucher disease have a decreased capacity to generate superoxide anion (O(2)(-)) on stimulation with opsonized S. aureus or formyl-methionyl-leucyl-phenylalanine. In this study, various forms of the cell-free assay system were used to probe the hypothesis that glucocerebroside (GC) accumulating in Gaucher patients' phagocytes may interfere with the activation of NADPH oxidase. Xanthine/xanthine oxidase assay was applied to explore the possibility that GC may scavenge O(2)(-). We found that addition of GC to the crude, semirecombinant or fully purified cell-free systems inhibited activation of NADPH oxidase in a concentration-dependent manner. The inhibitory effect of GC could be overcome by increased concentrations of p47(phox) and p67(phox). In contrast, O(2)(-) generation was not decreased by GC added to the assembled, catalytically active enzyme complex. In the xanthine/xanthine oxidase system, GC had no effect on the generation of O(2)(-). These data indicate that assembly of the respiratory burst oxidase of phagocytic cells may be a possible target of the pathologic actions of GC.     

Purification and characterization of a third cytosolic component of the superoxide-generating NADPH oxidase of macrophages.

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes in a cell-free system by anionic amphiphiles requires the participation of both membrane and cytosolic components. We reported that ammonium sulfate fractionation (Pick, E., Kroizman, T., and Abo, A. (1989) J. Immunol. 143, 4180-4187) and affinity chromatography on 2',5'-ADP-agarose (Shaag, D., and Pick, E. (1990) Biochim. Biophys. Acta 1037, 405-412) permit separation of cytosol in two fractions (sigma 1 and sigma 2) that support O2- production by solubilized membrane synergistically. We now describe the purification of sigma 1 to near homogeneity and demonstrate that it represents a cytosolic component distinct from p47-phox and p67-phox, that are both found in fraction sigma 2. Sigma 1 was absolutely required for the full expression of amphiphile-activated NADPH-oxidase activity. This requirement was evident whether sigma 1 was added to cell-free systems composed of: (a) solubilized membrane and a sigma 2-enriched cytosolic fraction, or (b) purified cytochrome b559, incorporated in liposomes, and purified sigma 2. Sigma 1 was purified by a sequence comprising ammonium sulfate fractionation, hydrophobic chromatography on phenyl-Superose, absorption with CM-Sepharose, anion exchange chromatography on DEAE-Sepharose, and gel filtration on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sigma 1 of maximal purity, under both reducing and nonreducing conditions, demonstrated the presence of two proteins, of 24 and 22 kDa. On gel filtration, sigma 1 was eluted as a symmetrical peak of 46 kDa that by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the presence of both 24- and 22-kDa bands. We suggest that, in its native form, sigma 1 might represent a complex of the 24- and 22-kDa proteins. The specific roles of each molecule in NADPH oxidase function remain to be determined.     

The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel.

The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.     

Reconstitution of the partially purified membrane component of the superoxide-generating NADPH oxidase of pig neutrophils with phospholipid

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin-agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 +/- 0.53 nmol/mg protein (mean +/- SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0-7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 +/- 3.5 mumol O2-.min-1.mg-1 membrane-derived protein (mean +/- SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids.     

A new superoxide-generating oxidase in murine osteoclasts

Superoxide production contributes to osteoclastic bone resorption. Evidence strongly indicates that NADPH oxidase is an enzyme system responsible for superoxide generation in osteoclasts. A membrane-bound subunit, p91, is the catalytic domain of NADPH oxidase. However, osteoclasts from p91 knockout mice still produce superoxide at a rate similar to that observed in wild type mice. This unexpected phenomenon prompted us to examine the osteoclasts for an alternative to the p91-containing oxidase. In this study, the cloning of a NADPH oxidase subunit (Nox 4) with 578 amino acids is reported. Nox 4 has 58% similarity in amino acids with the known p91 subunit of NADPH oxidase. Nox 4 is present and active in osteoclasts. Antisense oligonucleotides of Nox 4 reduced osteoclastic superoxide generation as well as resorption pit formation by osteoclasts. This new oxidase complex was present and functional in osteoclasts from p91 knockout mice, explaining the normal resorptive activity seen in the osteoclasts where no p91 is present.     

Activation of the superoxide-generating NADPH oxidase of intestinal lymphocytes produces highly reactive free radicals from sulfite.

The objective of this study was to investigate the ability of immune cells of the small intestine to produce highly reactive free radicals from the food additive sulfites. These free radicals were characterized with a spin-trapping technique using the spin traps 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the presence of glucose, purified lymphocytes from intestinal Peyer's patches (PP) and mesenteric lymph nodes (MLN) were stimulated with phorbol 12-myristate 13-acetate (PMA) to produce superoxide and hydroxyl DEPMPO radical adducts. The formation of these adducts was inhibited by superoxide dismutase or diphenyleneiodonium chloride, indicating that these cells produced superoxide radical during reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. With the treatment of sodium sulfite, PMA-stimulated PP lymphocytes produced a DEPMPO-sulfite radical adduct and an unknown radical adduct. When DEPMPO was replaced with DMPO, DMPO-sulfite and hydroxyl radical adducts were detected. The latter adduct resulted from DMPO oxidation by sulfate radical, which was capable of oxidizing formate or ethanol. Oxygen consumption rates were further increased after the addition of sulfite to PMA-stimulated lymphocytes, suggesting the presence of sulfiteperoxyl radical. Taken together, oxidants generated by stimulated lymphocytes oxidized sulfite to sulfite radical, which subsequently formed sulfiteperoxyl and sulfate radicals. The latter two radicals are highly reactive, contributing to increased oxidative stress, which may lead to sulfite toxicity, altered functions in intestinal lymphocytes, or both.     

Phospholipase D-dependent and-independent activation of the neutrophil NADPH oxidase

Stimulation of the respiratory burst of human neutrophils by fMet-Leu-Phe (in the absence of cytochalasin B) is largely unaffected when the activities of protein kinase C and phospholipase D are inhibited. This has been confirmed using three separate assays to measure the respiratory burst. However, whilst these enzymes are not required for the initiation or maximal rate of oxidant generation, they are required to sustain oxidase activity. In contrast, in the presence of cytochalasin B, fMet-Leu-Phe stimulated oxidase activity is much more dependent on phospholipase D activity. It is proposed that (in the absence of cytochalasin B) activation of the NADPH oxidase utilises cytochrome b molecules that are already present on the plasma membrane and activation occurs independently of phospholipase D and protein kinase C. Once these complexes are inactivated, then new cytochrome b molecules must be recruited from sub-cellular stores. This translocation and/or activation of these molecules is phospholipase D dependent. Some support for this model comes from the finding that the translocation of CD11b (which co-localises with cytochrome b) onto the cell surface is phospholipase D dependent.Abbreviations GM-CSF granulocyte-macrophage colony-stimulating factor - fMet-Leu-Phe N-formylmethionyl-leucyl-phenylalanine luminol 5-amino-2,3-dihydro-1,4-phthalazinedione, O₂,-superoxide radical     

Molecular basis of phosphorylation-induced activation of the NADPH oxidase

The multi-subunit NADPH oxidase complex plays a crucial role in host defense against microbial infection through the production of reactive oxygen species. Activation of the NADPH oxidase requires the targeting of a cytoplasmic p40-p47-p67(phox) complex to the membrane bound heterodimeric p22-gp91(phox) flavocytochrome. This interaction is prevented in the resting state due to an auto-inhibited conformation of p47(phox). The X-ray structure of the auto-inhibited form of p47(phox) reveals that tandem SH3 domains function together to maintain the cytoplasmic complex in an inactive form. Further structural and biochemical data show that phosphorylation of p47(phox) activates a molecular switch that relieves the inhibitory intramolecular interaction. This permits p47(phox) to interact with the cytoplasmic tail of p22(phox) and initiate formation of the active, membrane bound enzyme complex.     

Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils

The gp91phox subunit of flavocytochrome b₅₅₈ is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b₅₅₈. gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin–gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H₂O₂ generation by human neutrophils treated with the lipid raft disrupting agent methyl-β-cyclodextrin (MβCD). MβCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MβCD treatment either stimulated or inhibited H₂O₂ production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins.