The Respiratory Burst Oxidase and the Molecular Genetics of Chronic Granulomatous Disease

The NADPH-binding site of the respiratory burst oxidase system of neutrophils has been proposed to be either at a cytosolic component or at the beta-subunit of cytochrome b558. In this study, affinity labeling of resting and stimulated membranes, the latter having been assembled by all of the oxidase components from both membrane and cytosol, was carried out using [32P]NADPH dialdehyde (oNADPH). Stimulation of human neutrophils with PMA greatly increased O2(-)-generating activity and caused considerable translocation of the cytosolic components p47phox and p67phox. Nevertheless, PMA stimulation did not produce a labeled band which included positions at 47, 67, and approximately 32 kD. The most intense band reflected a molecular mass of 84 kD regardless of the state of activation, but a labeled band was never found near the beta-subunit (91 kD) of cytochrome b558. This 84-kD protein was further confirmed in neutrophils of 14 patients with gp91phox-deficient X-linked chronic granulomatous disease. These results indicate that the NADPH-binding component is not recruited from the cytosol, and also, that a membranous redox component besides cytochrome b558 must be involved in the NADPH oxidase system.

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The respiratory burst oxidase and the molecular basis of chronic granulomatous disease

American Journal of Hematology ◽

10.1002/ajh.2830370410 ◽

1991 ◽

Vol 37 (4) ◽

pp. 263-266 ◽

Cited By ~ 33

Author(s):

Bernard M. Babior

Keyword(s):

Chronic Granulomatous Disease ◽

Respiratory Burst ◽

Molecular Basis ◽

Granulomatous Disease ◽

Respiratory Burst Oxidase

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Cytosolic components of the respiratory burst oxidase: resolution of four components, two of which are missing in complementing types of chronic granulomatous disease.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.86.3.825 ◽

1989 ◽

Vol 86 (3) ◽

pp. 825-829 ◽

Cited By ~ 69

Author(s):

J. T. Curnutte ◽

P. J. Scott ◽

L. A. Mayo

Keyword(s):

Chronic Granulomatous Disease ◽

Respiratory Burst ◽

Granulomatous Disease ◽

Respiratory Burst Oxidase

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

Journal of Experimental Medicine ◽

10.1084/jem.178.6.2047 ◽

1993 ◽

Vol 178 (6) ◽

pp. 2047-2053 ◽

Cited By ~ 31

Author(s):

F E Maly ◽

C C Schuerer-Maly ◽

L Quilliam ◽

C G Cochrane ◽

P E Newburger ◽

...

Keyword(s):

Chronic Granulomatous Disease ◽

B Lymphocytes ◽

Respiratory Burst ◽

Function Analysis ◽

Genetically Engineered ◽

Granulomatous Disease ◽

Intact Cells ◽

Cytochrome B558 ◽

Respiratory Burst Oxidase ◽

O2 Production

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.

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Detection of carriers of the autosomal form of chronic granulomatous disease

Blood ◽

10.1182/blood.v71.2.505.bloodjournal712505 ◽

1988 ◽

Vol 71 (2) ◽

pp. 505-507 ◽

Cited By ~ 1

Author(s):

AJ Verhoeven ◽

ML van Schaik ◽

D Roos ◽

RS Weening

Keyword(s):

Chronic Granulomatous Disease ◽

Respiratory Burst ◽

Granulomatous Disease ◽

Mosaic Pattern ◽

Simple Method ◽

Nbt Test ◽

Activated Neutrophils ◽

Slide Test ◽

Microscopic Slide ◽

Stimulation Of

The NADPH:O2 oxidoreductase catalyzing the respiratory burst in activated phagocytes from healthy individuals is not operative in phagocytes from patients with chronic granulomatous disease (CGD). In a microscopic slide test using the dye nitroblue tetrazolium (NBT), carriers of X-linked CGD can be recognized by a mosaic pattern of NBT- positive and NBT-negative cells, governed by the expression of an unaffected or an affected X chromosome, respectively. Until now, it has not been possible to detect carriers of the autosomal form of CGD (other than by family studies) because all cells of these carriers stain positive in the NBT test. We have investigated whether neutrophils from carriers of autosomal CGD can be recognized by measurement of the rate of oxygen uptake upon stimulation of the cells. It was found that with the phorbol ester PMA as a stimulus, the respiratory burst is significantly lower in autosomal CGD carriers. With serum-treated zymosan as a stimulus, no difference between controls and carriers was observed. The addition of f-Met-Leu-Phe (1 microM) to PMA-activated neutrophils of control donors caused a transient increase in oxygen consumption of about 40%. Under these conditions, an increase of more than 100% was observed in neutrophils from carriers of autosomal CGD. These findings provide a simple method for the detection of carriers of the autosomal form of CGD.

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

Blood ◽

10.1182/blood.v73.6.1416.1416 ◽

1989 ◽

Vol 73 (6) ◽

pp. 1416-1420 ◽

Cited By ~ 106

Author(s):

CA Parkos ◽

MC Dinauer ◽

AJ Jesaitis ◽

SH Orkin ◽

JT Curnutte

Keyword(s):

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Autosomal Recessive ◽

Polyclonal Antibodies ◽

Granulomatous Disease ◽

Absorbance Spectrum ◽

Phagocytic Cells ◽

Inherited Disorders ◽

Western Blots ◽

Respiratory Burst Oxidase

Abstract 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.

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Expression of a chronic granulomatous disease-like defect by fluoride- exhausted neutrophils

Blood ◽

10.1182/blood.v60.4.822.822 ◽

1982 ◽

Vol 60 (4) ◽

pp. 822-826 ◽

Cited By ~ 21

Author(s):

Y Matzner ◽

LM Brass ◽

BJ McMurrich ◽

WA Peters ◽

J Andre-Schwartz ◽

...

Keyword(s):

Chronic Granulomatous Disease ◽

Respiratory Burst ◽

Normal Rate ◽

Granulomatous Disease

Abstract Neutrophils incubated with 20 mM F- express a respiratory burst without degranulating or performing phagocytosis. After 60 min of F- treatment, the burst is exhausted and cannot be restarted. Neutrophils so treated have a microbicidal defect similar to that of chronic granulomatous disease (CGD): they kill Str. mitis at a nearly normal rate, but show a marked impairment in the destruction of S. aureus. They differ from CGD neutrophils in that they also display a defect in motility. This defect, however, is not so severe as to seriously impair their ability to kill bacteria by mechanisms that are independent of endogenously generated microbicidal oxidants.

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Localization of the 47 kDa phosphoprotein involved in the respiratory-burst NADPH oxidase of phagocytic cells

Biochemical Journal ◽

10.1042/bj2600243 ◽

1989 ◽

Vol 260 (1) ◽

pp. 243-248 ◽

Cited By ~ 68

Author(s):

P G Heyworth ◽

C F Shrimpton ◽

A W Segal

Keyword(s):

Cytochrome B ◽

Respiratory Burst ◽

Peptide Mapping ◽

Granulomatous Disease ◽

Myristate Acetate ◽

Phagocytic Cells ◽

Response Curves ◽

Respiratory Burst Oxidase ◽

Cytosolic Factor ◽

Stimulation Of

A 47 kDa phosphoprotein is involved in the respiratory-burst oxidase of phagocytic cells. After stimulation of neutrophils with phorbol myristate acetate, this phosphoprotein was identified in both the cytosol and membranes. Peptide mapping of the two forms resulted in identical patterns of phosphopeptides. Dose-response curves for accumulation of phosphoprotein in the two sites were very similar, whereas the detection of the phosphoprotein in the cytosol preceded that in the membranes. The membrane-associated 47 kDa phosphoprotein was absent from the neutrophils of patients with X-chromosome-linked chronic granulomatous disease, which lack cytochrome b-245, and intermediate levels were detected in the membranes of their heterozygote carrier mothers. Activation of the neutrophil oxidase system appears to be dependent upon phosphorylation of the cytosolic 47 kDa protein and its association with cytochrome b-245 in the membranes. It is probably the cytosolic factor required for reconstitution of the active oxidase in cell-free systems.

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