Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease.

Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91- phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91- phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.

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p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox

Blood ◽

10.1182/blood.v84.8.2767.2767 ◽

1994 ◽

Vol 84 (8) ◽

pp. 2767-2775 ◽

Cited By ~ 44

Author(s):

CD Porter ◽

MH Parkar ◽

AJ Verhoeven ◽

RJ Levinsky ◽

MK Collins ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Cell Lines ◽

Cytochrome B ◽

Function Analysis ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Side Chains ◽

In Vitro Mutagenesis ◽

Granulomatous Disease ◽

P22 Phox

Abstract Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91- phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91- phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.

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Detection of gp91-phox precursor protein in B-cell lines from patients with X-linked chronic granulomatous disease as an indicator for mutations impairing cytochrome b558 biosynthesis

Biochemical Journal ◽

10.1042/bj3150571 ◽

1996 ◽

Vol 315 (2) ◽

pp. 571-575 ◽

Cited By ~ 15

Author(s):

Colin D. PORTER ◽

KURIBAYASHI KURIBAYASHI ◽

Mohamed H. PARKAR ◽

Dirk ROOS ◽

Christine KINNON

Keyword(s):

Nadph Oxidase ◽

B Cell ◽

Chronic Granulomatous Disease ◽

Cell Lines ◽

Binding Domain ◽

Granulomatous Disease ◽

Cytochrome B558 ◽

Stable Association ◽

P22 Phox ◽

Fad Binding

NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22-phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3.

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A newly recognized point mutation in the cytochrome b558 heavy chain gene replacing alanine57 by glutamic acid, in a patient with cytochrome b positive X-linked chronic granulomatous disease

European Journal of Pediatrics ◽

10.1007/bf01955051 ◽

1993 ◽

Vol 152 (6) ◽

pp. 469-472 ◽

Cited By ~ 14

Author(s):

T. Ariga ◽

Y. Sakiyama ◽

K. Tomizawa ◽

S. Imajoh-Ohmi ◽

S. Kanegasaki ◽

...

Keyword(s):

Glutamic Acid ◽

Chronic Granulomatous Disease ◽

Point Mutation ◽

Cytochrome B ◽

Heavy Chain ◽

Chain Gene ◽

Granulomatous Disease ◽

Heavy Chain Gene ◽

Cytochrome B558

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

Journal of Clinical Investigation ◽

10.1172/jci114898 ◽

1990 ◽

Vol 86 (5) ◽

pp. 1729-1737 ◽

Cited By ~ 225

Author(s):

M C Dinauer ◽

E A Pierce ◽

G A Bruns ◽

J T Curnutte ◽

S H Orkin

Keyword(s):

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Gene Structure ◽

Light Chain ◽

Human Neutrophil ◽

Autosomal Recessive ◽

Chromosomal Location ◽

Granulomatous Disease ◽

P22 Phox

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The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils

Biochemical Journal ◽

10.1042/bj2510887 ◽

1988 ◽

Vol 251 (3) ◽

pp. 887-891 ◽

Cited By ~ 120

Author(s):

J A Ellis ◽

S J Mayer ◽

O T Jones

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Oxidase Inhibitor ◽

Superoxide Production ◽

Human Neutrophils ◽

Granulomatous Disease ◽

Nadph Oxidase Inhibitor ◽

Specific Granules ◽

Aerobic And Anaerobic

NADPH-dependent superoxide production by intact human neutrophils is inhibited by DPI (diphenyleneiodonium), when stimulated by either FMLP (N-formylmethionyl-leucyl-phenylalanine) or PMA (phorbol 12-myristate 13-acetate). Addition of 10 microM-DPI abolished the reduction of both the FAD and the cytochrome b components of the NADPH oxidase. DPI inhibition of the oxidase was associated with defective aerobic killing of staphylococci by human neutrophils. Anaerobic killing, phagocytosis, chemotaxis and motility were relatively unaffected by 10 microM-DPI. Degranulation of the azurophil and specific granules, induced by the soluble stimuli FMLP or PMA, and by particulate stimuli was decreased by the presence of DPI. The above effects of DPI on human neutrophils are similar to those found in chronic granulomatous disease.

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Chronic granulomatous disease due to granulocytes with abnormal NADPH oxidase activity and deficient cytochrome-b

Blood ◽

10.1182/blood.v61.3.423.423 ◽

1983 ◽

Vol 61 (3) ◽

pp. 423-428 ◽

Cited By ~ 41

Author(s):

RA Seger ◽

L Tiefenauer ◽

T Matsunaga ◽

A Wildfeuer ◽

PE Newburger

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Maximum Velocity ◽

Granulomatous Disease ◽

Michaelis Constant ◽

Difference Spectra ◽

Oxidase Enzyme ◽

Reduced Activity ◽

Insight Into

Abstract A patient with an X-linked genetic disease resembling chronic granulomatous disease (CGD) but differing in several aspects from previously studied cases is described. The oxidase enzyme of the patient's granulocytes was normally activated, but had reduced activity as shown by an increased Michaelis constant and decreased maximum velocity of NADPH-dependent superoxide production. Cytochrome-b was undetectable in dithionite difference spectra. This CGD-like disease further implicates cytochrome-b as an important component of the microbicidal NADPH oxidase system and provides insight into its role in the enzyme complex.

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Chronic granulomatous disease due to granulocytes with abnormal NADPH oxidase activity and deficient cytochrome-b

Blood ◽

10.1182/blood.v61.3.423.bloodjournal613423 ◽

1983 ◽

Vol 61 (3) ◽

pp. 423-428 ◽

Cited By ~ 1

Author(s):

RA Seger ◽

L Tiefenauer ◽

T Matsunaga ◽

A Wildfeuer ◽

PE Newburger

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Maximum Velocity ◽

Granulomatous Disease ◽

Michaelis Constant ◽

Difference Spectra ◽

Oxidase Enzyme ◽

Reduced Activity ◽

Insight Into

A patient with an X-linked genetic disease resembling chronic granulomatous disease (CGD) but differing in several aspects from previously studied cases is described. The oxidase enzyme of the patient's granulocytes was normally activated, but had reduced activity as shown by an increased Michaelis constant and decreased maximum velocity of NADPH-dependent superoxide production. Cytochrome-b was undetectable in dithionite difference spectra. This CGD-like disease further implicates cytochrome-b as an important component of the microbicidal NADPH oxidase system and provides insight into its role in the enzyme complex.

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