Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation.
Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.
Li
Xing Jun
XJ
Groupe de Recherche et d'Etude du Processus Inflammatoire EA 2938 Université Joseph Fourier, Laboratoire Enzymologie, Centre Hospitalier Universitaire, 38043 Grenoble cedex 9, France.
Grunwald
Didier
D
Mathieu
Jacques
J
Morel
Françoise
F
Stasia
Marie-José
MJ
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004579:Electron Transport; D004789:Enzyme Activation; D006098:Granulocytes / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D010100:Oxygen / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; C036888:cytochrome b558; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; C028796:iodophenyl-nitrophenyl-phenyltetrazolium reductase; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D008058:Dihydrolipoamide Dehydrogenase; D010100:Oxygen