The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4

Abstract

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H(2)O(2) to O(2)(·-) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O(2)()-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O(2)(·-). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H(2)O(2) but increased O(2)(·-) formation by Nox4 without affecting Nox1-dependent O(2)(·-) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H(2)O(2). Mutation of this but not of four other conserved histidines also switched Nox4 from H(2)O(2) to O(2)(·-) formation. Thus, H(2)O(2) formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O(2)(·-) egress and/or provide a source for protons, allowing dismutation to form H(2)O(2).

FIGURE 1.

Function of the E-loop of Nox4. A, schematic illustration of the Nox protein and alignment of the amino acid sequence of the E-loop of human and murine Nox1, Nox2, and Nox4. B, schematic illustration of Nox4 and the deletion constructs generated. Boxes represent transmembrane domains (1–6), FAD binding site (FAD), and NADPH binding site (NADPH). Illustrations are not to scale. C, confocal microscopic fluorescent image of Nox4 del218–235+264–273 (red) stably expressed in HEK293 cells counterstained with GRP78 (green) and DAPI. D and E, determination of ROS production of HEK293 cells transiently transfected with the plasmids indicated. O₂^˙̄ generation was determined by L-012 (D), and H₂O₂ formation was determined by luminol + HRP chemiluminescence (E). ROS production was normalized against Nox protein expression determined by Western blot analysis. To facilitate better comparison of the constructs, the ROS formation of the most active construct was set to 100% (corresponding to mean 271,217 cpm (D) and 426,172 cpm (E)) n ≥ 3, mean ± S.E., *, p < 0.05 versus wild-type construct. % of Max., percentage of maximum.

FIGURE 2.

Role of the cysteines in the E-loop of Nox4 and Nox1. A–C, determination of ROS production of HEK293 cells transiently transfected with the plasmids indicated. O₂^˙̄ generation was determined by superoxide dismutase-sensitive cytochrome c reduction (A, left) or by L-012 chemiluminescence (B and C, left). H₂O₂ formation was determined by Amplex Red oxidation (A, right) or luminol + HRP chemiluminescence (B and C, right). The Nox1 plasmid was cotransfected with Noxo1 and Noxa1. Normalization was as in Fig. 1, n ≥ 3, mean ± S.E. *, p < 0.05 versus wild-type construct. 100% corresponds to mean 35,800 and 711,785 cpm (B, left and right) and to mean 734,017 and 59,462 cpm (C, left and right). % of Max., percentage of maximum.

FIGURE 3.

Effect of mutations in the E-loop on the ONOO⁻ production of Nox4. A–C, representative recording (A) and statistical analysis (B and C) of ONOO⁻ production of HEK293 cells transiently transfected with the plasmids indicated as well as protein expression of the constructs. ROS formation was determined by luminol chemiluminescence. After reading the cells in the absence of NO, the NO donor DetaNONOate (100 μmol/liter) was administered (first arrow). The subsequent signal was considered to arise from ONOO⁻ formation. At the end of the experiment, horseradish peroxidase (+HRP) was added (second arrow) to determine H₂O₂ formation. The Nox1 plasmid was cotransfected with Noxo1 and Noxa1. Normalization was as in Fig. 1. Exemplary Western blots of the construct are shown in B and C. n ≥ 3, mean ± S.E., *, p < 0.05 versus wild-type construct. 100% corresponds to mean 7625 and 7818 cpm (B and C, respectively). % of Max., percentage of maximum.

FIGURE 4.

Effect of an antibody directed against the E-loop on the H₂O₂ formation of Nox1 and Nox4. A, representative Western blot of HEK293 cells transfected with the plasmids indicated probed with the antibody mAB8E9, which is directed against the E-loop of Nox4. B and C, determination of H₂O₂ production by luminol + HRP chemiluminescence and O₂^˙̄ production in Cos7 cells transiently transfected with wild-type Nox4 or Nox1 (cotransfected with Noxo1 and Noxa1). The following substances indicated were added to the cells prior to measurements: monoclonal antibody directed against the E-loop (mAb8E9: 1 μg/ml), normal control mouse IgG (1 μg/ml), and the surface cross-linker bis-sulfosuccinimidyl suberate (BS3, 1 mmol/liter). Normalization was as in Fig. 1. n ≥ 3, mean ± S.E., *, p < 0.05 versus control. 100% corresponds to mean 1390 and 1355 cpm (B and C, respectively). % of Max., percentage of maximum.

FIGURE 5.

Role of conserved histidines on the ROS formation of Nox4. A, sequence alignment of Nox4 proteins from different species for conserved histidines on the extracytoplasmic side of the protein and schematic representation of the localization of the conserved histidines within the proteins (white circles). B, ROS production of HEK293 cells transiently transfected with the plasmids indicated. O₂^˙̄ generation was determined by L-012 chemiluminescence (left panel), and H₂O₂ formation was assessed by luminol + HRP chemiluminescence (right panel). Normalization was as in Fig. 1, n ≥ 3, mean ± S.E. *, p < 0.05 versus wild-type construct. 100% corresponds to mean 7647 (left) and 82,425 cpm (right). C, Western blot analysis of the expression of the different histidine mutants in HEK293 cells.

FIGURE 6.

Effect of mutations in the E-loop of Nox4 on Erk1/2 phosphorylation. HEK293 cells were transiently transfected with the plasmids indicated and subsequently subjected to SDS-PAGE and Western blot analysis with the antibodies indicated. A and B, representative blots (A) and statistical analysis (B) of the ratio of phosphorylated to non-phosphorylated Erk1/2 (pERK1/2). To allow better comparison, the signal of wild-type Nox4 was set to 100%. n ≥ 3, mean ± S.E. *, p < 0.05 versus Nox4 construct. % of Max., percentage of maximum.