scholarly journals X-Linked Chronic Granulomatous Disease: Mutations in the CYBB Gene Encoding the gp91-phox Component of Respiratory-Burst Oxidase

1998 ◽  
Vol 62 (6) ◽  
pp. 1320-1331 ◽  
Author(s):  
Julie Rae ◽  
Peter E. Newburger ◽  
Mary C. Dinauer ◽  
Deborah Noack ◽  
Penelope J. Hopkins ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Granulomatous Disease ◽  
Gene Encoding ◽  
Disease Mutations ◽  
Cybb Gene ◽  
Respiratory Burst Oxidase

scholarly journals NADPH-binding component of the respiratory burst oxidase system: studies using neutrophil membranes from patients with chronic granulomatous disease lacking the beta-subunit of cytochrome b558.

1994 ◽  
Vol 179 (1) ◽  
pp. 291-297 ◽  
Author(s):  
S Tsunawaki ◽  
H Mizunari ◽  
H Namiki ◽  
T Kuratsuji
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Beta Subunit ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Oxidase System ◽  
Cytochrome B558 ◽  
Respiratory Burst Oxidase ◽  
Stimulation Of ◽  
Binding Component

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.

scholarly journals Retrovirus-mediated reconstitution of respiratory burst activity in X- linked chronic granulomatous disease cells

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3311-3316 ◽  
Author(s):  
A Kume ◽  
MC Dinauer
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Myeloid Cell ◽  
Gene Replacement ◽  
Burst Activity ◽  
Granulomatous Disease ◽  
Wild Type ◽  
Respiratory Burst Activity ◽  
Gene Encoding ◽  
Functional Correction

X-linked chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a recombinant retrovirus vector was constructed and evaluated for its expression of human gp91phox in a human X-CGD myeloid cell line in which the endogenous gp91phox gene had been disrupted by gene targeting. The retrovirus construct, Zip/PGKgp91, was first introduced into the GP+envAm12 amphotropic packaging line and yielded virus producer clones with estimated titers of up to 1 x 10(5) cfu/mL. Coculture infection of X- CGD myeloid cells with Zip/PGKgp91 resulted in restoration of respiratory burst activity to 15% of the cells. Isolated clonal infectants expressed relatively low levels of recombinant gp91phox (< or = 12% of wild-type), but exhibited considerable superoxide- generating activity (up to nearly 60% of wild-type). These results show the feasibility of phenotypic correction of CGD using gene replacement therapy and suggest that even modest levels of gp91phox expression may lead to considerable functional correction of X-CGD neutrophils.

scholarly journals Retrovirus-mediated reconstitution of respiratory burst activity in X- linked chronic granulomatous disease cells

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3311-3316 ◽  
Author(s):  
A Kume ◽  
MC Dinauer
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Myeloid Cell ◽  
Gene Replacement ◽  
Burst Activity ◽  
Granulomatous Disease ◽  
Wild Type ◽  
Respiratory Burst Activity ◽  
Gene Encoding ◽  
Functional Correction

Abstract X-linked chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a recombinant retrovirus vector was constructed and evaluated for its expression of human gp91phox in a human X-CGD myeloid cell line in which the endogenous gp91phox gene had been disrupted by gene targeting. The retrovirus construct, Zip/PGKgp91, was first introduced into the GP+envAm12 amphotropic packaging line and yielded virus producer clones with estimated titers of up to 1 x 10(5) cfu/mL. Coculture infection of X- CGD myeloid cells with Zip/PGKgp91 resulted in restoration of respiratory burst activity to 15% of the cells. Isolated clonal infectants expressed relatively low levels of recombinant gp91phox (< or = 12% of wild-type), but exhibited considerable superoxide- generating activity (up to nearly 60% of wild-type). These results show the feasibility of phenotypic correction of CGD using gene replacement therapy and suggest that even modest levels of gp91phox expression may lead to considerable functional correction of X-CGD neutrophils.

scholarly journals Cloning of murine gp91phox cDNA and functional expression in a human X- linked chronic granulomatous disease cell line

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 2005-2010 ◽  
Author(s):  
H Bjorgvinsdottir ◽  
L Zhen ◽  
MC Dinauer
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cell Line ◽  
Respiratory Burst ◽  
Myeloid Cell ◽  
Functional Expression ◽  
Granulomatous Disease ◽  
Gene Encoding ◽  
Cytochrome B558 ◽  
Mammalian Expression ◽  
Gp91phox Subunit

Abstract The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.

scholarly journals Cloning of murine gp91phox cDNA and functional expression in a human X- linked chronic granulomatous disease cell line

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 2005-2010 ◽  
Author(s):  
H Bjorgvinsdottir ◽  
L Zhen ◽  
MC Dinauer
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cell Line ◽  
Respiratory Burst ◽  
Myeloid Cell ◽  
Functional Expression ◽  
Granulomatous Disease ◽  
Gene Encoding ◽  
Cytochrome B558 ◽  
Mammalian Expression ◽  
Gp91phox Subunit

The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.

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

1993 ◽  
Vol 178 (6) ◽  
pp. 2047-2053 ◽  
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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