Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes
© Kawahara et al; licensee BioMed Central Ltd. 2007
Received: 07 March 2007
Accepted: 06 July 2007
Published: 06 July 2007
NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.
We assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to canonical motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity, as verified by mutational analysis. The presence of these non-canonical, but highly conserved regions suggests that all Nox/Duox may possess a common biological function remained in a long history of Nox/Duox evolution.
This report provides the first comprehensive analysis of the evolution and conserved functions of Nox and Duox family members, including identification of conserved amino acid residues. These results provide a guide for future structure-function studies and for understanding the evolution of biological functions of these enzymes.
Reactive oxygen species (ROS) [e.g., superoxide anion (O2-), hydrogen peroxide (H2O2)] are thought of as cytotoxic and mutagenic; however, recent data point to important biological roles for ROS [1–4]. Phagocytes generate large amounts of O2- as part of their microbicidal activity, which results from activation of a membrane-associated NADPH oxidase. The key redox component of the oxidase is flavocytochrome b558, which is comprised of an O2--generating catalytic subunit, gp91phox (a.k.a., Nox2), and a non-catalytic subunit, p22phox [5–7]. Recent studies indicate that similar NADPH oxidase systems are present in a wide variety of non-phagocytic cells. While the nature of these non-phagocyte NADPH oxidases is still being defined, it is clear that they are functionally and structurally distinct from the phagocyte oxidases.
Genetic approaches have implicated Nox/Duox-derived ROS in biological roles and pathological conditions, including hypertension (Nox1), innate immunity (Nox2, Duox), suppression of pathogen-induced cell death (plant Nox), stomatal closure (plant Nox), otoconia formation in the inner ear (Nox3), biosynthesis of extracellular matrix (Duox), and thyroid hormone biosynthesis (Duox1/2) [18, 24, 29, 43–48]. Although widely expressed, little is known about evolutionary relationships among Nox proteins.
Herein, we analyzed Nox/Duox protein sequences from 14 vertebrates, one urochordate, one echinodermate, three insects, one nematode, four fungi, two red algae, one amoeba, and one green plant. Using this large data set, we report (i) a novel molecular taxonomy and phylogeny of Nox/Duox proteins, (ii) synteny of vertebrate Nox/Duox genes by genome annotation, (iii) evolutionary substitution rates of vertebrate Nox proteins and regulatory subunits, and (iv) identification of key amino acid residues and regions conserved among all Nox proteins.
Molecular taxonomy of Nox domains
We assembled deduced amino acid sequences from 101 Nox/Duox genes (see Additional file 4). Each Nox candidate was preliminarily aligned with human Noxes to check whether the sequences conserved canonical regions required for O2- generation, such as the four heme-ligating histidines corresponding to His-101, His-115, His-209, His-222 of human Nox2 (GenBank™ No. NM_000388). Nox genes are present in most eukaryotes including vertebrates, urochordates, echinodermates, nematodes, insects, fungi, plants amoeba, and red alga, but not in prokaryotes. Schematic domain structures of Nox/Duox family proteins of human, the green plant A. thaliana, fungus Magnaporthe grisea (M. grisea), the cellular slime mold amoeba (D. discoideum), and the red alga (C. crispus) are shown in Figure 1B. All members of Nox/Duox family expressed a Nox domain containing the six transmembrane segments and flavocytochrome moiety (Figure 1A). In addition, Nox5, Duoxes, At-rboh-D, fungal NoxC, and amoeba NoxC all contained an EF hand-containing calcium-binding domain (for details, see the section for EF-hand motif). Duoxes also contained a peroxidase homology domain; whereas, amoeba Noxes B and C also contained an asparagine-rich region (labled "N in Figure 1B). The malaria mosquito Anopheles gambiae (A. gambiae) genome encoded one unique Nox gene, Nox-mosquito (referred to here as "NoxM"), which encodes only the Nox domain and no calcium-binding domain (Figure 1B).
The Nox5 subgroup was composed of the orthologs (#'s 39–52 in Figure 2) present in vertebrates [except for Mus muscles (M. muscles) and Rattus norvegicus (R. norvegicus)], echinoderm, and insects; however, Nox5 was not found in the urochordate C. intestinalis, or the nematode C. elegans. A Nox5 ortholog of a green-spotted pufferfish Tetraodon nigroviridis (T. nigroviridis) was classified in the Nox5 subgroup, as shown in Figure 2 (# 47), but the predicted protein does not contain the N-terminal extension with the calcium-binding domain that is present in other Nox5 orthologs (amino acid sequence is shown in Additional file 4). However, the nucleotide sequence around the presumed start codon (gaggcaugc, methinone codon underlined) also did not match to a consensus Kozak sequence [cc(a/g)ccaugg] , suggesting that the reported sequence is likely to be incomplete. A Nox5 ortholog (# 45) of zebrafish Danio rerio (D. rerio) also did not contain the presumed start codon and the calcium-binding domain (sequence is shown in Additional file 4), suggesting that the sequence of this Nox5 ortholog is also incomplete. The genome of sea urchin S. purpuratus encodes two Nox5 isologs, Nox5A and Nox5B (Figure 2). The Duox subgroup showed broad expression in Bilateria, such as vertebrates, urochordates, echinoderms, nematodes and insects, but was not found in amoeba, fungi, or plants. Plant Nox homologs, previously termed "respiratory burst oxidase homologues (rboh)" , formed a distinct subgroup (Figure 2), and A. thaliana had 10 rboh homologues, suggesting specialized functions or tissue expression.
Noxes representing the NoxA/NoxB subgroup and the NoxC subgroup were present in fungi, and all fungi examined contained both NoxA and NoxB, except for Aspergillus nidulans (A. nidulans), which possessed only a NoxA gene. Yeast [Schizosaccharomyces pombe (S. pombe) and Saccharomyces cerevisiae (S. cerevisiae)] did not possess any Noxes. The domain structure of the NoxA ortholog is similar to that of Nox1-Nox4; whereas, NoxB proteins also have a short N-terminal extension that does not contain any recognizable domains or motifs (Figure 1B). Fungal NoxC, present in M. grisea and Fusarium graminearum (F. graminearum), has an N-terminal EF-hand domain (Figure 1B). The slime mold amoeba D. discoideum, a protozoan that straddles the boundary between animals and plants , contained three Nox isologs, NoxA, NoxB, and NoxC (Figure 1B). Amoeba NoxC has an EF-hand domain as well as an N-terminal extension containing an asparagine-rich region ("N", Figure 1B); however, NoxA and NoxB both lack the EF-hand domain (Figure 1B). The EF-hand-containing subfamilies (Nox5, Duox, NoxC, and plant Nox) were the most abundant of the Noxes, comprising well over half of the taxonomic tree (Figure 2). Unlike other members of the NoxC/NoxD family, algal Noxes (#s 100 and 101 in Figure 2) do not contain an EF-hand domain (Figure 1B), but branched from a root shared by amoeba and fungal NoxC (Figure 2). Therefore, we refer to these algal Nox proteins that lack an EF hand domain as NoxD, which together with NoxC form a distinct subgroup. Because they share structural features common to EF hand-containing Noxes, we speculate that that these Noxes may be regulated by an as-yet unknown calcium-binding protein.
Synteny of Nox/Duox genes in vertebrates
The human Nox3 gene is positioned following TIAM2, TFBIM and CLDN20 on chromosome 6 (Figure 3D), a synteny that was conserved in mammals and chickens Gallus gallus (G. gallus). Puffer fish, fugu T. rubripes and tetraodon T. nigroviridis lacked a Nox3 ortholog, and TIAM2, TFBIM and CLDN20 were followed instead by FILIP1, TMEM30A and COL12A1. In the genome of X. tropicalis, there was greater variation in synteny: TFBIM was present, but neither Nox3 nor other linked markers were seen. A Nox3 gene was not found in the genome of zebrafish D. rerio, but nucleotide fragments encoding these marker genes of D. rerio were too short to demonstrate the absence of Nox3 by syntenic analysis. Thus, Nox3 emerged during evolution sometime after the emergence of fish and amphibians from a common ancestor of birds and mammals.
The synteny of genetic markers surrounding Nox5 was highly conserved in human, dog Canis familliaris (C. familliaris), mouse, rat, chicken, cow Bos taurus (B. taurus), opossum Monodelphis domestica, (M. domestica, a Nox5 ortholog sequence DDBJ™ accession No. BR000304) and frog (Figure 3F). We also found Nox5 gene fragments in rabbit Oryctolagus cuniculus (O. cuniculus) and armadillo Dasypus novemcinctus (D. novemcinctus) draft-sequenced genomes (DDBJ accession No. BR000301 and BR000302, respectively). However, rodents (mouse and rat in this study) lacked Nox5, clearly demonstrating that this gene had been lost. Interestingly, pufferfish T. rubripes and T. nigroviridis had Nox5-like genes, but the gene markers were present on a different scaffold fragment (Figure 3F). Mammals and frog X. tropicalis each had two paralogs of Duox (Figure 3G), while chicken had only one. Fish genomes possessed a single Duox gene that followed a single NIP gene (Figure 3G). Like Duox, the NIP gene has also undergone gene duplication to form NIP1 and NIP2. Interestingly, NIP1 and NIP2 in Figure 3G are identical to DuoxA1 and DuoxA2, respectively, proteins that were recently described to participate in the activation and maturation of Duoxes . Due to the complexity of the tetraploid genomes of zebrafish and incomplete genomic sequence, however, we cannot rule out a second Duox in another chromosomal location.
Structural variations among Nox domains
Substitution rates of Nox domains and Nox regulatory subunits
Identification of critical amino acids common to all Nox/Duox proteins
Variations in calcium-binding domain structures
Phylogeny of Noxes
This report provides the first extensive analysis of Nox sequences and synteny throughout evolution and provides a conceptual framework for future structure/enzymatic function studies and for understanding the diversity of biological functions of these enzymes. Molecular taxonomy (Figure 2) revealed seven Nox/Duox subfamilies rather than the three that were previously identified based on the presence or absence of calcium-binding and peroxidase domains . Significantly, Noxes are not present in prokaryotes. One can speculate that while defense enzymes, such as superoxide dismutase, evolved very early to protect aerobic organisms to protect against accidentally-generated ROS, later organisms subsequently developed the capacity to generate ROS in a regulated, "deliberate" manner, with specific regulatory subunits co-evolving with specific Noxes. The earliest Nox2 ortholog seems to have appeared in the sea urchin S. purpuratus. A number of investigators have suggested that sea urchin has phagocytic cells that express an ortholog of the complement component C3 and can phagocytose against invading microbes [68–70]. Although it is unclear whether these cells produce ROS to kill microbes, the taxonomy shown in Figure 2 implies that the sea urchin expresses a Nox2 ortholog that might play a role in the innate immune response.
The synteny of each Nox/Duox member raises new questions about Nox/Duox evolution. For example, Nox3 in mouse inner ear is essential for formation of otoconia, mineralized structures that participate in the vestibular system in perception of gravity . Fish and amphibians also have otoconia (called otoliths in fish) but do not express Nox3 (Figure 3). This may implicate another Nox, for example Nox1 ortholog, in otoconia formation prior to Nox3 appearance in land vertebrates, or it may point to a unique function of Nox3 in land vertebrates. Kiss et al. have suggested that lactoperoxidase (LPO) functions in peroxidation of the lipid envelope of globular substance in the inner ear together with Nox3 . Interestingly, molecular taxonomy of animal heme peroxidases demonstrates that LPO orthologs emerged in birds and mammals, but not in fish (T. Kawahara and J. D. Lambeth, unpublished observation). It implies that Noxes and their physiological partners evolved simultaneously, resulting in gaining a new function. Mosquito has a unique Nox gene, NoxM. Although a physiological function of NoxM is completely unknown, a unique appearance of NoxM gene in the species imply a possible relationship between NoxM and sucking of blood or a playing role as a principal vector of the pathogen. While the function of Nox5 is not yet understood, its loss in rodents suggests that another Nox may compensate in these species, implying a certain degree of plasticity of Nox isoform function. Alternatively, Nox5 may not perform an essential function, at least in short-lived species.
It is of interest to compare residue substitution rates among Nox isologs, since all members possess fundamentally similar structures in their flavocytochrome domains, including a high degree of conservation of binding sites for prosthetic groups. Substitution rates vary among different proteins, such as EGF (~ 2.5), NGF (~ 1.0), lactate dehydrogenase (~ 0.5), cytochrome c (~ 0.3), and histone H3 (~ 0.014) . The substitution rates of Nox/Duox subfamilies ranged from 0.3 ~ 0.7 (Figure 5); whereas, those of p22phox, organizer proteins (p47phox and NOXO1), and activator proteins (p67phox and NOXA1) were 0.5 ~ 1.2. The substitution rates of Nox2 and its regulatory subunits, p47phox and p67phox, were relatively low, implying evolutionary changes in these proteins are more poorly tolerated. Such a result may be explained by the importance of the biological function of Nox2 in host defense and by the stringent regulation of this enzyme system to prevent inappropriate activation leading tissue damage [5, 58, 59]. In addition, the Nox2 system requires multiple protein interactions among catalytic and regulatory subunits with upstream regulatory subunits and lipids, and these undoubtedly impose strict limitations on the number of tolerated mutations. Although incompletely understood, substitution data point to the critical nature of Duox functions, since changes in the Duox sequence are also poorly tolerated over evolutionary time (Figure 5). Duoxes are implicated in thyroid hormone biosynthesis  and innate immunity in lung , and are distributed in a variety of other tissues where they perform unknown functions. Duox is also implicated in fertilization in sea urchin, where H2O2-supported cross-linking of fertilization envelope proteins prevents polyspermy .
In contrast to other eukaryotes, yeast (S. pombe and S. cerevisiae) did not possess Noxes. Yeast ferric reductase (Fre) has a domain structure similar to Noxes but participates in iron uptake rather than oxygen reduction . Alignment between human Nox2 and Fre proteins demonstrates that the sequence of Fre proteins is very different from the Noxes except for the VXGPFG-motif and NADPH-binding site residues (see Additional file 9). This suggests that this distant homolog has evolved in yeast to carry out an entirely different function, and it is debatable whether it should even be classified with the Nox family.
In addition to binding residues for prosthetic groups, the present study has identified four additional regions (B-loop, TM6-FAD, VXGPFG-motif, and C-terminus) as critical for function in all Noxes. The specific functions of these regions are not yet fully understood; however, mutational analysis demonstrates their importance (Figure 7B). The B-loop (Arg-80) and TM6-FAD (Gly-322) regions of Nox2 appear to participate directly or indirectly in binding to p22phox (Figure 7C), since their mutation prevented co-immunoprecipitation of Nox2 and p22phox. In addition, these mutations prevented glycosylation of Nox2 to form the mature 91 kDa form of the protein, supporting the concept that association with p22phox is a necessary pre-requisite for glycosylation . Nevertheless, these two amino acid residues are also conserved in Noxes that do not require p22phox (e.g., human Nox5). Thus, these residues might mediate another important interaction in Nox5 that is analogous to that with p22phox, and ongoing studies are investigating the roles of these residues in Nox5 function. The presence of non-canonical, but highly conserved residues and regions that are shown in Figure 7A suggests that Noxes might have an unknown common feature relevant to the mechanism of activation or a common biological function. Moreover, these conserved regions may also provide a key to identifying a novel common molecule that interacts and co-operates with all Nox/Duox proteins.
In summary, we report herein an exhaustive analysis of Nox/Duox protein family. The present studies provide a new molecular classification system in which Nox and Duox proteins are organized into seven distinct subfamilies. These studies also identify Nox3 as the most recently emerged Nox. Calcium-regulated, EF-hand-containing Noxes appeared very early during the evolution of eukaryotes. Two mosquitoes possess a unique Nox gene, NoxM, but not fruit fly or ant. Consistent with the physiological importance of Nox2 in innate immunity and Duox for hormone synthesis and host defense, these two Nox proteins are more stringently conserved of all Noxes. By comparison of amino acid sequences, 68 residues were identified as highly conserved among all Nox/Duox orthologs, and the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity. Thus, this report provides a conceptual basis for understanding the evolutionary history of Noxes and Duoxes and provides key structural information relevant to the activation mechanisms of modern Nox/Duox proteins.
Nox/Duox family gene sequences were assembled from the following species: Homo sapiens (H. sapiens, human), C. familliaris (dog), R. norvegicus (rat), M. musculus (mouse), B. taurus (cow to search for Nox5 ortholog), M. domestica, (opossum to search for Nox5 ortholog), D. novemcinctus (armadillo to search for Nox5 ortholog), O. cuniculus (rabbit to search for Nox5 ortholog), G. gallus (chicken), X. tropicalis (frog), D. rerio (zebrafish), T. rubripes (fugu), T. nigroviridis (tetraodon), Oryzias latipes (O. latipes, medaka), C. intestinalis, S. purpuratus (sea urchin), C. elegans, D. melanogaster (fruit fly), A. mellifera, (honeybee), A. gambiae (malaria mosquito), A. aegypti (yellow fever mosquito), A. thaliana as a green plant, D. discoideum (amoeba), Podospora anserina (P. anserina, fungus-Pa), A. nidulans (fungus-An), M. grisea (fungus-Mg), F. graminearum (fungus-Fg), C. crispus (alga-Cc), and P. yezoensis (alga-Py).
In addition to gene accession numbers that have been published in papers cited above [26, 28, 30, 31, 33, 39, 41, 42], existing homologues/orthologs of Nox/Duox were searched using NCBI HomoloGene . Human Nox1-Nox5, Duox1, and Duox2; dog Nox1, Nox4, Duox1, and Duox2; mouse Nox1-Nox4, Duox1, and Duox2; rat Nox1-Nox4, Duox1, and Duox2; chicken Nox3; and fruit fly Nox5 and Duox amino acid sequences were obtained from GenBank, and accession numbers are shown in Additional file 4. BLASTP searches were performed for amino acid sequences predicted computationally from genomes of C. familliaris, R. norvegicus, M. musculus, D. novemcinctus, O. cuniculus, G. gallus, X. tropicalis, D. rerio, T. rubripes, T. nigroviridis, O. latipes, C. intestinalis and C. elegans . Sequences that had >50% identity to the sequence of the closest template were selected.
To identify the Nox/Duox orthologs of sea urchin S. purpuratus and insects (D. melanogaster, A. mellifera, A. gambiae), BLASTP searches were performed using the NCBI sea urchin protein database  and the NCBI Eukaryotic Genome Database , respectively. Fungi Genome BLAST and NCBI BLASTP  and DictyBase BLASTP  were used to search for Nox homologs in fungi (P. anserina, A. nidulans, M. grisea, and F. graminearum) and amoeba (D. discoideum), respectively. The presence of 10 genes encoding A. thaliana Nox homologs has been predicted , which was confirmed by searching the TIGR A. thaliana Protein Database . Sequences and accession numbers of assembled Nox orthologs are listed in Additional file 4, and information on additional database searching is described in Additional file 12. Assembled sequences, including newly defined or previously mis-annotated sequences, were annotated based on molecular taxonomy analysis of the Nox domain (Figure 2), and these sequences were deposited as third party annotation (TPA) sequences in GenBank/EMBL/DDBJ database (accession No. BR000261–BR000304). All amino acid sequences analyzed in this study are shown in Additional file 4 (Nox/Duox proteins) and Additional file 9 (Fre proteins).
To estimate evolutionary substitution rates, we screened orthologs of the regulatory subunits, p22phox, p47phox, NOXO1, p67phox, and NOXA1 in vertebrates (H. sapiens, C. familliaris, R. norvegicus, M. musculus, G. gallus, X. tropicalis, D. rerio, T. rubripes, T. nigroviridis) using servers described above. In this study, partial Nox/Duox or regulatory subunit genes, which lacked either a presumed start codon or a stop codon, were assumed to be intact if they showed >50% identity to the closest homolog. All amino acid sequences of the regulatory subunits analyzed in this study are shown in Additional file 6.
Phylogenic analysis and synteny
Multiple sequence alignment and phylogenetic analyses were carried out with ClustalW [81, 82]. Phylogenetic trees were reconstructed by the neighbor-joining method [83, 84] implemented with Kimura 2-parameter distances . Each node of the phylogenetic tree was evaluated by 1,000 bootstrap replications . Amino acid sequences of Nox and Duox were trimmed and aligned to the length of human Nox2 (see Additional file 5). The additional 4 predicted transmembrane regions of the algal Nox orthologs [residues 372–676 of C. crispus NoxD GenBank™ No. AAZ73480.1 and residues 363–778 of P. yezoensis NoxD GenBank™ No. ABA18724.1] were also trimmed prior to phylogenetic analyses. To elucidate synteny, we used the AlignSliceView program , which provides genome annotation according to DNA-DNA similarity and selected conserved genes among the genomes of vertebrates  as markers. These marker genes are defined in Additional file 10.
Alignment of EF-hand regions
According to the motif search server PROSITE , single and multiple EF-hand motifs were found in the extreme N-terminal region of Nox5, plant Nox, NoxC, and a loop region between 1st and 2nd TM domains of Duox protein. After trimming the N-terminal extension from TM1 of Nox5, plant Nox, NoxC (e.g., residues 1–235 of human Nox5α) and the loop between TM1 and TM2 domain of Duox (e.g., residues 767–1074 of human Duox1), alignment was performed. The alignment is shown in Additional file 8, and amino acid residues corresponding to the EF-hand motifs are shown in Additional file 2.
SWISS-MODEL [65, 89] was utilized to predict protein structure of the EF-hand domain of human Nox5 (residues 1–235 of human Nox5α, GenBank™ No. AF353088) and human Duox1 (residues 767–1074 of human Duox1, GenBank™ No. NM_017434). Modeling of the N-terminal region of Nox5 was carried out based on the structures of calcineurin B subunit isoform 1 (PDB accession No. 1m63F), calcineurin B-like protein 2 (PDB No. 1uhnA), and calcineurin B-like protein 4 (PDB No. 1v1fA). Homology modeling of human Duox1 was performed using calcineurin B subunit isoform 1 (PDB No. 1m63B, 1m63F, and 1auiB). Structures were visualized using the program DeepView .
Estimates of substitution rates of vertebrate Nox/Duox and regulators
Using the estimated divergence time of species and number of identical amino acid residues , we calculate a substitution rate for each amino acid site per 109 years. Detailed methods to calculate rates and amino acid residues are described in Additional files 12 and 3, respectively.
Generation of point mutations of human Nox2
Measurement of ROS production by Nox2-transfected cells
Human embryonic kidney (HEK) 293 cells were grown for 24 hrs in 6-well plates and allowed to reach 50% confluency in 2 ml of culture medium. HEK 293 cells, which endogenously express p22phox, were co-transfected with vectors encoding Nox2, p47phox, p67phox, and an active form of the small GTPase Rac1 [Rac1(V12G)] using FuGENE 6 (Roche Applied Science, Indianapolis, IN). ROS was measured using luminol chemiluminescence, as previously described , and detailed procedures are described in Additional file 12.
Immunoprecipitation of Nox2 and p22phox proteins
Transfected cells were lysed for 20 min on ice with lysis buffer containing 50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% deoxycholate, 1 mM NaVO4, 10 mM NaF, protease inhibitor mixture (Complete™; Roche Applied Science, Indianapolis, IN), 1 mM phenylmethylsulfonyl fluoride, and 100 μM diisopropyl fluorophosphate. The lysates were centrifuged for 5 min at 10,000 × g, and protein G-sepharose (Sigma, St. Louis, MO) was added to the supernatants to remove non-specific binding proteins. After centrifugation, the supernatants were mixed with anti-Nox2 monoclonal antibody 54.1 . Protein G-sepharose was added to each mixture, and the precipitated protein from 5 × 106 cell equivalents was subjected to SDS-PAGE, followed by Western blot analysis.
Western blot analysis
To assess Nox2 expression and ability to bind p22phox, Western blot analysis was performed using monoclonal antibodies, 54.1 and 44.1 against human Nox2 and p22phox, respectively [92, 93]. Detailed procedures are described in Additional file 12.
GraphPad Prism (GraphPad software Inc.) was used for t-test statistical analysis to show significant differences of substitution rates.
List of abbreviations used
NADPH oxidase: Duox, dual oxidase
reactive oxygen species
respiratory burst oxidase homologue
Chronic Granulomatous Disease
phorbol 12-myristate 13-acetate
We express our appreciation to Dr. Al Jesaitis for the generous gift of monoclonal antibody against human p22phox and to Dr. Yasushi Okamura for suggesting of sea urchin genome research. We are grateful to Drs. Dirk Roos, Eunice Laurent and Mrs. Heather Jackson for reading and commenting on the manuscript. We thank Dr. Andreas Fritz and Mr. Robert Esternberg for their advice and help to search for zebrafish orthologs. We also thank Mrs. Asami Kawahara for great cooperation and patience during long nights and weekends of computational analysis at home. This work was supported by NIH grants CA105116, GM067717, AR42426, and RR020185.
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