Regulation of NADPH oxidase activity in phagocytes: relationship between FAD/NADPH binding and oxidase complex assembly

Abstract

The X(+)-linked chronic granulomatous disease (X(+)-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X(+)-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X(+)-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.

FIGURE 1.

Location and conservation of Nox2 amino acids involved in X⁺-CGD among Noxes and ferredoxin-NADP⁺ reductase family members. A, glycosylated asparagines are located in the external loops of Nox2 and numbered by their residue numbers. The four heme-binding histidines located in the third and fifth transmembrane domains are shown by single-letter code as H. The potential FAD and NADPH binding domains are illustrated by filled boxes. Mutations causing X⁺-CGD forms of CGD are preferentially located in the C-terminal cytosolic part of Nox2. Mutations written in black were not studied here. X⁺-CGD mutations in purple have been previously studied for their functional impact in transgenic PLB-985 cells. X⁺-CGD mutations studied here are in blue. Mutations in blue and green filled boxes are located in the potential NADPH- and FAD-binding sites, respectively. B, amino acids involved in X⁺-CGD and conserved in Nox1, Nox3, and Nox4 homologs and in FNRs are boxed in gray. The red boxes represent the potential FAD-binding sites, and the blue boxes represent the NADPH-binding sites of Noxes highly conserved in FNRs. FENR_ANASO means ferredoxin-NADP⁺ reductase from Anabaena sp., and FRN_SPINAC means ferredoxin-NADP⁺ reductase from S. oleacera.

FIGURE 2.

Expression of WT and mutated Nox2 in transfected X-CGD PLB-985 cells. 5 × 10⁵ differentiated transfected X-CGD PLB-985 cells were incubated with the Nox2 monoclonal antibody 7D5, combined with a phycoerythrin-conjugated anti-mouse IgG (H+L), as described under “Experimental Procedures.” Mouse IgG1 isotype was used as an irrelevant monoclonal antibody. For G408E and G408R mutated Nox2 PLB-985 cells, 7D5 positive clones were sorted by FACS. − and + represent Nox2 expression before and after sorting, respectively. Three positive clones were selected for each mutation and conserved in nitrogen.

FIGURE 3.

Cytochrome b₅₅₈ quantification in transfected X-CGD PLB-985 cells. A, immunodetection of both subunits of cytochrome b₅₅₈, Nox2 and p22^phox, was performed in 1% Triton X-100 soluble extract from PLB-985 transgenic cells, submitted to SDS-PAGE, blotted onto a nitrocellulose sheet, and revealed with monoclonal antibodies 54.1 and 44.1 against Nox2 and p22^phox respectively. Results shown are from one experiment representative of three. B, differential spectra analysis of cytochrome b₅₅₈ in 1% Triton X-100 soluble extract from transfected PLB-985 cells. Reduction was achieved by adding a few grains of sodium dithionite to the sample, and the reduced-minus-oxidized difference spectra were recorded at room temperature with a DU 640 Beckman spectrophotometer. The amounts of cytochrome b₅₅₈ expressed in nanomoles of cytb/mg protein represent the mean ± S.D. of three separate experiments.

FIGURE 4.

NADPH oxidase assembly in transfected PLB-985 cells stimulated by latex beads. A, control of translocation experiments in Nox2-transfected PLB-985 cells and X-CGD PLB-985 cells. Positive control of p47^phox and p67^phox translocation corresponds to WT-Nox2 cells, whereas PLB-985 CGD X⁰ cells were used as a negative control of p47^phox and p67^phox translocation. B, cytosolic mutants located in the active pocket site (FAD/NADPH-binding site); C, mutants located in regions closed to the FAD/NADPH-binding site. p47^phox and p67^phox translocation to the plasma and phagosomal membranes were evaluated by confocal microscopy analysis in WT and mutated Nox2 transfected PLB-985 cells stimulated by PMA-treated latex beads as described under “Experimental Procedures.” The same observations were obtained in four to six independent experiments done for each transfected-PLB-985 cells. D, 10⁷ X⁰-CGD and WT-Nox2 or mutated Nox2 transfected PLB-985 cells were activated with PMA (80 ng/ml) or without PMA and sonicated in ice-cold oxidase buffer as described previously (34). Plasma membranes were separated on a discontinuous sucrose gradient, and the presence of cytosolic factors was visualized by Western blotting as described under “Experimental Procedures.” − and + represent negative or positive cytosolic factors translocation to the plasma membranes, and +/– represents a diminished (or not optimal) cytosolic factors translocation. This is representative of one experiment of two.

FIGURE 5.

X⁺CGD mutation locations within the cytosolic C-terminal domain of Nox2. Surface and schematic representation of the three-dimensional model of the cytosolic C-terminal domain of Nox2. FAD and NADPH are represented as sticks using the CPK color code (carbons are in yellow and cyan for FAD and NADPH, respectively). A, side chain of residue mutated in X⁺-CGD are represented as sticks and are colored orange. B, expanded view on the active site pocket. P415H and C537R mutation are modeled. Side chains are represented as sticks and also as surfaces for mutated ones. C, expanded view on top of the active site pocket, at the theoretical interface with the membranous part of Nox2. C-terminal Phe-570 is represented as sticks and is colored green. D, zoom on the NADPH pyrophosphate interacting loop (408–412), highlighted in red, containing the X⁺CGD mutated residue Gly-408. E, expanded view on the 358–370 β-sheet involved in the binding of the FAD adenosine moiety. X⁺CGD mutation C369R is modeled as sticks (respectively colored in orange and white for original cysteine and arginine mutations). Figures were drawn using PyMol software.