Restitution of superoxide generation in autosomal cytochrome-negative chronic granulomatous disease (A22(0) CGD)-derived B lymphocyte cell lines by transfection with p22phax cDNA.

The respiratory burst oxidase of phagocytes and B lymphocytes is a multicomponent enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2-production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or physiologic stimuli, such as phagocytosis in phagocytes or cross-linking of surface immunoglobulin in B lymphocytes. The activity of this enzyme is greatly diminished or absent in patients with chronic granulomatous disease (CGD), an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every CGD patient studied so far, an abnormality has been found in a gene encoding one of the four components of the respiratory burst oxidase: the membrane proteins p22phox or gp91phox which together form the cytochrome b558 protein, or the cytosolic proteins p47phox or p67phox. Autosomal recessive cytochrome-negative CGD (A22(0) CGD) is associated with mutations in the gene coding for p22phox. We report here that the capacity for O2- production and cytochrome b558 protein expression were restored to Epstein-Barr virus-transformed B lymphocytes from two A22(0) CGD patients by transfection with an expression plasmid containing a p22phox cDNA. No detectable O2- was generated by untransfected p22phox-deficient lymphocytes. The genetic reconstitution of the respiratory burst in A22(0) CGD B lymphocytes by transfer of the wild-type p22phox cDNA represents a further step towards somatic gene therapy for this subgroup of A22(0) CGD. This system will also be useful for expression of genetically engineered mutant p22phox proteins in intact cells, facilitating the structure-function analysis of cytochrome b558.

Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2.

A major action of the microbicidal system of human neutrophils is the formation of superoxide anion (O2-) by a multicomponent oxidase that transfers electrons from the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen. The mechanism of assembly and activation of the oxidase from its cytosolic and membrane-bound components is unknown, but may require the activity of a guanosine 5'-triphosphate (GTP)-binding component. A cytosolic GTP-binding protein (Gox) that regulates the NADPH oxidase of neutrophils was identified. Gox was purified and shown to augment the rate of O2- production in a cell-free oxidase activation system. Sequence analysis of peptide fragments from Gox identified it as Rac 2, a member of the Ras superfamily of GTP-binding proteins. Antibody to a peptide derived from the COOH-terminus of Rac 2 inhibited O2- generation in a concentration-dependent manner. These results suggest that Rac 2 is a regulatory component of the human neutrophil NADPH oxidase, and provide new insights into the mechanism by which this oxygen radical-generating system is regulated.

Activation of human neutrophil nicotinamide adenine dinucleotide phosphate, reduced (triphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cell-free system.

Sonicates from unstimulated human neutrophils produce no measurable superoxide since the superoxide-generating enzyme, NADPH oxidase, is inactive in these preparations. Previous attempts to activate the oxidase in disrupted cells with conventional neutrophil stimuli have been unsuccessful. This report describes a cell-free system in which arachidonic acid (82 microM) was able to activate superoxide generation that was dependent upon the presence of NADPH and the sonicate. For activation to occur, both the particulate and supernatant fractions of the sonicate must be present. Calcium ions, which are required for activation of intact neutrophils by arachidonate, were not necessary in the cell-free system. In quantitative terms, the superoxide-generating activity in the cell-free system could account for at least 20-50% of the superoxide rate observed in intact neutrophils stimulated with arachidonate. Sonicates from patients with chronic granulomatous disease (CGD) could not be activated by arachidonic acid in the cell-free system. In three patients representing both genetic forms of CGD, the defect appeared to reside in the particulate fraction. The soluble cofactor was normal in all three patients and could be used to activate normal neutrophil pellets in the presence of arachidonic acid. Thus, at least a portion of the activation mechanism in the neutrophil, that residing in the soluble phase, appeared to be normal in patients with CGD.

Metabolism of procainamide to the cytotoxic hydroxylamine by neutrophils activated in vitro.

An almost universal side effect of long-term therapy with procainamide is the appearance of serum autoantibodies and less frequently a syndrome resembling lupus erythematosus. Previous studies demonstrated that procainamide-hydroxylamine (PAHA), a metabolite generated by hepatic mixed function oxidases, was highly toxic to dividing cells, but evidence that PAHA could be formed in the circulation was lacking. This study examines the capacity of neutrophils to metabolize procainamide to reactive forms. Neutrophils activated with opsonized zymosan were cytotoxic only if procainamide was present, whereas N-acetyl procainamide, which does not induce autoimmunity, was inert in this bioassay. PAHA was detected by HPLC in the extracellular medium if ascorbic acid was present. Generation of PAHA and cytotoxic procainamide metabolites was inhibited by NaN3 and catalase but not by superoxide dismutase, indicating that H2O2 and myeloperoxidase were involved. Nonactivated neutrophils and neutrophils from patients with chronic granulomatous disease did not generate cytotoxic PAHA, demonstrating that H2O2 was derived from the respiratory burst accompanying neutrophil activation. These conclusions were supported by results of a cell-free system in which neutrophils were replaced by myeloperoxidase and H2O2 or an H2O2 generating system. These studies demonstrate the capacity of neutrophils to mediate metabolism of procainamide and establish the role of myeloperoxidase released during degranulation and H2O2 derived from the respiratory burst in the direct cooxidation of procainamide to PAHA. The profound biologic activity of this metabolite and its possible generation within lymphoid compartments implicate this process in the induction of autoimmunity by procainamide.

Activation of H+ conductance in neutrophils requires assembly of components of the respiratory burst oxidase but not its redox function.

In phagocytes, superoxide generation by the NADPH oxidase is accompanied by metabolic acid production. Cytoplasmic acidification during this metabolic burst is prevented by a combination of H+ extrusion mechanisms, including a unique H+ conductance. NADPH oxidase is deficient in chronic granulomatous disease (CGD) patients. The burst of acid production is absent in CGD patients lacking the 47-kD (p47-phox) or the 91-kD (gp91-phox) subunits of the oxidase. Activation of the H+ conductance is also defective in these patients suggesting that (a) the oxidase itself undertakes H+ translocation or (b) oxidase assembly is required to stimulate a separate H+ conducting entity. To discern between these possibilities, three rare forms of CGD were studied. In neutrophils expressing nonfunctional cytochrome b, the conductance was activated to near-normal levels, implying that functional oxidase is not required to activate H+ extrusion. CGD cells expressing diminished amounts of cytochrome displayed H+ conductance approaching normal levels, suggesting that the oxidase itself does not translocate H+. Finally, the conductance was only partially inhibited in patients lacking the 67-kD subunit, indicating that this component is not essential for stimulation of H+ transport. We propose that normal assembly of the oxidase subunits is required for optimal activation of a closely associated but distinct H+ conducting entity.

Biological defense mechanisms. The effect of bacteria and serum on superoxide production by granulocytes.

We previously reported that granulocytes are able to produce superoxide (O(2) (-)), a highly reactive compound formed by the one-electron reduction of oxygen. The demonstration of O(2) (-) production was based on the observation that the reduction of extra-cellular cytochrome c by granulocytes was greatly diminished by superoxide dismutase, an enzyme catalyzing the conversion of O(2) (-) to hydrogen peroxide and oxygen. In the present report, studies concerning the effect of bacteria and serum on O(2) (-)-dependent cytochrome c reduction by granulocytes are described.In the absence of bacteria, the O(2) (-)-dependent reduction of extracellular cytochrome c by granulocytes under optimal assay conditions amounted to 9.2+/-2.8 SD nmol/3 x 10(6) cells/20 min. When bacteria (100 organisms/cell) were present, the O(2) (-)-dependent cytochrome c reduction under otherwise similar conditions increased by a factor of nearly four (34.5+/-9.4). There was no effect of albumin or catalase on cytochrome c reduction, and boiled dismutase had only a small effect. Omission of granulocytes or substitution of live cells by cells by cells killed by heat abolished O(2) (-)-dependent cytochrome c reduction. Bacteria killed by autoclaving were almost as effective as live bacteria in stimulating granulocyte O(2) (-) production. Measurements of particle uptake and O(2) uptake by granulocytes indicated that superoxide dismutase did not affect granulocyte metabolism nonspecifically, supporting the conclusion that the diminution of cytochrome c reduction in the presence of dismutase was due to the destruction of O(2) (-) by this enzyme. Stimulation of O(2) (-) production by bacteria was strongly dependent on the presence of serum in the incubation mixture. Serum heated to 56 degrees C for 45 min was as effective as unheated serum in stimulating O(2) (-) production in the presence of bacteria, but boiled serum had no effect. Other experiments suggested that incubation of bacteria with serum resulted in the release of a nonparticulate heat-labile substance capable of stimulating O(2) (-) production in the absence of bacteria. Certain characteristics of the O(2) (-)-dependent cytochrome c reduction by granulocytes were studied, including the dependence of this process on granulocyte, cytochrome c, and bacterial concentrations. In addition, O(2) (-)-dependent cytochrome c reduction was followed as a function of time. A constant rate was found with resting granulocytes. With bacteria the time course was more complex. A well-defined lag was followed by a fairly brief period of extremely vigorous cytochrome c reduction. During this period, the maximum rate of cytochrome c reduction exceeded the rate observed in the absence of bacteria by a factor of 12. The rate then decreased until by 40 min, it had slowed to the rate observed in the absence of bacteria. From the above results, it was concluded that the exposure of the granulocyte to bacteria plus serum initiates a process in which a defined quantity of O(2) (-) is formed in a rapid burst lasting 20-30 min. It is conceivable that the O(2) (-) generated by this process may be involved in the killing of bacteria by the granulocytes.

Functional defect in neutrophil cytosols from two patients with autosomal recessive cytochrome-positive chronic granulomatous disease.

The kinetics of activation of the respiratory burst oxidase in the cell-free oxidase-activating system have been explained by a three-stage mechanism in which the membrane-associated oxidase components M: (a) take up a cytosolic factor S to form a complex M.S that is (b) slowly converted in the second stage to a precatalytic species [M.S]*, which finally (c) takes up two more (possibly identical) cytosolic components, C alpha and C beta, to successively generate [M.S]*C alpha, a low-activity (i.e., high Km) oxidase, and finally [M.S]*C alpha C beta, the ordinary (i.e., low Km) oxidase (Babior, B.M., R. Kuver, and J.T. Curnutte. 1988. J. Biol. Chem. 263:1713-1718). Studies with the cell-free oxidase-activating system from normal neutrophils and from neutrophils obtained from two patients with type II (autosomal recessive cytochrome-positive) chronic granulomatous disease (CGD) have suggested that (a) the defective element in the cytosol from patient neutrophils is S; (b) in normal neutrophil cytosol, S is limiting with respect to M; and (c) C alpha and C beta interact cooperatively with the activated precursor complex [M.S]*. It was further speculated that S might be identical to the nonphosphorylated progenitor of the phosphorylated 48-kD proteins that are missing in certain forms of CGD, and that other forms of type II CGD besides the one described in this report remain to be discovered.

Fluoride-mediated activation of the respiratory burst in human neutrophils. A reversible process.

Fluoride ion (F-) is an effective activator of the respiratory burst in neutrophils, as indicated by its ability to induce O2- production by these cells. Other halide ions did not activate the burst, Cl-, in particular appeared to antagonize the effect of F- on O2- production. F- stimulated O2- production showed a requirement for Ca++, but was independent of other exogenous cations. Neither phagocytosis nor degranulation were necessary for respiratory burst activation by F-. The effect of F- on the respiratory burst was reversible. Washing the cells after treatment with F-, while they were still producing large amounts of O2-, returned them to the resting state. They could then be stimulated again to produce O2- in amounts equivalent to those originally produced. Our experiments indicated that restimulation did not represent the activation of a population of cells that had not been activated during the initial exposure to F-, nor did it represent serial activation of different subpopulation of the O2- forming enzyme molecules present in a given cell. Rather, our data suggest that the entire population of O2- forming enzyme molecules was activated in a reversible fashion by F-.

The particulate superoxide-forming system from human neutrophils. Properties of the system and further evidence supporting its participation in the respiratory burst.

Studies were performed to characterize the previously reported particulate O2--forming system from human neutrophils. Of eight reducing agents examined, including glutathione, ascorbic acid, and intermediates of the glycolytic and hexose monophosphate shunt pathways, only the pyridine nucleotides could serve as electron donors. At 0.1 mM pyridine nucleotide, O2- production was relatively independent of pH. The Km for NADH was approximately 0.7 mM regardless of pH, while with NADPH the Km varied from 0.02 mM at pH 6.0 to 0.3 mM at pH 7.5. The molar ratio of NADPH oxidized to O2- produced was consistent with the reaction: NADPH + 2 O2- leads to NADP+ H+; the product nucleotide was shown enzymatically to be NADP. O2- production was not inhibited by CN-, Na-, EDTA, or 1,10-phenanthroline. Particulate O2- production accounted for 35% of the oxygen taken up during the respiratory burst by an equivalent number of intact neutrophils. Greatly diminished O2- production was seen with particles prepared from cells obtained from three patients with chronic granulomatous disease, with 2.5 mM NADPH as electron donor. With 5.0 mM NADH similar observations were made with particles from two of the patients, but with this nucelotide, O2- production was only slightly reduced in the third case. The evidence available suggests that this particulate O2- -forming system is the one responsible for the respiratory burst in activated neutrophils. The relationship between this system and other O2- -forming system found in human neutrophils is discussed.

Manganese-dependent NADPH oxidation by granulocyte particles. The role of superoxide and the nonphysiological nature of the manganese requirement.

Recent work has indicated that superoxide is involved in the manganese-stimulated oxidation of NADPH by crude granule preparations of guinea pig neutrophils. The characteristics of a model manganese-requiring NADPH-oxidizing system that employs a defined O2-generator have now been compared to the original neutrophil-granule system. With respect to pH dependence, cyanide sensitivity, and reduced pyridine nucleotide specificity, the properties of the two systems are very similar. Additional information has been obtained concerning cation specificity and the kinetics of the metal-catalyzed NADPH oxidation. From the similarities between the properties of the model and neutrophil particle systems, we postulate that the manganese-dependent NADPH oxidation observed in the presence of neutrophil granules represents in large part of nonenzymatic free radical chain involving the oxidation of NADPH to NADP, with O2- as both the chain initiator and one of the propagating species. In this reaction, the neutrophil particles serve only as a source of O2-. Further, the same changes in kinetics (decrease in apparent Km for NADPH) observed previously when granules from phagocytizing rather than resting cells were employed could be mimicked by varying the rate of O2-generation by the model system. We conclude from these results that it is unnecessary to invoke a manganese-requiring enzyme as a component of the phagocytically stimulated respiratory system of the neutrophil.

Pyridine nucleotide-dependent superoxide production by a cell-free system from human granulocytes.

Using an assay that measured superoxide dismutase-inhibitable nitro blue tetrazolium reduction, we studied superoxide (O2-) production by a cell-free system from human granulocytes. At 40 muM NADPH and a protein concentration of 0.12 mg/ml, lysates prepared from human granulocytes formed O2- at a rate of 18. 4 +/- 4.6 SE nmol/ml reaction mixture per donor, but not with glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, sodium lactate, glutathione, or ascorbic acid. The Km's for NADPH and NADH were 8.6 +/- 4.6 muM and 0.83 +/- 0.30 mM, respectively, suggesting that NADPH is the physiological electron donor in this system. O2- production was not inhibited by 1mM KCN. The rate of O2- production by the cell-free system was comparable to the rate of O2-production by an equivalent quantity of intact granulocytes incubated under similar conditions. O2- production by lysates from granulocytes preincubated with serum under conditions previously shown to stimulate O2- production in the intact cells was no different than its production by lysates from unstimulated cells. O2- production at 0.2 mM and 0.02 mM NADPH by lysates from the granulocytes of two patients with chronic granulomatous disease was similar to O2- production by control lysates. This finding was interpreted in terms of the possibility that the metabolic lesion in chronic granulomatous disease may lie outside the oxygen-metabolizing enzyme system of the granulocyte, or alternatively, that the granulocytes may contain two O2- - forming enzymes, one of which is inactive in chronic granulomatous disease.

Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent.

As a highly reactive substance produced in biological systems by the one-electron reduction of oxygen, superoxide (O(2) (-)) seemed a likely candidate as a bactericidal agent in leukocytes. The reduction of cytochrome c, a process in which O(2) (-) may serve as an electron donor, was found to occur when the cytochrome was incubated with leukocytes. O(2) (-) was identified as the agent responsible for the leukocyte-mediated reduction of cytochrome c by the demonstration that the reaction was abolished by superoxide dismutase, an enzyme that destroys O(2) (-), but not by boiled dismutase, albumin, or catalase. Leukocyte O(2) (-) production doubled in the presence of latex particles. The average rate of formation of O(2) (-) in the presence of these particles was 1.03 nmol/10(7) cells per 15 min. This rate, however, is only a lower limit of the true rate of O(2) (-) production, since any O(2) (-) which reacted with constituents other than cytochrome c would have gone undetected. Thus. O(2) (-) is made by leukocytes under circumstances which suggest that it may be involved in bacterial killing.

A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease.

A membrane-bound cytochrome b, a heterodimer formed by a 91-kD glycoprotein and a 22-kD polypeptide, is a critical component of the phagocyte NADPH-oxidase responsible for the generation of superoxide anion. Mutations in the gene for the 91-kD chain of this cytochrome result in the X-linked form of chronic granulomatous disease (CGD), in which phagocytes are unable to produce superoxide. Typically, there is a marked deficiency of the 91-kD subunit and the cytochrome spectrum is absent (X- CGD). In a variant form of CGD with X-linked inheritance, affected males have a normal visible absorbance spectrum of cytochrome b, yet fail to generate superoxide (X+ CGD). The size and abundance of the mRNA for the 91-kD subunit and its encoded protein were examined and appeared normal. To search for a putative mutation in the coding sequence of the 91-kD subunit gene, the corresponding RNA from an affected X+ male was amplified by the polymerase chain reaction and sequenced. A single nucleotide change, a C----A transversion, was identified that predicts a nonconservative Pro----His substitution at residue 415 of the encoded protein. Hybridization of amplified genomic DNA with allele-specific oligonucleotide probes demonstrated the mutation to be specific to affected X+ males and the carrier state. These results strengthen the concept that all X-linked CGD relates to mutations affecting the expression or structure of the 91-kD cytochrome b subunit. The mechanism by which the Pro 415----His mutation renders the oxidase nonfunctional is unknown, but may involve an impaired interaction with other components of the oxidase.

Subcellular localization of the superoxide-forming enzyme in human neutrophils.

The subcellular distribution of the superoxide (O2-)-forming enzyme in human neutrophils was investigated. Cells were activated by phorbolmyristate acetate or by opsonized zymosan, and were then fractionated by zonal-rate sedimentation at two different speeds. At high speed, the specific granules were resolved from the azurophils and the membrane fraction, while at low speed, the azurophil granules could be separated from fast-sedimenting particle aggregates. Under both conditions, the major portion of the O-2--forming activity (60--70% of the total) was found to be associated with the membrane fraction which was characterized by the presence of alkaline phosphatase, alkaline phosphodiesterase I, and acid aryl phosphatase. No significant O-2--forming activity was found in either specific or azurophil granules. Some activity was present in the fastest sedimenting fractions which, as shown by electron microscopy, were heterogeneous and contained aggregated material which included membrane fragments. These fractionation results provide strong additional support for the current view that the activable O-2--forming system is localized in the plasma membrane of human neutrophils.

Identification of a thermolabile component of the human neutrophil NADPH oxidase. A model for chronic granulomatous disease caused by deficiency of the p67-phox cytosolic component.

Mild heating of human neutrophils inactivates the respiratory burst oxidase, producing a defect in superoxide production and bacterial killing comparable to that seen in patients afflicted with chronic granulomatous disease (CGD). We have now investigated the mechanism and specificity of this inactivation by examining the effect of mild heating on the known oxidase components: the membrane-bound subunits of the cytochrome b558 (gp91-phox and p22-phox) and the two cytosolic oxidase factors (p47-phox and p67-phox). Heating (46 degrees C for 7.5 min) caused intact neutrophils to lose greater than 85% of their capacity to produce superoxide, a defect which was localized to the cytosolic, but not the membrane, fraction. Complementation studies with CGD cytosols deficient in either p47-phox or p67-phox suggested that the defective component of heat-inactivated cytosol was p67-phox. This was confirmed by experiments showing that recombinant p67-phox, but not p47-phox, exhibited lability at 46 degrees C and completely reconstituted oxidase activity of heat-treated cytosol. These studies indicate that mild heating of either intact neutrophils or normal neutrophil cytosol results in a selective inactivation of p67-phox, providing a model oxidase system for the extremely rare p67-phox-deficient form of CGD.

Chronic granulomatous disease due to a defect in the cytosolic factor required for nicotinamide adenine dinucleotide phosphate oxidase activation.

The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is present in a dormant state in unstimulated neutrophils. It can be converted to an active form in a cell-free system if both the plasma membrane and cytosol fractions are incubated together in the presence of arachidonic acid. This system was used to determine the nature of the biochemical defect in seven patients with the autosomal recessive, cytochrome b-positive form of chronic granulomatous disease (CGD). A severe deficiency in the cytosol factor was identified in each patient. The defective activity was not caused by the presence of an inhibitor, nor could it be restored to normal by combining cytosol fractions from different patients. In contrast, the membrane fractions from all seven patients contained normal levels of NADPH oxidase when activated in the presence of control cytosol. Of family members tested (obligate heterozygotes for this disorder), seven of eight had intermediate levels of cytosol factor activity. The respiratory burst defect in this form of CGD is caused by an abnormality in the cytosolic factor required for NADPH oxidase activation.

The p67-phox cytosolic peptide of the respiratory burst oxidase from human neutrophils. Functional aspects.

Most cases of cytosol-defective chronic granulomatous disease are due to the deficiency of a 47-kD protein (p47-phox) whose phosphorylation normally accompanies the activation of the respiratory burst oxidase. Recently, a form of chronic granulomatous disease was described in which the failure of O2- production was associated with the absence of a 67-kD polypeptide (p67-phox) from the cytosol of affected neutrophils. Using neutrophils obtained from a patient with this form of the disease, we examined the function of p67-phox in the activation of the oxidase. Our studies showed that in whole p67-phox-deficient neutrophils, p47-phox was phosphorylated in a normal fashion. In the cell-free oxidase-activating system, the ability of the p67-phox-deficient cytosol to support oxidase activation was partly restored by the addition of p47-phox-deficient cytosol; the p67-phox-deficient cytosol, however, was not complemented by cytosol inactivated with NADPH dialdehyde, an affinity label previously found to block the NADPH-binding component of the oxidase. Despite these differences, the kinetic properties of the p67-phox-deficient cytosol closely resembled those of the p47-phox-deficient cytosol. Taken together with earlier findings, these results suggest that (a) in the neutrophil cytosol, p67-phox is at least partly complexed to p47-phox; (b) it is in the form of this complex that p67-phox participates in oxidase activation; and (c) p47-phox appears to be translocated from the cytosol to the plasma membrane during oxidase activation, but complexation to p67-phox is not necessary for this translocation, nor for the accompanying extra protein phosphorylation.

Mutations in the promoter region of the gene for gp91-phox in X-linked chronic granulomatous disease with decreased expression of cytochrome b558.

We examined the molecular defect in two kindreds with "variant" X-linked chronic granulomatous disease (CGD). Western blots of neutrophil extracts showed decreased immunoreactive cytochrome b558 components gp91-phox and p22-phox. Analysis of mRNA demonstrated reduced gp91-phox transcripts, with relative preservation of an alternative mRNA species created by transcription initiation in the third exon of the gene. Single strand conformation polymorphism analysis of the 5' flanking region of the patients' gp91-phox genes revealed an electrophoretic abnormality not detected in 40 other gp91-phox genes. Genomic sequencing demonstrated a single base change associated with CGD in each kindred: in one, adenine to cytosine at base pair-57 and in the other, thymidine to cytosine at -55. These mutations are located between the "CCAAT" and "TATA" box consensus sequences involved in eukaryotic gene transcription. Gel shift assays revealed two specific DNA-protein complexes formed between phagocyte nuclear extracts and an oligonucleotide probe representing bases -31 to -68 of the gp91-phox promoter region; the faster-migrating complex could not be formed with oligonucleotides containing either of the promoter mutations. Thus, these promoter region mutations appear to be causally related to the loss of association of a DNA-binding protein and lead to diminished gp91-phox expression, abnormal transcription initiation, and the development of CGD.

Human neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease.

A membrane-bound cytochrome b, a heterodimer formed by a 91-kD glycoprotein (heavy chain) and a 22-kD polypeptide (light chain), is an essential component of the phagocyte NADPH-oxidase responsible for superoxide generation. Cytochrome b is absent in two subgroups of chronic granulomatous disease (CGD), an inherited disorder characterized by the lack of oxidase activity. Mutations in the cytochrome heavy chain gene, encoded by the CYBB locus in Xp21.1, result in the X-linked form of CGD. A rare subgroup of autosomal recessive CGD also lacks cytochrome b (A- CGD), but the genetic defect has not previously been identified. In order to search for possible mutations in the cytochrome light chain locus, CYBA, the structure of this gene was characterized. The CYBA locus was localized to 16q24, and the approximately 600-bp open reading frame determined to be encoded by six exons that span approximately 8.5 kb. Three unrelated patients with A- CGD were studied for evidence of mutations in the light chain gene. One patient, whose parents were first cousins, was homozygous for a large deletion that removed all but the extreme 5' coding sequence of the gene. The other two patients had a grossly normal light chain transcript on Northern blot of mononuclear cell RNA. The light chain transcript was amplified by the polymerase chain reaction and sequenced. One patient was a compound heterozygote for two alleles containing point mutations in the open reading frame that predict a frame shift and a nonconservative amino acid replacement, respectively. The second patient, whose parents were second cousins, was homozygous for a different single-base substitution resulting in another nonconservative amino acid change. These results indicate that A- CGD can results from defects in the gene encoding the 22-kD light chain of the phagocyte cytochrome b.

Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils.

Resting and phorbol-activated human neutrophils were separated by treatment with Triton X-100 into detergent-extractable and cytoskeleton fractions. Respiratory burst oxidase activity was restricted entirely to the cytoskeleton. The cytoskeleton also contained approximately 15% of the neutrophil cytochrome b558, an oxidase-associated heme protein, as well as most of the oxidase-related cytosolic polypeptide p67phox. In contrast, the components of the oxidase-associated phosphoprotein family p47phox were found almost exclusively in the detergent extract, suggesting that p47phox is needed for oxidase activation but not for O2- production by the activated oxidase. Activation of the oxidase had no apparent effect on the distribution of any of these species between the cytoskeleton and the detergent extract. Our results support earlier studies implying that the cytoskeleton participates in an important way in regulating the activity of the O2(-)-forming respiratory burst oxidase of neutrophils.