Crucial Role of Two Potential Cytosolic Regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH Oxidase Activation

Xing Jun Li,Didier Grunwald,Jacques Mathieu,Françoise Morel,Marie-José Stasia

doi:10.1074/jbc.m500226200

Xing Jun Li, Didier Grunwald + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m500226200

Copy DOI

Abstract

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.

Highlights

Baldridge and Gerard [1, 2] discovered the respiratory burst in which dramatic oxygen consumption occurred in neutrophils during bacteria phagocytosis
Leusen and co-workers found that cytosolic factors p47phox and p67phox did not translocate to the plasma membrane of phagocytes from an XϩCGD case because of the D500G missense mutation located in this sequence of gp91phox
We produced two mutants known to disturb cytosolic factor translocation to the plasma membrane, the D500G mutant, which had reproduced a human XϩCGD case [30], and the RR9192EE mutant of the B loop of Nox2 previously studied by BiberstineKindade et al [35]

Summary

Introduction

Baldridge and Gerard [1, 2] discovered the respiratory burst in which dramatic oxygen consumption occurred in neutrophils during bacteria phagocytosis. Access of NADPH into the binding site could potentially be regulated by interaction of this loop with cytosolic oxidase factors. A peptide mimicking 491–504 residues of Nox inhibited the translocation of p47phox and p67phox to the membrane [30] In another XϩCGD case consisting of a ⌬488–497 gp91phox deletion in the ␣-helical loop, translocation of p47phox and p67phox to the plasma membrane occurred normally [40]. Dinauer and co-workers have studied the role of a second cytosolic region of Nox, the B loop, on oxidase activity [35]. They found that two Arg at positions 91 and 92 were essential for NADPH oxidase activity and assembly. Nox, and Nox resemble Nox in that they consist of six ␣-helix transmembrane regions in the N-terminal, with two intracytosolic loops

Objectives

Results

Conclusion