title: Identification of NOX2 regions for normal biosynthesis of cytochrome b558 in phagocytes highlighting essential residues for p22phox binding.
abstract: Cytochrome b558, the redox core of the NADPH oxidase (NOX) complex in phagocytes, is composed of NOX2 and p22phox, the synthesis of which is intimately connected but not fully understood. We reproduced 10 rare X-minus chronic granulomatous disease (CGD) mutations of highly conserved residues in NOX1-NOX4, in X0-CGD PLB-985 cells in order to analyse their impact on the synthesis of cytochrome b558. According to the impact of these mutations on the level of expression of NADPH oxidase 2 (NOX2) and its activity, mutants were categorized into group A (W18C, E309K, K315del and I325F), characterized by a linear relationship between NOX2 expression and NOX activity, and group B (H338Y, P339H, G389A and F656-F570del), showing an absence of NOX activity associated with variable levels of NOX2 expression. These last residues belong to the FAD-binding pocket of NOX2, suggesting that this functional domain also plays a role in the structural integrity of NOX2. Finally, we observed an abnormal accumulation of p65 (65-kDa monomer), the NOX2 precursor and p65-p22phox dissociation in the W18C, E309K, I325F and G389A mutants, pointing out a possible role of the first transmembrane domain (Trp18), and the region between the membrane and the dehydrogenase domain of NOX2 (Glu309, Ile325 and Gly389), in the binding with p22phox.
authors.affiliation: *Chronic Granulomatous Disease Diagnosis and Research Centre, Therex-TIMC/Imag, UMR CNRS 5525, Université Grenoble Alpes, Grenoble, F-38041, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Beaumel
authors.affiliation: ‡iRTS/CEA, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Grunwald
authors.affiliation: §Université Grenoble Alpes, Institut de Biologie Structurale (IBS), CNRS, F-38044 Grenoble, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Fieschi
authors.affiliation: *Chronic Granulomatous Disease Diagnosis and Research Centre, Therex-TIMC/Imag, UMR CNRS 5525, Université Grenoble Alpes, Grenoble, F-38041, France.
authors.forename: Marie José
authors.initials: MJ
authors.lastname: Stasia
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D003573:Cytochrome b Group / Q000096:biosynthesis*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010586:Phagocytes / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human