scholarly journals 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

1996 ◽  
Vol 315 (2) ◽  
pp. 571-575 ◽  
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.

scholarly journals X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2196-2202 ◽  
Author(s):  
CD Porter ◽  
MH Parkar ◽  
RJ Levinsky ◽  
MK Collins ◽  
C Kinnon
Keyword(s):  
Gene Therapy ◽  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Large Subunit ◽  
Granulomatous Disease ◽  
Barr Virus ◽  
Inherited Immunodeficiency ◽  
Epstein Barr

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X- linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

scholarly journals X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2196-2202 ◽  
Author(s):  
CD Porter ◽  
MH Parkar ◽  
RJ Levinsky ◽  
MK Collins ◽  
C Kinnon
Keyword(s):  
Gene Therapy ◽  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Large Subunit ◽  
Granulomatous Disease ◽  
Barr Virus ◽  
Inherited Immunodeficiency ◽  
Epstein Barr

Abstract Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X- linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

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

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2767-2775 ◽  
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

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.

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

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2767-2775 ◽  
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.

scholarly journals Altered expression of neutrophil peripheral benzodiazepine receptor in X-linked chronic granulomatous disease

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 184-188
Author(s):  
F Zavala ◽  
F Veber ◽  
B Descamps-Latscha
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Host Defense ◽  
Covalent Binding ◽  
Benzodiazepine Receptor ◽  
Mononuclear Phagocytes ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Peripheral Benzodiazepine Receptor ◽  
Cytochrome B558

This study was aimed at determining whether the peripheral benzodiazepine receptor (PBZDR), which is abundantly expressed on mononuclear phagocytes, is involved in host defense mechanisms depending on phagocyte membrane-associated NADPH-oxidase complex. Analysis by reversible and covalent binding of PBZDR expression on human neutrophils shows that it is modulated during NADPH-oxidase activation with phorbol 12-myristate 13-acetate. Based on a series of 17 patients with chronic granulomatous disease (CGD), results show that PBZDR expression is dramatically impaired in X-linked CGD, an inherited disorder due to a mutation on the gene coding for cytochrome b558 NADPH- oxidase component, whereas it is unaffected in autosomal recessive CGD where cytochrome b558 is normally expressed, suggesting a link between PBZDR and cytochrome b558 expressions. PBZDR can be assigned by covalent binding to an 18-Kd membrane protein. These results suggest that the neutrophil PBZDR, which can accommodate the widely prescribed anxiolytic drug Valium (diazepam), is involved in host defense against pathogens, a function that could be affected by neuroimmune interactions.

scholarly journals Restoration of superoxide generation to a chronic granulomatous disease- derived B-cell line by retrovirus mediated gene transfer

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1125-1129 ◽  
Author(s):  
A Thrasher ◽  
M Chetty ◽  
C Casimir ◽  
AW Segal
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Line ◽  
B Lymphocyte ◽  
Single Gene ◽  
Granulomatous Disease ◽  
Inherited Disorders ◽  
Barr Virus ◽  
Epstein Barr

Failure of a superoxide generating system, the NADPH oxidase, present in neutrophils and other phagocytes gives rise to chronic granulomatous disease (CGD), a group of single-gene inherited disorders all characterized by an extreme susceptibility to pyogenic infection, with potentially fatal consequences. About 30% of CGD cases are caused by an autosomally inherited deficiency of a 47-Kd cytoplasmic component of the oxidase (p47-phox). Epstein-Barr virus (EBV) immortalized B- lymphocyte lines established from these CGD patients also express this NADPH oxidase defect and consequently are rendered incapable of generating superoxide on stimulation. We have used a p47-phox-deficient EBV-transformed B-cell line as a recipient for retroviral transfer of a functional p47-phox cDNA. The presence and activity of the retrovirally encoded p47-phox in the transduced cells is demonstrated and we show that this restores their capacity to generate superoxide.

scholarly journals Chronic granulomatous disease and glutathione peroxidase deficiency, revisited

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3861-3869 ◽  
Author(s):  
PE Newburger ◽  
SE Malawista ◽  
MC Dinauer ◽  
T Gelbart ◽  
RC Woodman ◽  
...  
Keyword(s):  
Glutathione Peroxidase ◽  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Normal Female ◽  
Granulomatous Disease ◽  
Gene Encoding ◽  
Cytochrome B558 ◽  
Peroxidase Enzyme ◽  
Peroxidase Deficiency

We have restudied two kindreds that formed the basis of the original report of autosomal recessive chronic granulomatous disease (CGD) associated with leukocyte glutathione peroxidase deficiency. Case 1 from the original study and the surviving brother of the originally reported case 2 both have severe CGD, with no detectable respiratory burst activity in purified intact neutrophils. However, their leukocytes exhibit normal glutathione peroxidase enzyme activity and gene expression. Examination of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase components known to be defective in CGD reveals no detectable cytochrome b558 nor any membrane activity in a cell-free NADPH oxidase assay system. Molecular analysis of the genes encoding cytochrome b558 subunits shows, in case 1, a C-->T substitution at nucleotide 688 of the gene encoding the gp91-phox subunit of cytochrome b558, resulting in a termination signal in place of Arginine-226. Levels of gp91-phox mRNA are markedly decreased despite normal levels of gene transcription, indicating a post- transcriptional effect of the nonsense mutation on mRNA processing or stability. The X-linked form of CGD developed in this cytogenetically normal female due to the uniform inactivation of the normal X chromosome in her granulocytes, indicated by the expression in her granulocyte mRNA of only one allele of a glucose-6-phosphate dehydrogenase polymorphisms for which she is heterozygous in genomic DNA. Case 2 (of the present study) has distinct mutations in each allele of the p22-phox gene.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Hypoxic induction of gene expression in chronic granulomatous disease- derived B-cell lines: oxygen sensing is independent of the cytochrome b558-containing nicotinamide adenine dinucleotide phosphate oxidase

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 756-761 ◽  
Author(s):  
RH Wenger ◽  
HH Marti ◽  
CC Schuerer-Maly ◽  
I Kvietikova ◽  
C Bauer ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
Cell Lines ◽  
Nicotinamide Adenine Dinucleotide ◽  
Oxygen Sensing ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Wild Type ◽  
Superoxide Generation ◽  
Cytochrome B558 ◽  
Nadph Oxidase Complex

Reduced oxygenation of a variety of cells results in transcriptional upregulation of several genes, including the hematopoietic hormone erythropoietin, the angiogenic vascular endothelial growth factor (VEGF), and glycolytic enzymes such as aldolase. Recently, the heme protein cytochrome b558 of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex has been proposed as a key component of the oxygen-sensing mechanism. Cytochrome b558 consists of the p22phox and gp91phox subunits and is essential for superoxide generation in phagocytes and B lymphocytes. Mutations in these subunits result in cytochrome b558-negative chronic granulomatous disease (cytb- CGD), an inherited disorder in humans characterized by reduced microbicidal activity due to deficient superoxide generation. To test whether NADPH oxidase is involved in oxygen sensing, we exposed wild- type B-cell lines as well as cytb- CGD-derived B cell lines, deficient in either p22phox or gp91phox, to hypoxia (1% oxygen) or CoCl2 (100 mumol/L) and compared the mRNA levels of VEGF and aldolase with the untreated controls. Northern blot analysis revealed unimpaired basal and inducible expression of VEGF and aldolase mRNA in all four cytb- CGD-derived B-cell lines compared with wild-type cells. Furthermore, reconstitution of cytochrome b558 expression in cytb- CGD-derived B cells by transfection with p22phox or gp91phox expression vectors did not modify VEGF and aldolase mRNA expression. Thus, cytochrome b558 of the NADPH oxidase complex appears not to be essential for hypoxia- activated gene expression and can be excluded as a candidate for the putative universal oxygen sensor.

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