flavin-adenine-dinucleotide and Granulomatous-Disease--Chronic

Topics: Amino Acid Sequence; Cell Membrane; Cytochrome b Group; Cytosol; Electron Spin Resonance Spectroscopy; Electron Transport; Flavin-Adenine Dinucleotide; Granulomatous Disease, Chronic; Humans; Molecular Sequence Data; Molecular Structure; NADH, NADPH Oxidoreductases; NADP; NADPH Oxidases; Sequence Homology, Amino Acid

Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two X-linked CGD patients with mutations in gp91(phox) that alter the regions in this membrane-bound NADPH oxidase component involved in docking of the cytosolic component p47(phox).. Hydrogen peroxide and superoxide generation, bactericidal activity, and NADPH oxidase protein expression by the patients' neutrophils were measured, and genetic analysis was performed.. We report two patients, each with a novel missense mutation in CYBB, the gene that encodes gp91(phox). Surprisingly, neutrophils from these patients showed total absence of superoxide production, although they retained 13-30% of the hydrogen peroxide production capability. We speculate that this is due to direct electron transfer from flavin adenine dinucleotide (FAD) in gp91(phox) to oxygen, leading to inefficient hydrogen peroxide formation instead of efficient superoxide production.. X-linked CGD patients with mutations that alter the gp91(phox) protein in regions involved in docking of the cytosolic NADPH oxidase component p47(phox) may have higher than expected hydrogen peroxide generation capability.

Topics: Child, Preschool; Flavin-Adenine Dinucleotide; Genetic Diseases, X-Linked; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Male; Membrane Glycoproteins; Middle Aged; NADPH Oxidase 2; NADPH Oxidases; Neutrophils; Oxygen; Superoxides

The X(+)-linked chronic granulomatous disease (X(+)-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X(+)-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X(+)-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.

Topics: Binding Sites; Cell Line; Flavin-Adenine Dinucleotide; Gene Expression Regulation, Enzymologic; Granulomatous Disease, Chronic; Humans; Membrane Glycoproteins; Mutation, Missense; NADP; NADPH Oxidase 2; NADPH Oxidases; Phagocytes; Protein Structure, Tertiary

Mutations in leukocyte NADPH oxidase genes lead to defective respiratory burst in leukocytes and cause chronic granulomatous diseases (CGD) in humans. The most common form of CGD is caused by mutations in the membrane-bound oxidase component gp91phox, which is encoded by the CYBB gene on the X chromosome. We previously reported on a patient with a CYBB mutation (H338Y) that prevents the intracellular trafficking and expression of gp91phox on leukocytes. The capacity of the leukocytes to produce reactive oxygen species (ROS) was rescued by treatment with thapsigargin and flavin adenine dinucleotide (FAD). The increase in ROS production was not due to the increase in cytoplasmic calcium induced by thapsigargin because the treatment of calcium ionophore did not have the same effect. Protein and cellular analyses on leukocytes and cells transfected with GFP-tagged gp91phox mutant showed that treated cells expressed more Endo H-resistant gp91phox protein on the cell surface and are more effective in killing bacteria. Thapsigargin- and FAD-treated CGD leukocytes had enhanced activity in protecting mice from Staphylococcus-induced peritoneal abscess formation in a mouse model of CGD. These results indicate that thapsigargin-FAD ex vivo treatment is effective in rescuing the ROS-producing activity of leukocytes in selected CGD patients.

Topics: Animals; Cytochrome b Group; Disease Models, Animal; Female; Flavin-Adenine Dinucleotide; Granulomatous Disease, Chronic; Humans; Leukocytes; Membrane Glycoproteins; Mice; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidases; Reactive Oxygen Species; Staphylococcus aureus; Thapsigargin

We report here two atypical cases of X-linked CGD patients (first cousins) in which cytochrome b(558) is present at a normal level but is not functional (X91+). The mutations were localized by single-strand conformational polymorphism of reverse transcriptase-polymerase chain reaction amplified fragments and then identified by sequence analysis. They consisted in two base substitutions (C919 to A and C923 to G), changing His303 to Asn and Pro304 to Arg in the cytosolic gp91phox C-terminal tail. Mismatched polymerase chain reaction and genomic DNA sequencing showed that mothers had both wild-type and mutated alleles, confirming that this case was transmitted in an X-linked fashion. A normal amount of FAD was found in neutrophil membranes, both in the X91+ patients and their parents. Epstein-Barr virus-transformed B lymphocytes from the X91+ patients acidified normally upon stimulation with arachidonic acid, indicating that the mutated gp91phox still functioned as a proton channel. A cell-free translocation assay demonstrated that the association of the cytosolic factors p47phox and p67phox with the membrane fraction was strongly disrupted. We concluded that residues 303 and 304 are crucial for the stable assembly of the NADPH oxidase complex and for electron transfer, but not for its proton channel activity.

Topics: Cell Membrane; Cytochrome b Group; Cytosol; Flavin-Adenine Dinucleotide; Granulomatous Disease, Chronic; Humans; Infant; Male; Membrane Glycoproteins; Mutation, Missense; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidase 2; NADPH Oxidases; Neutrophils; Polymorphism, Single-Stranded Conformational; RNA, Messenger; Tetradecanoylphorbol Acetate

Defective NADPH oxidase components prevent superoxide (O-2) generation, causing chronic granulomatous disease (CGD). X-linked CGD patients have mutations in the gene encoding the gp91(phox) subunit of cytochrome b558 and usually lack gp91(phox) protein completely (X91(0)). gp91(phox) is considered to be a flavocytochrome that contains binding sites for NADPH, FAD, as well as heme. We here report a rare X-linked CGD patient whose neutrophils entirely failed to produce O-2, but presented a diminished expression of gp91(phox) containing about one-third of the heme present in normal individuals by Soret absorption. Translocation of cytosolic factors p67(phox) and p47(phox) was normal. However, the FAD content in his neutrophil membranes was as low as that of X91(0) patients, suggesting complete depletion of FAD in his gp91(phox). This was in agreement with the finding that a single base substitution (C1024 to T) changed His-338 to Tyr in gp91(phox) in a predicted FAD-binding domain of the flavocytochrome model. The loss of FAD could not be corrected even after addition of reagent FAD or a FAD-rich dehydrogenase fraction isolated from normal neutrophils to the patient's membranes, in a reconstitution in vitro with normal cytosol. These results indicate that His-338 is a very critical residue for FAD incorporation into the NADPH oxidase system. This is the first such mutation found in CGD.

Topics: Amino Acid Sequence; Binding Sites; Biological Transport; Child, Preschool; Cytochrome b Group; Cytosol; Ferredoxin-NADP Reductase; Flavin-Adenine Dinucleotide; Genetic Linkage; Granulomatous Disease, Chronic; Heme; Histidine; Humans; Male; Membrane Glycoproteins; Membrane Transport Proteins; Molecular Sequence Data; Mutation; NADPH Dehydrogenase; NADPH Oxidase 2; NADPH Oxidases; Neutrophils; Phosphoproteins; Sequence Homology, Amino Acid; Sex Chromosome Aberrations; Superoxides; X Chromosome

The NADPH-binding component of the neutrophil superoxide-generating oxidase was studied in the particulate oxidase fractions obtained from the neutrophils of normal and chronic-granulomatous-disease (CGD) patients. The molecular mass of the NADPH-binding component of the stimulated human neutrophils, which was labelled with the 2',3'-dialdehyde derivative of NADPH and sodium cyanoboro[3H]hydride, was 66 kDa. The 66 kDa component was also labelled in monocytes, but not in red blood cells, platelets and lymphocytes. The particulate oxidase fractions obtained from the patients with CGD had a diminished amount of FAD, whether they contained cytochrome b558 or not. The fractions labelled with the NADPH analogue showed that CGD patients had the NADPH-binding component in the neutrophils. The molecular mass of the component was identical with that of the normal neutrophils. The patients are thought to have an intact NADPH-binding domain of the oxidase in the neutrophils in spite of a diminished amount of FAD in the particulate fractions. The component of the oxidase in the resting neutrophils was also labelled with the analogue. The molecular mass of the component in the resting neutrophils was identical with that of the stimulated neutrophils, and the component was not phosphorylated during the activation process. These results indicate that the NADPH-binding component of the oxidase, which is specific to phagocytes, is present in the resting neutrophils and that the component does not change with respect to molecular mass during the activation process.

Topics: Cytochrome b Group; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Granulomatous Disease, Chronic; Humans; NADH, NADPH Oxidoreductases; NADP; NADPH Oxidases; Neutrophils; Phosphorylation; Photofluorography

The nitroblue tetrazolium (NBT) test is the primary screening test for chronic granulomatous disease (CGD). Neutrophils and other phagocytic cells readily reduce NBT to blue formazan after oxidative stimulation whereas CGD cells remain colorless. In the present study purified eosinophil populations were obtained from CGD patients and normals by exposing peripheral blood to the peptide N-formyl-methionyl-leucyl-phenylalanine and then centrifuged over a discontinuous Percoll gradient. The eosinophils were then incubated in 0.1% NBT (37 degrees C, 15 min) with either phorbol myristate acetate or buffer alone (HEPES with calcium and magnesium). Blue staining characteristic of NBT reduction occurred in the phorbol myristate acetate-stimulated eosinophils of half (4/8) of the CGD patients tested. The staining was most intense between the nuclear lobes of the eosinophil with little staining near the cell periphery. The staining pattern was present in 75.5 +/- 5.3% of the purified eosinophils in those patients in which the phenomenon occurred and was reproducible in the same patients over a 6-month period. Eosinophils from normal individuals tested with NBT and phorbol myristate acetate showed intense staining over the entire cell cytoplasm as did normal neutrophils. Purified CGD eosinophils that did show the staining pattern were not able to produce superoxide (as measured by cytochrome C reduction) when stimulated by phorbol myristate acetate indicating the staining was probably not related to superoxide production. Patients with CGD have a mild eosinophilia (4.6 +/- 0.7% in 11 patients at the National Institutes of Health) and eosinophil staining may account for the small number of positive NBT cells reported in some patients.

Topics: Adolescent; Adult; Child; Child, Preschool; Cytochrome b Group; Eosinophils; Female; Flavin-Adenine Dinucleotide; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Male; Nitroblue Tetrazolium; Staining and Labeling; Tetradecanoylphorbol Acetate; Tetrazolium Salts

Twenty-five patients suffering from chronic granulomatous disease (CGD) and their families were investigated. Defects in the superoxide generating system were characterized at the level of the heme-containing cytochrome b and of the FAD-containing flavoprotein, both localized in the plasma membrane of granulocytes. It was confirmed that in most of the typical cases (18 of 22), the complete inability of superoxide generation was associated with the absence of detectable cytochrome b. Mothers but not fathers of such male patients were characterized by a diminished content of cytochrome b, confirming that the affected gene is localized on the X chromosome. In contrast, the granulocytes of four other typical patients (two female and two male) contained normal amounts of cytochrome b, whereas oxidative activity was absent. Since no abnormality of oxidative activity as well as of cytochrome b was found in granulocytes of the mothers and fathers of these patients, an autosomal recessive mode of inheritance of the disease is probable. The flavoprotein deficiency found in the granulocytes of four male patients was always associated with an absence of detectable cytochrome b. This could indicate a structural relationship between flavoprotein and cytochrome b (e.g., a flavocytochrome). Three further patients with mild X-linked CGD contrasted with the patients with severe or classic X-linked disease; the oxidative activity of their phagocytes was diminished but not absent, and the cytochrome b present, albeit in small amounts.

Topics: Adolescent; Child, Preschool; Cytochrome b Group; Female; Flavin-Adenine Dinucleotide; Flavoproteins; Genes, Recessive; Genetic Linkage; Granulocytes; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Infant; Infant, Newborn; Male; Nitroblue Tetrazolium; Superoxides; X Chromosome

Chronic granulomatous disease (CGD), an immunodeficiency syndrome characterized by extreme susceptibility to bacterial infections, is due to a defect of the respiratory burst in human phagocytes. NADPH oxidase, the enzyme that catalyzes the reduction of oxygen and the release of oxidative radicals, was studied in polymorphonuclear leucocytes (PMNs) in a family affected by an x-linked inheritance form at high penetrance of the disease. The contents of cytochrome b, suggested as the terminal component of the oxidase electron transport chain, and FAD, the hypothetical proximal component of the chain, were determined in patients and in carriers. Cytochrome b showed the typical behaviour of x-linked CGD: total absence in patients, intermediate values in carriers. FAD content evaluated on plasma membranes was less decreased than cytochrome b. Carriers also showed a decrease of this flavoprotein. Cytochrome b and FAD contents were compared to NBT test and superoxide production: a clear correlation was observed for the cytochrome b, but FAD plasma membrane evaluation could also be an interesting tool for the metabolic characterization of the disease in patients and in carriers.

Topics: Adult; Child; Cytochrome b Group; Female; Flavin-Adenine Dinucleotide; Genetic Linkage; Granulomatous Disease, Chronic; Humans; Infant; Male; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Nitroblue Tetrazolium; Oxidation-Reduction; Tetradecanoylphorbol Acetate; X Chromosome

Chronic granulomatous disease (CGD) is a genetically heterogeneous syndrome characterized by a microbial killing defect of polymorphonuclear leukocytes (PMNs) due to lack of superoxide O2-. 2 generation. Recent studies indicate that the neutrophil O2-.-generating system consists of at least two components, flavoprotein--flavin adenine dinucleotide (FAD)--and cytochrome b. We evaluate the cytochrome b and FAD content in PMN from 30 CGD patients. The method for quantitating cytochrome b was modified by using PMN sonicates incubated with azide plus hydrogen peroxide. With this approach, several absorption peaks corresponding to myeloperoxidase and eosinophil peroxidase, which overlap with peaks of cytochrome b, were obliterated from reduced-minus-oxidized spectra, whereas the peaks of cytochrome b were not and could be readily quantitated. Cytochrome b was detected in PMNs from all 24 normal adults (47.4 +/- 2.9 pmol/7.5 X 10(6) cells), was absent in PMNs from 11 male CGD patients and one female CGD patient but was present in normal amounts in PMNs from nine male and nine female CGD patients. Stimulated nitroblue tetrazolium (NBT) tests performed on PMNs from mothers of CGD patients indicated that cytochrome b deficiency was associated with X-linked inheritance, except in one case in which probable autosomal recessive inheritance was demonstrated. The PMN NBT test of the mother of another male patient without cytochrome b deficiency suggested an X-linked form of inheritance. In related studies, the FAD content in PMN particulate fractions was reduced in 4 of 28 CGD patients studied. All four CGD patients with reduced FAD lacked cytochrome b. However, three patients with cytochrome b deficiency had normal FAD. Thus, the results indicate that PMN cytochrome b deficiency is observed in most X-linked and in some autosomal recessive CGD, that cytochrome b deficiency may be associated with FAD deficiency, and that cytochrome b and FAD are normal in most patients with non-X-linked CGD.

Topics: Adult; Chromosome Aberrations; Chromosome Disorders; Cytochrome b Group; Female; Flavin-Adenine Dinucleotide; Genes, Recessive; Granulomatous Disease, Chronic; Humans; Male; Neutrophils; Spectrometry, Fluorescence; Subcellular Fractions

Chronic granulomatous disease (CGD) is a genetically transmitted disorder thought to result from defect(s) in the activation or turnover of the NADPH dependent O2- generating oxidase enzyme system of human neutrophils and monocytes. The normal oxidase may be a flavoprotein-cytochrome b559 complex; therefore, these components of the oxidase were quantitated in the neutrophils from patients and family members of two unrelated CGD kindreds. The male propositus from an X-linked recessive kindred had a neutrophil oxidase fraction with low FAD content (26 pmol/mg protein) and undetectable cytochrome b559 (less than 5 pmol/mg protein). The male propositus from an autosomal recessive kindred had a neutrophil oxidase fraction with low FAD content (34 pmol FAD/mg protein), but normal cytochrome b559 content (170 pmol cytochrome b559/mg protein). Both parents of this latter CGD patient had normal FAD and cytochrome b559 content in their neutrophil oxidase fraction. We conclude that the carrier state in certain X-linked recessive female carriers of CGD can be detected by partial deficiencies of both flavoprotein and cytochrome b559 components of the oxidase, whereas presumed heterozygous carriers of certain autosomal recessive CGD kindreds cannot be detected by this means.

Topics: Child; Cytochrome b Group; Female; Flavin-Adenine Dinucleotide; Free Radicals; Granulomatous Disease, Chronic; Heterozygote; Humans; Male; Neutrophils; Nitroblue Tetrazolium; Phagocytes; Photosystem II Protein Complex; Superoxides

The NADPH-dependent O2-.-generating oxidase in subcellular fractions from the neutrophils of three male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an approximately 0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied.

Topics: Adolescent; Adult; Cytochrome b Group; Flavin-Adenine Dinucleotide; Granulomatous Disease, Chronic; Humans; Infant; Male; NADH, NADPH Oxidoreductases; NADP; NADPH Oxidases; Neutrophils; Oxidation-Reduction; Photosystem II Protein Complex; Spectrometry, Fluorescence; Subcellular Fractions

A subcellular particulate fraction from normal neutrophils that was enriched in NADPH-dependent O-.2-generating activity (Gabig, T. G., Schervish, E. W., and Santinga, J. T. (1982) J. Biol. Chem. 257, 4114-4119) has been further characterized. This preparation contained 0.25 +/- 0.02 nmol of flavin adenine dinucleotide/mg of protein and 0.28 +/- 0.01 nmol of cytochrome b/mg of protein. Measurable amounts of riboflavin or flavin mononucleotide were not present. The flavoprotein was completely resolved from the cytochrome b by selective bile salt extraction of the particulate oxidase fraction. The identical subcellular particulate fraction was studied in the neutrophils from two male patients with chronic granulomatous disease. The neutrophil oxidase fraction from one of the chronic granulomatous disease patients had a cytochrome b component that was spectrally abnormal, but a normal content of flavin adenine dinucleotide. The fraction from this patient's neutrophils corresponding to the resolved flavoprotein from normal cells had fluorescence excitation and emission spectra that were identical to the normal flavoprotein. The neutrophil oxidase fraction from the second chronic granulomatous disease patient had a quantitatively and spectrally normal cytochrome b but less than 8% of the normal amount of flavin adenine dinucleotide. The fraction from the latter patient's neutrophils corresponding to the resolved flavoprotein from normal cells had no detectable flavoprotein by fluorescence excitation and emission spectroscopy. It is postulated that these two patients represent distinct mutants in two separate components of the neutrophil NADPH-dependent O-.2-generating oxidase system, flavoprotein and cytochrome b.

Topics: Cytochrome b Group; Flavin-Adenine Dinucleotide; Flavoproteins; Granulomatous Disease, Chronic; Humans; Male; NADP; Neutrophils; Oxidoreductases; Spectrometry, Fluorescence; Superoxides

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b(-245) at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b(-245) the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b(-245) and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b(-245) of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b(-245) and support a scheme for electron transport thus: [Formula: see text]

Topics: Cell Membrane; Cytochrome b Group; Electron Transport; Flavin-Adenine Dinucleotide; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Light; Neutrophils; Oxidation-Reduction; Phagocytosis; Spectrometry, Fluorescence