title: Four novel mutations in the gene encoding gp91-phox of human NADPH oxidase: consequences for oxidase assembly.
abstract: The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients with chronic granulomatous disease (CGD) are defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox or p22-phox, which constitute the membrane-bound cytochrome b(558). In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal amounts of nonfunctional cytochrome b(558) in the patients' neutrophils. In phorbol-myristate-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369-->Arg, Gly408-->Glu, and Glu568--> Lys substitutions was strongly disturbed. Only a Thr341-->Lys substitution, residing in a region of gp91-phox involved in flavin adenine dinucleotide (FAD) binding, supported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b(558). Based on mutagenesis studies of structurally related flavin-dependent oxidoreductases, we propose that the Thr341-->Lys substitution results in impaired hydride transfer from NADPH to FAD. Because we found no electron transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical for electron transfer. Apparently, an intimate relation exists between domains of gp91-phox involved in electron transfer and in p47/p67-phox binding. (Blood. 2000;95:666-673)
authors.affiliation: Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, the Laboratory of Experimental and Clinical Immunology, and the Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: J H
authors.initials: JH
authors.lastname: Leusen
authors.forename: C
authors.initials: C
authors.lastname: Meischl
authors.forename: M H
authors.initials: MH
authors.lastname: Eppink
authors.forename: P M
authors.initials: PM
authors.lastname: Hilarius
authors.forename: M
authors.initials: M
authors.lastname: de Boer
authors.forename: R S
authors.initials: RS
authors.lastname: Weening
authors.forename: A
authors.initials: A
authors.lastname: Ahlin
authors.forename: L
authors.initials: L
authors.lastname: Sanders
authors.forename: D
authors.initials: D
authors.lastname: Goldblatt
authors.forename: H
authors.initials: H
authors.lastname: Skopczynska
authors.forename: E
authors.initials: E
authors.lastname: Bernatowska
authors.forename: J
authors.initials: J
authors.lastname: Palmblad
authors.forename: A J
authors.initials: AJ
authors.lastname: Verhoeven
authors.forename: W J
authors.initials: WJ
authors.lastname: van Berkel
authors.forename: D
authors.initials: D
authors.lastname: Roos
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002474:Cell-Free System; D002675:Child, Preschool; D003600:Cytosol / Q000201:enzymology; D006105:Granulomatous Disease, Chronic / Q000097:blood / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D007223:Infant; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D012016:Reference Values; D016897:Respiratory Burst; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013481:Superoxides / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D008562:Membrane Glycoproteins; D013481:Superoxides; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate