title: Identification of a region in p47phox/NCF1 crucial for phagocytic NADPH oxidase (NOX2) activation.
abstract: A point mutation in the mouse Ncf1(m1J) gene decreases production of ROS by the phagocytic NOX2 complex. Three mRNA splice variants are expressed, but only one is expressed as a protein, although at lower levels than the WT NCF1 (also known as p47phox). Our aim was to investigate whether the mutant p47phox, lacking 8 aa, is active, but as a result of its low expression, ROS production is decreased in Ncf1(m1J) mice, or whether the mutant p47phox completely lacks the capability to activate the NOX2 complex. The p47phox mutant (Δ228-235), which was equal to the protein in Ncf1(m1J) mice, failed to activate the NOX2 complex. When the deleted region was narrowed down to 2 aa, the p47phox protein remained inactive and failed to translocate to the membrane upon activation. Single amino acid substitutions revealed Thr233 to be vital for ROS production. Residues Tyr231 and Val232 also seemed to be important for p47phox function, as p47phox_Y231G and p47phox_V232G resulted in a >50% decrease in ROS production by the NOX2 complex. In addition, we identified the epitope of the D-10 anti-p47phox mAb. In conclusion, the p47phox protein variant expressed in Ncf1(m1J) mice is completely defective in activating the NOX2 complex to produce ROS, and the effect is dependent on SH3 region amino acids at positions 231-233, which are vital for the proper assembly of the NOX2 complex.
authors.affiliation: MediCity Research Laboratory, University of Turku, Turku, Finland.
authors.forename: Outi
authors.initials: O
authors.lastname: Sareila
authors.forename: Noora
authors.initials: N
authors.lastname: Jaakkola
authors.forename: Peter
authors.initials: P
authors.lastname: Olofsson
authors.forename: Tiina
authors.initials: T
authors.lastname: Kelkka
authors.forename: Rikard
authors.initials: R
authors.lastname: Holmdahl
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008817:Mice, Mutant Strains; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000276:immunology*; D010587:Phagocytosis / Q000235:genetics / Q000276:immunology*; D017354:Point Mutation; D017382:Reactive Oxygen Species / Q000276:immunology*
chemical_list: D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1