Form Of Chronic Granulomatous Disease Research Articles

Prenatal diagnosis in a family with X‐linked chronic granulomatous disease with the use of the polymerase chain reaction

In the X-linked form of chronic granulomatous disease (X91 degrees CGD), the genetic defect is linked to the CYBB locus on the X chromosome. We studied a family with a genetic defect in this gene, consisting of a G----A substitution at the fifth base of the 5' donor splice site of intron 3. This mutation leads to skipping of exon 3 after transcription of the gene. The expectant mother was diagnosed as a carrier. Analysis of polymerase chain reaction (PCR)-amplified genomic DNA from a chorionic villus biopsy (CVB) showed the same mutation in the male fetus. After termination of the pregnancy, the diagnosis was confirmed by conventional methods. This is the first time that PCR has been used for prenatal diagnosis of CGD.

Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A

Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane- derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b- negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

Reconstitution of Defective Respiratory Burst Activity With Partially Purified Human Neutrophil Cytochrome B in Two Genetic Forms of Chronic Granulomatous Disease: Possible Role of Rap1A

Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane-derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b-negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

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.

Activation of O2(-)-generating oxidase in an heterologous cell-free system derived from Epstein-Barr-virus-transformed human B lymphocytes and bovine neutrophils. Application to the study of defects in cytosolic factors in chronic granulomatous disease.

Epstein-Barr-virus-transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4 beta-phorbol 12-myristate 13-acetate exhibit an NADPH-dependent oxidase activity capable of generating the superoxide anion O2-, similar to, but less efficient than that of activated neutrophils. A cell-free system of oxidase activation consisting of a membrane fraction and cytosol from EBV B lymphocyte homogenate supplemented with guanosine 5'-[gamma-thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O2-, assessed by the superoxide-dismutase-sensitive reduction of cytochrome c in the presence of NADPH. However, cytochrome c reduction was slow and largely insensitive both to superoxide dismutase, and to iodonium biphenyl, a powerful inhibitor of the oxidase activity in neutrophils. A markedly faster reduction of cytochrome c in the presence of NADPH was obtained with a heterologous system consisting of cytosol from EBV B lymphocytes and bovine neutrophil membranes, GTP[S], arachidonic acid and Mg2+; in this system, reduction of cytochrome c was totally inhibited by superoxide dismutase and iodonium biphenyl. These results show that EBV B lymphocytes contain a substantial amount of cytosolic factors of oxidase activation, and that the limiting factors for O2- production in B lymphocytes are the membrane components of the oxidase complex. The heterologous system of EBV B lymphocyte cytosol and bovine neutrophil membranes provided a rapid and convenient method to diagnose cytosolic defects in autosomal forms of chronic granulomatous disease. In addition, it might be a useful tool to explore the mechanism of action of the cytosolic factors in oxidase activation.

Analysis of various types of chronic granulomatous disease with the monoclonal antibody 7D5 directed against the small subunit of surface cytochrome b558

Four different types of chronic granulomatous disease (CGD) were analysed with the monoclonal antibody, 7D5, directed against the small 23 kD subunit of cytochrome b558 using a flow cytometric fluorescence analytical method. 7D5 immunofluorescent surface staining of granulocytes was absent in 12 patients with X-linked cytochrome b558 deficiency, in 2 patients with variant X-linked CGD with residual (X-forming activity and in 2 patients with autosomal recessive cytochrome b558 deficiency. The mothers of the patients with the X-linked form of CGD had 2 cell populations, one 7D5 negative or weakly positive and one 7D5 positive. The granulocytes of both parents of one patient with autosomal recessive cytochrome b558 deficiency had slightly reduced fluorescence intensity comparable to their reduced cytochrome b558 content. Three CGD patients with normal cytochrome b558 and their parents had granulocytes normally stained with antibody 7D5. 7D5 antibody enables rapid detection and classification of CGD patients with cytochrome b558 deficiency as well as rapid identification of heterozygous carriers.

Identification of the NADPH-binding subunit of the respiratory burst oxidase.

The respiratory burst oxidase is a multicomponent membrane-bound enzyme that uses NADPH to reduce oxygen to O2-. When oxidase-containing membranes from activated neutrophils are treated with 0.3 M KCl, the NADPH-binding component of the oxidase elutes from the membranes in an active form. Treatment of this eluate with [32P]NADPH dialdehyde labels an approximately 32-kDa protein that is absent from eluates obtained from normal resting membranes or from resting or activated membranes from patients with one form of chronic granulomatous disease. We propose that this approximately 32-kDa protein is the NADPH-binding component of the oxidase.

Purification and characterization of an oxidase activating factor of 63-kilodaltons from bovine neutrophils

A 63-kDa protein, which behaves as an oxidase activating factor in bovine neutrophils, has been purified to electrophoretic homogeneity. The protein was isolated from the cytosol of resting bovine neutrophils after several steps, including ammonium sulfate precipitation and chromatography on AcA44, DE-52 cellulose, Mono Q, and Superose 12 in the presence of dithiothreitol. The oxidase activating potency of the protein was assayed with a cell-free system consisting of neutrophil membranes, GTP gamma S, arachidonic acid, and a complementary cytosolic fraction. The purification factor was 200 and the yield 3%. During the course of gel filtration on calibrated Superose 12, the 63-kDa protein eluted as a dimer. Its isoelectric point was 6.4 +/- 0.1. Antibodies raised in rabbits against the 63-kDa protein reacted with a protein of similar size in human neutrophils and in HL60 promyelocytic cells induced to differentiate into granulocytes. No immune reaction was observed in cytosol from undifferentiated HL60 cells, in extracts from bovine skeletal muscle, liver, and brain, or in cytosol prepared from neutrophils derived from a patient with an autosomal cytochrome b positive form of chronic granulomatous disease lacking the 67-kDa oxidase activating factor. Immunoblotting with the 63-kDa bovine protein antiserum demonstrated that activation of bovine neutrophil oxidase by phorbol myristate acetate induced the translocation of the 63-kDa protein from cytosol to the membrane.

Origin of Mutations in Two Families With X-Linked Chronic Granulomatous Disease

The most common X-linked recessive form of chronic granulomatous disease (X-CGD) is characterized by the absence of cytochrome b558 in neutrophils. In a rare variant form of X-CGD, cytochrome b558 is present but not functional. The gene (locus symbol CYBB) was localized to band Xp21 by studies of patients with small chromosome deletions. The gene was cloned based on its location and found to encode the 91-Kd subunit of the cytochrome b558 complex. Most female carriers for X-CGD can be identified by their X-inactivation mosaicism; on average 50% of their neutrophils express the mutant phenotype and fail to reduce nitroblue tetrazolium (NBT). In 2 of 4 families studied, the maternal grandmothers had normal NBT tests, suggesting either nonrandom X-inactivation or new mutations. Restriction fragment length polymorphism analysis using closely linked flanking markers or the NsiI polymorphism detected by the CYBB probe itself, allowed us to identify the X chromosome carrying the mutation as derived from a healthy NBT-positive maternal grandfather. The mothers of the affected boys must have received a paternal X chromosome carrying a new mutation, consistent with the maternal grandmothers' normal NBT tests. In all of eight potential carriers studied, the results of the NBT and DNA marker testing were in complete agreement. Prenatal diagnosis by DNA testing can be performed in early gestation obviating the need for fetal blood sampling.

Origin of mutations in two families with X-linked chronic granulomatous disease

The most common X-linked recessive form of chronic granulomatous disease (X-CGD) is characterized by the absence of cytochrome b558 in neutrophils. In a rare variant form of X-CGD, cytochrome b558 is present but not functional. The gene (locus symbol CYBB) was localized to band Xp21 by studies of patients with small chromosome deletions. The gene was cloned based on its location and found to encode the 91-Kd subunit of the cytochrome b558 complex. Most female carriers for X-CGD can be identified by their X-inactivation mosaicism; on average 50% of their neutrophils express the mutant phenotype and fail to reduce nitroblue tetrazolium (NBT). In 2 of 4 families studied, the maternal grandmothers had normal NBT tests, suggesting either nonrandom X- inactivation or new mutations. Restriction fragment length polymorphism analysis using closely linked flanking markers or the NsiI polymorphism detected by the CYBB probe itself, allowed us to identify the X chromosome carrying the mutation as derived from a healthy NBT-positive maternal grandfather. The mothers of the affected boys must have received a paternal X chromosome carrying a new mutation, consistent with the maternal grandmothers' normal NBT tests. In all of eight potential carriers studied, the results of the NBT and DNA marker testing were in complete agreement. Prenatal diagnosis by DNA testing can be performed in early gestation obviating the need for fetal blood sampling.

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.

Update on Chronic Granulomatous Diseases of Childhood

SELECTED CASE A 19-YEAR-OLD black man presented with an autosomal recessive form of chronic granulomatous disease (CGD) of childhood (47-kd cytosol protein deficiency). At age 5 years, he was evaluated for a prolonged febrile illness and was found to have a positive tuberculin skin test. He responded to therapy with isoniazid and ethambutol for presumptive tuberculosis. He did well until age 8 years, when he presented with malaise, fever, and an infiltrate in the upper part of his right lung. Bronchoscopy did not yield a diagnosis. He was treated with broad-spectrum antibacterial therapy without altering his signs and symptoms. An immunologic workup that included a nitroblue tetrazolium dye reduction test indicated a failure of his blood neutrophils to produce superoxide. Subsequent studies confirmed the diagnosis of CGD, as indicated by a failure of his stimulated neutrophils to consume oxygen or produce hydrogen peroxide and by defective in vitro killing of

Regulation of macrophage function by interferon-gamma. Somatic cell genetic approaches in murine macrophage cell lines to mechanisms of growth inhibition, the oxidative burst, and expression of the chronic granulomatous disease gene.

The importance of oxidative cytocidal mechanisms of phagocytic cells in immune protection against microbial pathogens is uniquely revealed by chronic granulomatous disease (CGD), a genetic deficiency disease of man. This cytocidal response in mononuclear phagocytes is principally regulated by IFN-gamma. A somatic cell genetic approach was taken to select oxidative variants from a cloned murine macrophage cell line, J774.16, which formally permitted us to dissociate three regulatory effects of IFN-gamma on these cells: the antiproliferative effect, the antiviral effect, and production of superoxide anion. Half of the variants defective in O-2 production after phorbol myristate acetate stimulation were also resistant to the antiproliferative effects of IFN-gamma. This result suggests that IFN-gamma-induced growth inhibition and production of cytocidal oxygen intermediates are mediated via a common pathway. The somatic cell genetic approach has allowed us to develop in vitro macrophage models for several forms of CGD. One variant characterized in detail, D9, was unable to produce superoxide after stimulation by phorbol esters. At the molecular level, Northern blot analysis revealed that the mRNA encoding the large subunit of the putative CGD gene product, cytochrome b558, was absent in this variant. Another class of variants constitutively unable to produce O-2 or the cytochrome b558 mRNA could be induced to do so by IFN-gamma. These somatic mutants may be useful models in clarifying the role of the CGD gene product and its regulation in the production of cytocidal oxygen intermediates.

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.

Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease

Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytic cells fail to generate antimicrobial oxidants. The various forms of CGD can be classified in terms of the mode of inheritance (either X-linked or autosomal recessive), and whether the neutrophils display the absorbance spectrum of a unique b-type cytochrome important for the function of the respiratory burst oxidase. The finding that purified neutrophil cytochrome b is a heterodimer consisting of a 91kD glycosylated and a 22kD nonglycosylated polypeptide has raised the question of which subunits are absent (or defective) in the various types of CGD. To address this question we have studied the expression of the cytochrome b subunits in three genetically distinct forms of CGD: X-linked/cytochrome b-negative (X-), autosomal recessive/cytochrome b-negative (A-), and autosomal recessive/cytochrome b-positive (A+). Using polyclonal antibodies to each of the two subunits, we prepared Western blots of lysates of intact neutrophils from ten CGD patients. In the controls and three patients with A+ CGD, both cytochrome subunits were easily detected. Consistent with the previously reported finding in five X- patients, neither subunit could be identified in neutrophils from three additional X- patients. Both subunits were also undetectable in four patients with A- CGD (three females, one male). This latter group of patients most likely bears a normal 91kD gene, since the patients are genetically distinct from the 91kD-defective X- group. The mutation in A- CGD, therefore, probably involves the 22kD gene and the eventual expression of the 22kD subunit. Furthermore, the expression of the 91kD subunit in this group of patients appears to be prevented due to the 22kD mutation in a manner converse to that seen in the X- CGD patients. Based on these studies, we hypothesize that the stable of expression of either of the two cytochrome subunits is dependent upon the other.

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.

A phosphoprotein of Mr 47,000, defective in autosomal chronic granulomatous disease, copurifies with one of two soluble components required for NADPH:O2 oxidoreductase activity in human neutrophils.

The NADPH:O2 oxidoreductase (NADPH oxidase) of human neutrophils is converted from a dormant to an active state upon stimulation of the cells. We have studied the soluble fraction that is required for NADPH oxidase activation in a cell-free system. Human neutrophils were separated in a membrane-containing and a soluble fraction. The soluble fraction was separated on carboxymethyl (CM) Sepharose in 10 mM 4-morpholino-ethanesulfonic acid buffer of pH 6.8. Reconstitution of the NADPH oxidase activity, measured as O2 consumption, was only found when the membrane fraction was combined with the flowthrough of the CM Sepharose column as well as with a fraction that eluted at 125 mM NaCl. This result indicates that at least two soluble components are necessary for reconstitution of the NADPH oxidase activity: one that does not bind to CM Sepharose and one that does bind. These components were designated soluble oxidase component (SOC) I and SOC II, respectively. Boiling destroyed the activity in both fractions. In the soluble fraction of human lymphocytes and thrombocytes neither SOC I nor SOC II activity was found. SOC II copurified with a 47-kD phosphoprotein, previously found defective in patients with the autosomal form of chronic granulomatous disease (CGD). Inactive soluble fractions of cells from autosomal CGD patients were reconstituted with a SOC II fraction from control cells. The result of this experiment indicates that autosomal CGD patients are normal in SOC I but defective in SOC II.

The 47-kDa protein involved in the NADPH:O 2 oxidoreductase activity of human neutrophils is phosphorylated by cyclic AMP-dependent protein kinase without induction of a respiratory burst

When human neutrophilic granulocytes are stimulated with chemoattractants or phorbol esters, these cells respond with a so-called respiratory burst: such stimuli induce the activation of a NADPH:O 2 oxidoreductase, which converts oxygen into superoxide. This activation coincides with the phosphorylation of a number of proteins, amongst which a 47-kDa phosphoprotein. Neutrophils from patients with the autosomal form of chronic granulomatous disease (CGD) fail to mount a respiratory burst and concomitantly lack phosphorylation of the 47-kDa protein. We have shown this protein to be a substrate for protein kinase C. In the present paper we describe the phosphorylation of the 47-kDa phosphoprotein by cyclic AMP-dependent protein kinase. For these studies, we used neutrophil cytoplasts, i.e., neutrophils devoid of nucleus and granules, but with an intact NADPH:O 2 oxidoreductase. Addition of dibutyryl cyclic AMP (Bt 2cAMP) to intact human neutrophil cytoplasts resulted in an increase in protein phosphorylation. Among the phosphorylated proteins is a 47-kDa phosphoprotein. Increased protein phosphorylation was also observed upon addition of Bt 2cAMP to neutrophil cytoplast lysates. In lysates of neutrophil cytoplasts from patients with the autosomal form of CGD, phosphorylation of the 47-kDa protein was absent. This finding (confirmed by analysis on two-dimensional gels) indicates that the 47-kDa phosphoprotein, relevant for the NADPH:O 2 oxidoreductase, is a substrate for the cAMP-dependent protein kinase. Unlike phorbol ester-induced phosphorylation, Bt 2cAMP-induced phosphorylation is not accompanied by initiation of a respiratory burst. This observation demonstrates that 47-kDa phosphoprotein phosphorylation can be uncoupled from respiratory burst activity and indicates that other modifications of the NADPH:O 2 oxidoreductase are required for induction of activity.

Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease.

The mechanisms regulating activation of the respiratory burst enzyme, NADPH oxidase, of human neutrophils (PMN) are not yet understood, but protein phosphorylation may play a role. We have utilized a defect in a cytosolic factor required for NADPH oxidase activation observed in two patients with the autosomal recessive form of chronic granulomatous disease (CGD) to examine the role of protein phosphorylation in activation of NADPH oxidase in a cell-free system. NADPH oxidase could be activated by SDS in reconstitution mixtures of cytosolic and membrane subcellular fractions from normal PMN, and SDS also enhanced phosphorylation of at least 16 cytosolic and 14 membrane-associated proteins. However, subcellular fractions from CGD PMN plus SDS expressed little NADPH oxidase activity, and phosphorylation of a 48-kD protein(s) was selectively defective. The membrane fraction from CGD cells could be activated for NADPH oxidase when mixed with normal cytosol and phosphorylation of the 48-kD protein(s) was restored. In contrast, the membrane fraction from normal cells expressed almost no NADPH oxidase activity when mixed with CGD cytosol, and phosphorylation of the 48-kD protein(s) was again markedly decreased. Protein kinase C (PKC) activity in PMN from the two patients appeared to be normal, suggesting that a deficiency of PKC is not the cause of the defective 48-kD protein phosphorylation and that the cytosolic factor is not PKC. These results demonstrate that the cytosolic factor required for activation of NADPH oxidase also regulates phosphorylation of a specific protein, or family of proteins, at 48 kD. Although the nature of this protein(s) is still unknown, it may be related to the functional and phosphorylation defects present in CGD PMN and to the activation of NADPH oxidase in the cell-free system.

Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. Defects in the phosphorylation of a group of closely related 48-kDa proteins in two forms of chronic granulomatous disease.

When 32P-labeled human neutrophils were activated by exposure to phorbol myristate acetate, three 48-kDa proteins (designated pp48/6.8, pp48/7.3, and pp48/7.8, from their isoelectric points) were found to have become labeled. With maximal stimulation, labeling was complete by 30 s. With lesser degrees of stimulation, the extent of labeling at 2 min correlated with rates of production by the phorbol-treated cells. Increased labeling of these 48-kDa proteins was also seen in cells exposed to f-Met-Leu-Phe. In phorbol-treated neutrophils from patients with X-linked cytochrome b558-negative chronic granulomatous disease, pp48/7.8 was labeled in a normal fashion, but pp48/6.8 and pp48/7.3 failed to take up 32P. In cells from patients with autosomal recessive cytochrome b558-positive chronic granulomatous disease, however, none of the three proteins took up 32P in response to phorbol. The three proteins appear to be very closely related, as indicated by the findings that phosphoserine was the only phosphoamino acid found in any of the three, and all three yielded identical one-dimensional phosphopeptide maps after digestion with either chymotrypsin or staphylococcal proteinase V8. These results reconcile earlier observations on protein phosphorylation in chronic granulomatous disease and provide further evidence for a relationship between the phosphorylation of this group of 48-kDa proteins and the activation of the respiratory burst oxidase.