Regulation of NADPH oxidase activity in phagocytes: relationship between FAD/NADPH binding and oxidase complex assembly.
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.
Debeurme
Franck
F
From the Chronic Granulomatous Disease Diagnosis and Research Center, University Hospital Grenoble, Therex-TIMC/Imag UMR CNRS 5525, Université Joseph Fourier, 38043 Grenoble Cedex 9.
Picciocchi
Antoine
A
From the Chronic Granulomatous Disease Diagnosis and Research Center, University Hospital Grenoble, Therex-TIMC/Imag UMR CNRS 5525, Université Joseph Fourier, 38043 Grenoble Cedex 9.
Dagher
Marie-Claire
MC
From the Chronic Granulomatous Disease Diagnosis and Research Center, University Hospital Grenoble, Therex-TIMC/Imag UMR CNRS 5525, Université Joseph Fourier, 38043 Grenoble Cedex 9.
Grunwald
Didier
D
iRTSV/CEA, 17 Rue des Martyrs, 38054 Grenoble Cedex 9, France.
Beaumel
Sylvain
S
From the Chronic Granulomatous Disease Diagnosis and Research Center, University Hospital Grenoble, Therex-TIMC/Imag UMR CNRS 5525, Université Joseph Fourier, 38043 Grenoble Cedex 9.
Fieschi
Franck
F
Institut de Biologie Structurale, CEA, 41 Rue Jules Horowitz, Grenoble F-38027; CNRS, UMR 5075, Grenoble F-38027; Université Joseph Fourier, Grenoble F-38041.
Stasia
Marie-José
MJ
From the Chronic Granulomatous Disease Diagnosis and Research Center, University Hospital Grenoble, Therex-TIMC/Imag UMR CNRS 5525, Université Joseph Fourier, 38043 Grenoble Cedex 9. Electronic address: mjstasia@chu-grenoble.fr.
D001665:Binding Sites; D002460:Cell Line; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006105:Granulomatous Disease, Chronic / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009249:NADP / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010586:Phagocytes / Q000201:enzymology*; D017434:Protein Structure, Tertiary
D008562:Membrane Glycoproteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases