Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gauss, K. A.
Right arrow Articles by Quinn, M. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gauss, K. A.
Right arrow Articles by Quinn, M. T.
(Journal of Leukocyte Biology. 2002;71:319-328.)
© 2002 by Society for Leukocyte Biology

Cloning and sequencing of rabbit leukocyte NADPH oxidase genes reveals a unique p67phox homolog

Katherine A. Gauss*, Patrice L. Mascolo*, Daniel W. Siemsen*, Laura K. Nelson*, Peggy L. Bunger*, Patrick J. Pagano{dagger} and Mark T. Quinn*

* Department of Veterinary Molecular Biology, Montana State University, Bozeman; and
{dagger} Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan

Correspondence: Mark T. Quinn, Ph.D., Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717. E-mail: mquinn{at}montana.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The NADPH oxidase plays an important role in immune and nonimmune cell functions. Because rabbits represent an established model for studying a number of important disease processes that involve NADPH oxidase activity, we carried out studies to clone and sequence all five rabbit leukocyte NADPH oxidase genes. Comparison of the rabbit sequences with those of other species showed that, with the exception of p67phox, the rabbit phox proteins were highly conserved. In contrast, rabbit p67phox had a very divergent C-terminus and was 17 amino acids longer than any other known p67phox homolog. This was surprising, given the high degree of conservation among all of the phox proteins sequenced previously. To evaluate the functional consequences of this difference, wild-type rabbit p67phox and a mutated rabbit p67phox missing the C-terminal 17 amino acids were expressed and analyzed in a cell-free assay. Our results show that the full-length and truncated rabbit p67phox proteins were able to support oxidase activity, although the truncated form reproducibly supported a higher level of activity than full-length p67phox. These studies contribute to our understanding of the nature of the leukocyte NADPH oxidase in different species and will be valuable in future research using the rabbit model.

Key Words: neutrophil • superoxide anion • host defense


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polymorphonuclear leukocytes or neutrophils play an important role in the body’s host defense against bacterial and fungal pathogens [1 ]. These phagocytic cells respond to the presence of a pathogen by generating microbicidal oxidants designed to kill the pathogen [1 ]. The generation of microbicidal oxidants by neutrophils results from the activation of a multiprotein enzyme complex known as the NADPH oxidase (reduced nicotinamide adnenine dinucleotide phosphate), which is responsible for transferring electrons from NADPH to O2, resulting in the formation of superoxide anion (O2-; reviewed in refs. [2 , 3 ]). O2- is rapidly converted to secondary toxic oxygen species, such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), which can efficiently kill microorganisms and in combination with the primary and secondary granule contents, comprise the primary host-defense mechanism used by neutrophils [3 ]. The importance of the NADPH oxidase to host immunity is demonstrated by the recurrent infections that occur in individuals with chronic granulomatous disease (CGD), resulting from genetic defects of the NADPH oxidase [4 ].

Activation of the NADPH oxidase system involves the assembly of several neutrophil proteins, some located in the plasma membrane and others in the cytosol (reviewed in refs. [2 , 3 ]). The key membrane-associated proteins comprise flavocytochrome b558 (known as flavocytochrome b), which functions as the redox center of the oxidase and is a heterodimer of two tightly associated polypeptides (gp91phox and p22phox) [5 ]. The cytosolic NADPH oxidase proteins include p40phox, p47phox, p67phox, as well as a low molecular-weight GTPase, Rac2 (reviewed in ref. [6 ]).

Through analysis of neutrophils from patients with CGD, p22phox, p47phox, p67phox, and gp91phox have been shown to be absolutely required for NADPH oxidase activity [4 ]. However, the mechanism by which the cytosolic proteins assemble with flavocytochrome b is complex and not completely understood. Previous studies by several groups have demonstrated that p47phox and p67phox exist in a complex in the cytosol [7 ], however p47phox seems to be the primary cytosolic component to interact with flavocytochrome b during the assembly process, and its association with flavocytochrome b is a prerequisite for binding p67phox [8 ]. During NADPH oxidase activation, p47phox has been shown to undergo extensive phosphorylation [9 , 10 ], and inhibition of phosphorylation during neutrophil activation markedly decreases O2- generation and translocation of p47phox and other cytosolic oxidase proteins [11 ]. Less is understood regarding the role of p67phox in NADPH oxidase. It has been shown that p67phox is phosphorylated during neutrophil activation [12 ], and recently Forbes et al. [13 ] mapped the major p67phox phosphorylation site to Thr233. In addition, it has been proposed that p67phox contains the NADPH-binding site of the NADPH oxidase [14 ] and that p67phox may be able to bind directly to flavocytochrome b through a Rac-dependent mechanism [15 ]. This point is controversial, however, because previous studies by several groups demonstrated that flavocytochrome b itself contained the NADPH-binding site (e.g., see ref. [16 ]). Nevertheless, it may be possible that the two NADPH oxidase components cooperate in the formation of the NADPH-binding site. In addition to p47phox and possibly flavocytochrome b, p67phox has also been shown to bind to Rac (Rac1 and/or Rac2) [17 ] and p40phox [18 ] and clearly plays an important role in NADPH oxidase assembly [19 ].

Although the NADPH oxidase of human neutrophils is by far the best characterized, this system is also important for host defense against infectious disease in essentially all other vertebrates. It is also clear that inflammatory responses, including NADPH oxidase activity, in neutrophils from various species are not necessarily identical to those of human cells and that it is important to study these processes in different species to provide a broader understanding of host defense in these animals. This is especially important for animals used as model organisms in studies relevant to inflammation and, more specifically, NADPH oxidase function. In addition, a comparison of the structure and function of neutrophil NADPH oxidase proteins from other species can lead to a better understanding of the role of these proteins in human cells.

Rabbits have been used extensively as model animals for studying a number of inflammatory processes (reviewed in ref. [20 ]). For example, much of our understanding of the N-formyl peptide chemoattractant receptor and its role in inflammation has been derived from studies on rabbit neutrophils (e.g., see ref. [21 ]). Rabbits have also been used as experimental models for a number of diseases, including pulmonary diseases [22 ], atherosclerosis [23 ], and hypertension [24 ]. These diseases appear to involve NADPH oxidase activity to some degree, yet very little is known about the structure of this enzyme system in rabbits. Consequently, the role of the NADPH oxidase in these diseases is not well understood. Previously, Hitt and Kleinberg [25 ] used comparative immunoblotting to show that homologous NADPH oxidase proteins were present in rabbit neutrophils, however not much else is known about this system in rabbits. To address this issue, we initiated studies to investigate the nature of the rabbit NADPH oxidase.

In the studies shown here, we cloned and sequenced all five rabbit leukocyte NADPH oxidase phox proteins and provide a comparative analysis of the rabbit sequences with other known phox sequences. Because rabbit p67phox was much longer than any other p67phox homolog sequenced to date, we also expressed rabbit p67phox and a truncated rabbit p67phox missing the additional C-terminal 17 amino acids and analyzed both proteins in a cell-free assay system to evaluate functional consequences of this difference. These studies contribute to our understanding of the structure and function of the leukocyte NADPH oxidase in different species and will be valuable in future research efforts using the rabbit model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Histopaque 1077 and 1119 were purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco’s phosphate-buffered saline (DPBS) and oligonucleotide primers were purchased from Gibco-BRL (Grand Island, NY). Alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG and alkaline phosphate substrate kits were from BioRad (Hercules, CA). PCR sequencing kits were from Applied Biosystems (Foster City, CA). Magnagraph nylon membranes were from Micron Separations (Westboro, MA). The rabbit leukocyte cDNA library used in these studies was a kind gift from Dr. Javier Navarro (University of Texas Medical Branch, Galveston).

Polymerase chain reaction (PCR) and sequencing
PCR reactions were performed as described previously [26 ] using a Robocycler 40 (Stratagene, La Jolla, CA) or DNA engine (MJ Research, Watertown, MA) and Taq Plus enzyme (Stratagene). For cycle sequencing, PCR was performed on a Perkin Elmer 2400 thermal cycler using Big Dye terminators, and the samples were sequenced on an ABI 310 genetic analyzer (Perkin-Elmer ABI, Foster City, CA). The nucleotide sequence translation and analyses were performed with DNAStar software (Madison, WI).

Cloning of rabbit NADPH oxidase genes
Rabbit NADPH oxidase proteins p22phox, p40phox, p47phox, p67phox, and gp91phox were cloned from a rabbit neutrophil cDNA library [27 ]. Briefly, cDNAs corresponding to the full-length open-reading frame (ORF) of bovine p22phox (576 bp), p40phox (1020 bp), and gp91phox (1713 bp) and partial ORF of bovine p47phox (584 bp representing nucleotides 12–560) were generated by PCR from bovine cDNAs [26 , 28 ]. Additionally, cDNA corresponding to the partial ORF of rabbit fibroblast p67phox (714 bp homologous to nucleotides 221–935 of the human sequence) was generated from rabbit aortic-advential fibroblast total RNA using RT-PCR as described previously [29 ]. All cDNAs were purified by agarose gel electrophoresis, labeled with 32P using a DECAprime II kit (Ambion, Austin, TX), and used to screen the rabbit neutrophil cDNA library using standard protocols. Approximately 5 x 105 clones were screened under high stringency using the following plaque hybridization conditions: 50% formamide, 6x saline sodium citrate (SSC), 2x Denhardt’s solution, 50 mM NaH2PO4, pH 7.0, 0.1 mg/ml tRNA, and 50% dextran sulfate at 42°C for 16 h. The filters were washed with 2x SSC, 0.1% sodium dodecyl sulfate (SDS), at room temperature three times for 15 min and then with 0.1x SSC, 0.1% SDS, at 50°C two times for 30 min. After tertiary screening, positive clones were obtained for rabbit p22phox (three clones), p40phox (three clones), p47phox (one clone), p67phox (one clone), and gp91phox (three clones). Phage lysates from the positive clones were amplified by PCR using {lambda}gt11 primers 5'-GGTGGCGACGACTCCTGGAGCC-3' and 5'-AACTGTGGTCTGGTTGACCATTAC-3' for the forward and reverse primers, respectively, as well as internal primers as described previously [26 , 28 ]. Three different PCR products from each clone were inserted into a pT-Adv sequencing vector using the AdvanTAge PCR cloning kit (Clontech, Palo Alto, CA) and sequenced to generate consensus sequences. Vector primers were used to sequence the 5' and 3' ends of each insert via cycle sequencing as described above. All clones were sequenced completely in forward and reverse directions to confirm the sequence.

The rabbit p67phox clone isolated by cDNA library screening was significantly longer than all other known p67phox homologs. To confirm this difference was not a result of an artifact in the library, we prepared total RNA from purified rabbit leukocytes using TRIzol reagent (Gibco-BRL) and performed RT-PCR using the reverse primer 5'-CCGCACGTCCTAATCTCAC-3' to amplify the 3' end of rabbit p67phox. The RT reaction was carried out at 42°C for 30 min, and the products were amplified by PCR using 5'-GAATGGCTGGAAGGCGAGTGCAGG-3' and 5'-CCGCACGTCCTAATCTCAC-3' as forward and reverse primers, respectively. PCR amplification conditions were 94°C for 1 min, 64°C for 1 min, and 72°C for 1 min at 35 cycles. The PCR product was gel-purified using a QIAquick gel extraction kit (Qiagen, Valencia, CA), and the insert was cloned into the pT-Adv sequencing vector for sequencing. Sequence analyses confirmed the extra 51 bases at the 3' end.

Expression and purification of full-length and truncated rabbit p67phox
The full-length rabbit p67phox clone was digested out of pT-Adv using EcoRI. Truncated rabbit p67phox was prepared from the full-length rabbit p67phox cDNA using 5'-CCGGAATTCATGTCCCTGGTGGAGGCC-3' and 5'-GCACGCCCAGCGCCGGCTGAGAATTCC-3' as forward and reverse primers, respectively, to remove 51 bases at the 3' end and add EcoRI restriction sites to the 5' and 3' ends of the truncated product. PCR was performed using pfu Turbo Taq (Stratagene) with conditions of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s at 25 cycles. The product was gel-purified using QIAquick gel extraction kit and digested with EcoRI. The full-length and truncated forms of rabbit p67phox were then ligated in-frame into the EcoRI site of the pET30 expression vector (Novagen, Madison, WI). The sequences and orientation of both inserts were confirmed by cycle sequencing as described above.

Full-length and truncated rabbit p67phox were expressed as His tagged-fusion proteins in Escherichia coli BL-21 (DE3) cells and were purified on His-Bind columns following the manufacturer’s protocol (Novagen pET System Protocols). The concentrations of all proteins (>95% pure) were determined with a BioRad (Richmond, CA) protein assay using bovine serum albumin as a standard.

Fractions containing the highest levels of recombinant p47phox and p67phox were used in functional assays as described below.

Preparation and fractionation of leukocytes
Neutrophils were purified from human blood using dextran sedimentation followed by hypotonic lysis of red blood cells and Histopaque gradient separation, as previously described [30 ]. Rabbit leukocytes were purified using hetastarch sedimentation followed by hypotonic lysis of red blood cells [31 ]. Rabbit and human cells were disrupted by N2 cavitation, and membrane and cytosolic fractions were prepared from the cavitates by sequential centrifugation [30 ].

Cell-free NADPH oxidase reconstitution assay
Amphiphile-activated NADPH oxidase activity was measured in a cell-free NADPH oxidase assay system, as described previously [30 ]. Briefly, assays were performed in 200 µl buffer containing 10 mM potassium phosphate (pH 6.7), 130 mM NaCl, 1 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA), 10 µM flavin adenine dinucleotide, 2 mM NaN3, 50 µM cytochrome c, and 10 µM GTP{gamma}S. Each well contained 6 µg human neutrophil membrane protein, 8 µg human p47phox, the indicated concentrations of rabbit or human p67phox, and 2 µg Rac2. Assays were incubated with 30 µM arachidonic acid for 5 min, followed by the addition of 200 µM NADPH to initiate the reaction, and the change in absorbance at 550 nm was measured continuously over 15 min at 25°C in a microtiter plate reader. Results are presented as Vmax rates, calculated using an {varepsilon} = 1.85 mM-1 cm-1 for cytochrome c, and expressed as nmoles O2-/min/mg membrane protein. Duplicate reactions containing 100 µg/ml superoxide dismutase (SOD) were conducted in parallel and subtracted from the results to obtain SOD-inhibitable activity.

Electrophoresis and Western blot analysis
Samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on polyacrylamide gradient gels and transferred to nitrocellulose membrane as described previously [32 ]. For reference, prestained molecular-weight standards (BRL, Bethesda, MD) were included on all gels. Western blots were probed with previously characterized monoclonal and polyclonal antiphox protein antibodies [32 , 33 ] followed by the appropriate alkaline phosphatase-conjugated secondary antibody (BioRad). The blots were developed using a BioRad alkaline-phosphatase development kit. Image analysis for molecular-weight determination was performed with an IS-1000 Alpha Imager digital imaging system and Alpha Image software (Alpha Innotech, San Leandro, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and sequencing of rabbit NADPH oxidase genes
To clone the rabbit NADPH oxidase genes, full-length cDNAs of bovine p22phox, p40phox, gp91phox, a partial cDNA of bovine p47phox, and a partial cDNA of rabbit fibroblast p67phox were labeled with 32P and used individually to screen the {lambda}gt11 rabbit neutrophil cDNA library with a standard plaque hybridization assay. Positive plaques were picked, and each clone was amplified by PCR. The rabbit cDNAs were then by PCR cloned into a PCR cloning vector and sequenced completely using vector primers. For sequence verification, the clones were sequenced twice from both strands.

The cDNA obtained in our p22phox screen was 666 nucleotides long and contained an ORF of 582 nucleotides encoding 194 amino acids (GenBank accession #AF323787). Comparison of the rabbit p22phox amino acid sequence with the other known p22phox sequences demonstrated considerable similarity with the human (81.5%), bovine (80.2%), porcine (81.3%), rattus (81.3%), murine (81.0%), and dolphin (78.1%) homologs (Fig. 1 ).



View larger version (53K):
[in this window]
[in a new window]
  		Figure 1. Comparison of rabbit p22phox amino acid sequence with other known p22phox sequences. The predicted p22phox amino acid sequences were aligned using MEGALIGN software (DNAStar). Amino acids identical to the rabbit sequence are indicated by dashes (–), and amino acids absent are indicated by dots (.). The predicted transmembrane hydrophobic regions are enclosed in rectangles, and the location of the invariant histidine residue in all p22phox homologs is indicated by a star (*). The putative p47phox-binding domain mapped to the human p22phox protein is underlined in bold (labeled A). See text for further details.

 
The cDNA obtained in our gp91phox screen was 1857 nucleotides long and contained an ORF of 1713 nucleotides encoding 570 amino acids (GenBank accession #AF323788). Comparison of the rabbit gp91phox amino acid sequence with the other known gp91phox sequences also demonstrated considerable similarity with the human (91.4%), bovine (90.5%), porcine (92.6%), murine (91.2%), and dolphin (90.0%) homologs (Fig. 2 ).



View larger version (47K):
[in this window]
[in a new window]
  		Figure 2. Comparison of rabbit gp91phox amino acid sequence with other known gp91phox sequences. The predicted gp91phox amino acid sequences were aligned using MEGALIGN software. Amino acids identical to the rabbit sequence are indicated by dashes (–), and amino acids absent are indicated by dots (.). The predicted transmembrane hydrophobic regions are enclosed in rectangles, and putative heme-coordinating histidines located in these transmembrane regions are indicated by a star (*). Potential N-linked glycosylation sites in the rabbit amino acid sequence with the consensus sequence N-X-(S/T) are indicated by arrowheads ({wedge}{wedge}{wedge}). Other functional regions mapped to the human gp91phox protein are underlined in bold and include putative p47phox-binding sites (labeled 1–4), flavin-binding domains (labeled A and B), and NADPH-binding domains (labeled C–F). See text for further details.

 
The cDNA obtained in our p40phox screen was 1270 nucleotides long and contained an ORF of 1020 nucleotides encoding 340 amino acids (GenBank accession #AF323790). Comparison of the rabbit p40phox amino acid sequence with the other known p40phox sequences demonstrated considerable similarity with the human (89.7%), murine (86.5%), and dolphin (85.3%) homologs (Fig. 3 ).



View larger version (44K):
[in this window]
[in a new window]
  		Figure 3. Comparison of rabbit p40phox amino acid sequence with other known p40phox sequences. The predicted p40phox amino acid sequences were aligned using MEGALIGN software. Amino acids identical to the rabbit sequence are indicated by dashes (–), and amino acids absent are indicated by dots (.). The SH3 domain and PC motif are shown in boxes, and the locations of consensus sites of phosphorylation among all homologs are indicated by a star (*). See text for further details.

 
The cDNA obtained in our p47phox screen was 1458 nucleotides long and contained an ORF of 1173 nucleotides encoding 391 amino acids (GenBank accession #AF324409). Comparison of the rabbit p47phox amino acid sequence with the other known p47phox sequences demonstrated considerable similarity with the human (83.6%), bovine (83.9%), murine (81.3%), and dolphin (80.3%) homologs (Fig. 4 ). The cDNA obtained in our p67phox screen was 1757 nucleotides long and contained an ORF of 1629 nucleotides encoding 543 amino acids (GenBank accession #AF323789). Comparison of the rabbit p67phox amino acid sequence with the other known p67phox sequences demonstrated considerable similarity with the human (77.6%), bovine (78.0%), murine (75.1%), and dolphin (77.2%) proteins, however rabbit p67phox was 16–17 amino acids longer than all other p67phox homologs (Fig. 5 ). Consequently, the predicted molecular weight of rabbit p67phox (60,302 Da) is also larger than that of the other homologs. This difference in p67phox was surprising, considering the rabbit homologs for all other NADPH oxidase proteins were highly conserved. To verify that this difference in p67phox length was not because of an artifact in our rabbit cDNA library, we prepared total RNA from rabbit leukocytes and used RT-PCR to isolate a fragment containing the 3' end of rabbit p67phox. Sequence analysis of this fragment again confirmed that rabbit p67phox contained these additional residues (unpublished results).



View larger version (54K):
[in this window]
[in a new window]
  		Figure 4. Comparison of rabbit p47phox amino acid sequence with other known p47phox sequences. The predicted p47phox amino acid sequences were aligned using MEGALIGN software. Amino acids identical to the rabbit sequence are indicated by dashes (–), and amino acids absent are indicated by dots (.). Both SH3 domains are shown in boxes, and the locations of putative sites of phosphorylation are indicated by a star (*). Consensus sites of phosphorylation among all homologs are also indicated in bold. The putative N-terminal, proline-rich SH3 domain-binding domain (A), p67phox/flavocytochrome b-binding region (B), and C-terminal proline-rich, p67phox SH3 domain-binding site (C) are underlined in bold. See text for further details.

 


View larger version (57K):
[in this window]
[in a new window]
  		Figure 5. Comparison of rabbit p67phox amino acid sequence with other known p67phox sequences. The predicted p67phox amino acid sequences were aligned using MEGALIGN software. Amino acids identical to the rabbit sequence are indicated by dashes (–), and amino acids absent are indicated by dots (.). Both SH3 domains are shown in boxes, and the location of a putative MAPK phosphorylation site is indicated by a star (*). The putative Rac-binding region (A), activation domain (B), and proline-rich, SH3 domain-binding site (C) are underlined in bold. See text for further details.

 
Western blot analysis of rabbit NADPH oxidase proteins
To further analyze the rabbit NADPH oxidase proteins, we purified rabbit neutrophil membranes and cytosol, as well as comparative samples of human neutrophil membranes and cytosol, and analyzed them using SDS-PAGE and Western blotting. Monoclonal anti-human p22phox (44.1) recognized the corresponding rabbit protein and stained a single band of approximately 21.6 kDa in samples of rabbit neutrophil plasma membranes (Fig. 6 ). This is slightly smaller than the human protein of 22 kDa but correlated well with the shorter amino acid sequence and the predicted molecular weight of 20.7 kDa for the rabbit homolog. In contrast, polyclonal anti-human p22phox (R3179) showed little cross-reactivity with the rabbit homolog, suggesting it recognizes a region of divergence between the two homologs (unpublished results).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 6. Western blot analysis of rabbit NADPH oxidase proteins. Human (lane 1) and rabbit (lane 2) neutrophil membranes (p22phox and gp91phox panels) and human (lane 1) and rabbit (lane 2) neutrophil cytosol (p40phox, p47phox, and p67phox panels) were analyzed by SDS-PAGE (7–18% polyacrylamide gradient gels) and Western blotting as described. Open and solid arrows indicate the human and rabbit proteins, respectively. Representative of three separate experiments.

 
Monoclonal anti-human gp91phox (54.1) and polyclonal anti-human gp91phox peptide (R2085; not shown) antibodies recognized gp91phox in samples of rabbit neutrophil plasma membranes, resulting in a broad band centered at approximately 74.3 kDa (Fig. 6) . The broad staining pattern of gp91phox in human and rabbit samples is consistent with the heterogeneous glycosylation of this protein [5 ]. Note, however, the molecular weight of rabbit gp91phox is smaller than that of the human counterpart (~91.4 kDa), which is likely because of a lower level of posttranslational glycosylation (see Discussion).

Rabbit p40phox was recognized in samples of rabbit neutrophil cytosol by monoclonal anti-human p40phox (1.9) and polyclonal anti-human p40phox peptide (not shown) antibodies. Although rabbit p40phox is one amino acid shorter than human p40phox, it migrated as a band of approximately 40.4 kDa, which was almost 1 kDa smaller than that of the human protein (~41.2 kDa). The reason for this difference in migration patterns is not clear but is likely a result of differences in amino acid composition.

Rabbit p47phox was recognized in samples of rabbit neutrophil cytosol by mouse polyclonal anti-bovine p47phox antibodies (MB47; Fig. 6 ), goat polyclonal anti-human p47phox (not shown), and rabbit polyclonal (R360) anti-human p47phox antibodies (data not shown). Rabbit p47phox migrated as a band of ~48 kDa, which is slightly larger than that of the human protein (~47 kDa).

Rabbit p67phox was recognized in samples of rabbit neutrophil cytosol by mouse polyclonal antibovine p67phox antibodies (MB8; Fig. 6 ) and by rabbit polyclonal (R1497) anti-human p67phox antibodies but with lower affinity (data not shown). Rabbit p67phox migrated as a band of ~73.1 kDa, which is larger than that of the human protein (~66 kDa) and is consistent with rabbit p67phox being 17 amino acids longer than the human counterpart.

Functional analysis of recombinant wild-type and truncated rabbit p67phox
To investigate the functional consequences of the C-terminal 17 amino acid extension on rabbit p67phox, we expressed and purified recombinant full-length rabbit p67phox and a truncated rabbit p67phox missing this extension. Characterization of the purified recombinant proteins by SDS-PAGE (Fig. 7 ) and Western blotting (unpublished results) verified the identity of these proteins as well as the expected difference in electrophoretic mobility, which was similar to that observed between rabbit and human p67phox (see Fig. 6 ). As shown in Figure 8 , the full-length and truncated forms of rabbit p67phox proteins were functionally active in reconstituting NADPH oxidase activity in semirecombinant assays that contained human neutrophil membranes and the complementary recombinant human cytosolic factors (see Materials and Methods). These results demonstrate that the rabbit homolog can functionally substitute for human p67phox, further confirming the highly conserved nature of the proteins making up the NADPH oxidase [26 ]. It is interesting that truncated rabbit p67phox produced, in general, higher activity than the full-length form, suggesting the possibility that this extra domain might play a functional role in regulating the level of NADPH oxidase assembly and/or activity. Further studies will be necessary to evaluate this issue in vivo as well as consider why this domain was not conserved among other vertebrates.



View larger version (79K):
[in this window]
[in a new window]
  		Figure 7. Characterization of recombinant full-length and truncated rabbit p67phox. Recombinant full-length (lane 1) and truncated (lane 2) rabbit p67phox were analyzed by SDS-PAGE on 4–20% polyacrylamide gradient gels as described in Materials and Methods. Open and solid arrows indicate the full-length and truncated proteins, respectively. Prestained molecular-weight standards are shown (STD). Representative of two separate experiments.

 


View larger version (22K):
[in this window]
[in a new window]
  		Figure 8. Functional analysis of recombinant full-length and truncated rabbit p67phox. SOD-inhibitable O2- production was measured in a heterologous, cell-free NADPH oxidase assay system as described. The results are expressed as nmol O2-/min/mg membrane protein for assays consisting of human neutrophil membranes, 8 µg recombinant human p47phox, 2 µg GTP{gamma}S-loaded Rac2, and the indicated concentrations of recombinant human p67phox ({blacksquare}), full-length rabbit p67phox ({circ}), and truncated rabbit p67phox ({blacktriangleup}). The results are expressed as mean ± SD; N = 3. Representative of three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The leukocyte NADPH oxidase has long been recognized as one of the primary components of the antimicrobial repertoire invoked during the innate host-defense response (reviewed in ref. [3 ]). However, recent work by a number of groups has demonstrated that an analogous NADPH oxidase is also present in nonphagocytic cells and that this system, although structurally similar, is regulated differently than the phagocyte oxidase (reviewed in ref. [34 ]).

The majority of studies investigating the function of nonphagocyte NADPH oxidase systems is based on studies using nonhuman animal models, such as mice, rats, and rabbits. In particular, rabbits have been used extensively as experimental models in studies of various diseases (see Introduction), where NADPH oxidase activity (phagocyte or nonphagocyte in origin) has been implicated. Yet, little is known about the structure of the NADPH oxidase in rabbits. To address this discrepancy, we have cloned and fully sequenced five rabbit leukocyte NADPH oxidase genes (p22phox, p40phox, p47phox, p67phox, and gp91phox) and provide evidence for the presence of a unique p67phox homolog in rabbits. Although there was a high degree of homology among all NADPH oxidase protein sequences analyzed to date, there were distinct differences unique to the rabbit homologs, which provide important clues relative to various structural and functional features of these proteins.

The rabbit p22phox sequence encodes a protein 194 amino acids long, which is one amino acid shorter than human p22phox, two amino acids longer than pig, rat, mouse, and dolphin p22phox, and three amino acids longer than the bovine homolog. The predicted molecular weight for this open reading frame is 20.7 kDa, which correlates well with our Western blot data, showing rabbit p22phox to be about 21.7 kDa and the human homolog to be 22 kDa. Comparison of rabbit p22phox with other known p22phox sequences revealed that the highest degree of variability occurred in two areas of the protein located at residues 58–90 and 168–188, the latter being the location of missing residues required for alignment with the human sequence. Further analysis of rabbit p22phox revealed the presence of only one histidine (His94), which is conserved among all species, and represents a putative heme coordination site [35 ]. The absence of a second histidine (His72), which has been shown to be polymorphic in human p22phox [35 ] and is absent in p22phox from all other species, provides further support for the conclusion that p22phox is not capable of coordinating a heme by itself. A highly conserved stretch of proline-rich sequence near the C-terminus (residues 155–160), representing a putative-binding region for one or both SH3 domains of p47phox [36 ], is also present in rabbit p22phox and is completely conserved across all species, further confirming the importance of this domain in p22phox function.

The rabbit gp91phox sequence encodes a protein 570 amino acids long, which is identical in length to human, bovine, and murine gp91phox and one amino acid longer than the dolphin homolog. Overall, rabbit gp91phox was the most highly conserved of all rabbit NADPH oxidase proteins (average 91.1±1.0% similarity). All gp91phox homologs, including rabbit, contained the same number of histidines in identical locations, suggesting that transmembrane orientation and heme coordination in gp91phox are very similar or identical regardless of the species of origin of gp91phox. Further analysis of rabbit gp91phox showed conserved regions of homology with the putative NADPH-binding domains [16 , 37 ] and p47phox-binding domains (reviewed in ref. [2 ]) proposed for human gp91phox. Analysis of rabbit gp91phox for potential N-linked glycosylation sequences revealed unique differences in the putative glycosylation sites compared with other gp91phox homologs. Rabbit gp91phox contains consensus glycosylation motifs (N-X-S/T) at Asn97, Asn132, and Asn149, which are present in all gp91phox sequences except mouse (missing Asn132) and dolphin (missing Asn149). Although human (Asn240), bovine (Asn247), dolphin (Asn247), and pig (Asn247) gp91phox contain a third putative external glycosylation site, this site is absent in rabbit gp91phox, which does contain a consensus glycosylation motif at Asn430, conserved in human, mouse, and dolphin gp91phox. However, this region of the protein is adjacent to several p47phox-binding domains (e.g., see ref. [30 ]) and consequently, would be located in the cytosol. Our Western blot results show that rabbit gp91phox migrates as a broad band of ~73 kDa, which is smaller than that of human gp91phox (91 kDa). Thus, based on the smaller size of the rabbit protein and the location of putative glycosylation sites, we conclude that rabbit gp91phox is glycosylated only at Asn132 and Asn149.

The rabbit p40phox sequence encodes a protein of 340 amino acids, which is one amino acid longer than the human, mouse, and dolphin homologs. The predicted molecular weight for this open reading frame is 39.0 kDa, which correlates well with our Western blot data showing rabbit p40phox to be about 40.4 kDa and the human homolog to be 41.2 kDa. Rabbit p40phox is also well-conserved when compared with other known p40phox homologs (average 86.9±2.0% similarity). Rabbit p40phox contains one Src homology 3 (SH3) domain (residues 175–225), which is highly conserved among species (average 91.3±2.3% identity). The high degree of sequence conservation in this area of p40phox is consistent with the functional role it plays in binding to p47phox [38 ], and recent studies by Cross [39 ] suggest that this interaction may play a role in increasing the affinity of p47phox for flavocytochrome b. Additionally, a region in the C-terminus of p40phox has been shown to participate in association with p67phox through a non-SH3-related interaction involving a conserved domain (residues 282–309) [38 ], which has been called a PC motif [40 ]. Consistent with its postulated role in p40phox function, this motif is highly conserved in rabbit p40phox.

The rabbit p47phox sequence encodes a protein of 391 amino acids, which is one amino acid longer than human and murine p47phox and one amino acid shorter than the bovine homolog. The predicted molecular weight for this open reading frame is 44.9 kDa, which is slightly lower than observed by SDS-PAGE and Western blot analysis (~48 kDa). This relative difference between predicted and observed molecular weights is similar to that found with the human and bovine homologs [32 ]. Comparison of rabbit p47phox with other p47phox sequences revealed a high degree of divergence among all species near the C-terminus (residues 339–361), with an average of ~22% homology among p47phox homologs in this region of the protein.

Rabbit p47phox contains two SH3 domains as well as a proline-rich motif near the carboxyl-terminus of the protein. Both SH3 domains are highly conserved, because the first (residues 163–211) and second (residues 227–281) SH3 domains share an average of 84.4 ± 2.7% and 85.6 ± 1.8% identity, respectively, with comparable SH3 domains in all other known p47phox sequences. The C-terminal proline-rich region of p47phox, corresponding to residues 361–371 of rabbit p47phox, represents a consensus SH3 domain-binding site and is identical in all p47phox homologs, which is consistent with the key role this domain plays in binding a p67phox SH3 domain [41 ]. Previously, we found that p67phox and flavocytochrome b used a common binding site in p47phox (residues 323–332), presumably at distinct stages of the activation process [32 ]. These residues are located within a highly cationic p47phox domain that also contains multiple, potential phosphorylation sites. During activation, phosphorylation and subsequent conformation changes in p47phox appear to result in exposure of this domain for binding to other oxidase proteins [32 ]. The high level of conservation among p47phox homologs from different species in this region is consistent with the critical function proposed for this domain in the NADPH oxidase assembly process [32 ].

Cytosolic p47phox becomes multiply phosphorylated during NADPH oxidase activation [9 , 10 ]. This phosphorylation has been proposed to neutralize a highly cationic region of p47phox encompassing residues 314–347, initiating a conformational change within the protein and allowing the protein to interact with the membrane and/or target proteins [42 ]. El Benna et al. [10 ] demonstrated that p47phox, isolated from phorbol 12-myristate 13-acetate-stimulated human neutrophils, was phosphorylated on serines 303, 304, 320, 328, 345, and 348 and at least one of the serines at 359, 370, and 379. By sequence comparison, rabbit p47phox contains serines at all of these sites, except for the residues corresponding to human p47phox Ser345 and Ser359 (where a conserved Thr is substituted). Consistent with previous studies showing that phosphorylation of Ser379 played an essential role in p47phox translocation to the membrane and NADPH oxidase activity [43 ], this serine and flanking consensus phosphorylation site are conserved in rabbit p47phox. Previously, El Benna et al. [44 ] demonstrated that the p47phox Ser345 and Ser348 (human sequence) were located in consensus sequences (-PXSP-), which are recognized by mitogen-activated protein kinase (MAPK), and postulated that p47phox was regulated by MAPK-mediated phosphorylation. However, this consensus sequence was not conserved in murine or bovine p47phox. Furthermore, this consensus site is also not conserved in rabbit p47phox, providing even greater support to the conclusion that p47phox is probably not phosphorylated by a MAPK or another proline-directed kinase during NADPH oxidase activation.

The rabbit p67phox sequence encodes a protein of 543 amino acids, which is 17 amino acids longer than human and dolphin p67phox, 16 amino acids longer than bovine p67phox, and 18 amino acids longer than the murine homolog. Given the high degree of conservation shown for the other four rabbit NADPH oxidase proteins, the presence of a unique p67phox was quite surprising. Nevertheless, this sequence was verified by sequencing samples isolated from freshly isolated rabbit leukocytes. The predicted molecular weight for the rabbit ORF is 60.3 kDa, which is lower than that observed by SDS-PAGE and Western blot analysis (~73 kDa). This difference in predicted molecular weight and observed molecular weight on SDS-PAGE is characteristic for this protein. Comparative immunoblots of rabbit p67phox and the human and bovine homologs further confirmed the increased size of the rabbit protein. Previously, Pagano et al. [29 ] isolated and sequenced a fragment of rabbit fibroblast p67phox corresponding to amino acids 74–312 of the human sequence. It is interesting that comparison of rabbit leukocyte p67phox with this fragment rabbit fibroblast p67phox suggests that they are not identical gene products (79.5% homology). Thus, these results provide evidence for the possibility that there may be a family of homologous p67phox genes and perhaps other NADPH oxidase genes, which are expressed differentially in various tissues. This is not unlikely, given the wide differences observed in the activity and regulation of nonphagocyte systems when compared with the phagocyte oxidase (e.g., see refs. [24 , 45 ]). In support of this idea, previously, we found that human fibroblast and neutrophil flavocytochrome b displayed characteristics of being genetically distinct from each other [46 ]. In addition, recently, several gp91phox isoforms have been identified in nonphagocyte tissues [47 ], although the role of all of these isoforms in NADPH oxidase-like function is not yet clear. Furthermore, the absence of a functional leukocyte NADPH oxidase in animal models of CGD does not appear to cause major phenotypic abnormalities, however this issue is still controversial and needs clarification. For example, Hsich et al. [48 ] showed that the absence of a functional NADPH oxidase in p47phox -/- mice did not influence basal blood pressure significantly, yet recent studies by Wang et al. [49 ] showed significantly lower basal blood pressure in gp91phox -/- mice, suggesting that NADPH oxidase-derived O2- actually plays a role in basal blood-pressure regulation. The absence of a functional NADPH oxidase also does not appear to promote the progression of atherosclerosis in p47phox -/-/apoE-/- [48 ] or gp91phox -/-/apoE-/- [50 ] mice, yet NADPH oxidase-derived O2- has definitely been implicated in the pathogenesis of this disease [51 ]. Thus, further analysis of these CGD animal models and how they relate to the human disease are clearly necessary to address these discrepancies. Furthermore, the absence of a functional leukocyte NADPH oxidase in CGD patients has not been shown to cause noticeable alterations in vascular or pulmonary physiology (e.g., see ref. [52 ]), although the evidence points to a role for an NADPH oxidase in these systems. Taken together, it seems reasonable to conclude that there may be tissue-specific isoforms of at least some of the key NADPH oxidase proteins, such as gp91phox and p67phox. Further sequencing of NADPH oxidase proteins from different tissues in the same species will be necessary to address this issue.

Overall, rabbit p67phox was the least conserved of all rabbit NADPH oxidase proteins (average 77.0±1.3% similarity) when compared with other known p67phox sequences. Furthermore, rabbit p67phox showed the greatest degree of variability among all species in the C-terminal half of the protein (residues 310–526), with an average of ~62% homology among p67phox homologs in this region of the protein. Rabbit p67phox contains two SH3 domains, and the location and size of these domains appear to be fairly well-conserved across species. Note, however, that the second p67phox SH3 domain (residues 465–513) showed more diversity in sequence (average 76.5±1.9% similarity) than the first SH3 domain (residues 247–295), which was almost identical among all species (average 97.9±1.7% similarity). In addition to the SH3 domains, rabbit p67phox also contains a conserved proline-rich SH3 domain-binding motif (residues 219–231), which has been proposed to mediate binding to the second SH3 domain of p47phox during NADPH oxidase assembly [19 ]. Comparison of p67phox homologs shows that this region is essentially identical in rabbit p67phox, supporting its putative role in NADPH oxidase function. An "activation domain" has also been identified in human p67phox (residues 199–210) [53 ], and comparison of this domain in rabbit p67phox shows that this sequence is identical, supporting its putative role in oxidase activation. The interaction between p67phox and Rac is also essential for NADPH oxidase activation and appears to be mediated primarily by binding Rac to the amino-terminal region of p67phox (residues 1–199) [17 ]. It is interesting that residues 1–199 of rabbit p67phox fall within the most conserved region of p67phox, and it has been demonstrated that the tetratricopeptide repeat (TPR) motifs located in the N-terminus of p67phox participate in the interaction with Rac [54 ]. Recently, Ahmed et al. [55 ] further narrowed down the location of the Rac-binding site to residues 171–199 of p67phox, a region that is almost identical between rabbit p67phox and all other known p67phox homologs.

The function of the 17 additional C-terminal amino acids found in rabbit p67phox is still unclear. Comparative oxidase assays using full-length and a truncated form of rabbit p67phox missing this C-terminal extension showed that the truncated form was slightly more active in supporting NADPH oxidase activity in vitro. This sequence of amino acids does not appear to have any consensus protein motifs, however it does display homology to regions in two different proteins that have both been shown to be calcineurin-binding proteins. Calcineurin is a serine/threonine phosphatase that is activated by elevated Ca2+ and the Ca2+-calmodulin complex (reviewed in ref. [56 ]). The calcineurin-binding proteins having regions of homology to the rabbit p67phox C-terminus include human calcineurin-binding protein 1 (Cabin 1; residues 2086–2094), a negative regulator for calcineurin signaling in T lymphocytes [57 ], and human nuclear factor of activated T cells (NF-AT; residues 982–992), which is a cytosolic transcription factor that translocates to the nucleus after dephosphorylation by calcineurin and induces cytokine gene transcription [58 ]. It is well-established that changes in cytosolic-free Ca2+ play a central role in triggering neutrophil responses [59 ], including regulation of the NADPH oxidase [60 ]. Activation-induced changes in neutrophil Ca2+ flux are also associated with intracellular movements of several Ca2+-binding proteins, including calreticulin [61 ] and calcineurin [62 ]. Thus, a putative association of a Ca2+-binding protein such as calcineurin with p67phox could serve to integrate these systems. It is interesting that Lawson and Maxfield [62 ] found that calcineurin also played a role in integrin recycling to the leading edge of migrating neutrophils. Whether calcineurin contributes to the known link between the integrin system and the NADPH oxidase [63 ] is an interesting possibility that awaits further studies.


   ACKNOWLEDGEMENTS
 
This work was supported in part by USDA/NRICGP 9502274, USDA/NRICGP 9703677, National Institutes of Health (NIH) grant RO1 AR42426, and the Montana State University Agricultural Experimental Station. K. A. G. is supported by an Arthritis Foundation Postdoctoral Fellowship. M. T. Q. is an Established Investigator of the American Heart Association. This is manuscript 2001-25 from the Montana Agricultural Experiment Station, Montana State University-Bozeman. We thank Dr. Javier Navarro (University of Texas Medical Branch, Galveston) for kindly providing the rabbit leukocyte cDNA library and Dr. Thomas L. Leto (NIAID, NIH, Bethesda, MD) for kindly providing samples of goat polyclonal anti-human p47phox.

Received June 1, 2001; revised July 10, 2001; accepted July 12, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Witko-Sarsat, V., Rieu, P., Descamps-Latscha, B., Lesavre, P., Halbwachs-Mecarelli, L. (2000) Neutrophils: molecules, functions and pathophysiological aspects Lab. Investig. 80,617-653[Medline]
  2. DeLeo, F. R., Quinn, M. T. (1996) Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins J. Leukoc. Biol. 60,677-691[Abstract]
  3. Clark, R. A. (1999) Activation of the neutrophil respiratory burst oxidase J. Infect. Dis. 179,S309-S317
  4. Meischl, C., Roos, D. (1998) The molecular basis of chronic granulomatous disease Springer Semin. Immunopathol. 19,417-434[Medline]
  5. Parkos, C. A., Allen, R. A., Cochrane, C. G., Jesaitis, A. J. (1987) Purified cytochrome b from human granulocyte plasma membrane is composed of two polypeptides with relative molecular weights of 91,000 and 22,000 J. Clin. Investig. 80,732-742
  6. Nauseef, W. M. (1999) The NADPH-dependent oxidase of phagocytes Proc. Assoc. Am. Physicians 111,373-382[Medline]
  7. Iyer, S. S., Pearson, D. W., Nauseef, W. M., Clark, R. A. (1994) Evidence for a readily dissociable complex of p47phox and p67phox in cytosol of unstimulated human neutrophils J. Biol. Chem. 269,22405-22411[Abstract/Free Full Text]
  8. Heyworth, P. G., Curnutte, J. T., Nauseef, W. M., Volpp, B. D., Pearson, D. W., Rosen, H., Clark, R. A. (1991) Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558 J. Clin. Investig. 87,352-356
  9. Rotrosen, D., Leto, T. L. (1990) Phosphorylation of neutrophil 47-kDa cytosolic oxidase factor. Translocation to membrane is associated with distinct phosphorylation events J. Biol. Chem. 265,19910-19915[Abstract/Free Full Text]
  10. El Benna, J., Faust, L. P., Babior, B. M. (1994) The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases J. Biol. Chem. 269,23431-23436[Abstract/Free Full Text]
  11. Nauseef, W. M., Volpp, B. D., Mccormick, S., Leidal, K. G., Clark, R. A. (1991) Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components J. Biol. Chem. 266,5911-5917[Abstract/Free Full Text]
  12. Dusi, S., Rossi, F. (1993) Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox Biochem. J. 296,367-371
  13. Forbes, L. V., Truong, O., Wientjes, F. B., Moss, S. J., Segal, A. W. (1999) The major phosphorylation site of the NADPH oxidase component p67phox is Thr233 Biochem. J. 338,99-105
  14. Smith, R. M., Connor, J. A., Chen, L. M., Babior, B. M. (1996) The cytosolic subunit p67phox contains an NADPH-binding site that participates in catalysis by the leukocyte NADPH oxidase J. Clin. Investig. 98,977-983[Medline]
  15. Dang, P. M. C., Cross, A. R., Babior, B. M. (2001) Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67PHOX and cytochrome b558 Proc. Natl. Acad. Sci. USA 98,3001-3005[Abstract/Free Full Text]
  16. Segal, A. W., West, I., Wientjes, F., Nugent, J. H. A., Chavan, A. J., Haley, B., Garcia, R. C., Rosen, H., Scarce, G. (1992) Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes Biochem. J. 284,781-788
  17. Diekmann, D., Abo, A., Johnston, C., Segal, A. W., Hall, A. (1994) Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity Science 265,531-533[Abstract/Free Full Text]
  18. Sathyamoorthy, M., de Mendez, I., Adams, A. G., Leto, T. L. (1997) p40phox down-regulates NADPH oxidase activity through interactions with its SH3 domain J. Biol. Chem. 272,9141-9146[Abstract/Free Full Text]
  19. de Mendez, I., Adams, A. G., Sokolic, R. A., Malech, H. L., Leto, T. L. (1996) Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells EMBO J 15,1211-1220[Medline]
  20. Matsukawa, A., Yoshinaga, M. (1998) Sequential generation of cytokines during the initiative phase of inflammation, with reference to neutrophils Inflamm. Res. 47,S137-S144
  21. Ye, R. D., Quehenberger, O., Thomas, K. M., Navarro, J., Cavanagh, S. L., Prossnitz, E. R., Cochrane, C. G. (1993) The rabbit neutrophil N-formyl peptide receptor: cDNA cloning, expression, and structure/function implications J. Immunol. 150,1383-1394[Abstract]
  22. Frevert, C. W., Matute-Bello, G., Martin, T. R. (2000) Rabbit models of pneumonia, peritoneal sepsis, and lung injury Methods Mol. Biol. 138,319-330[Medline]
  23. Aliev, G., Burnstock, G. (1998) Watanabe rabbits with heritable hypercholesterolaemia: a model of atherosclerosis Histol. Histopathol. 13,797-817[Medline]
  24. Pagano, P. J., Clark, J. K., Cifuentes-Pagano, M. E., Clark, S. M., Callis, G. M., Quinn, M. T. (1997) Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: enhancement by angiotensin II Proc. Natl. Acad. Sci. USA 94,14483-14488[Abstract/Free Full Text]
  25. Hitt, N. D., Kleinberg, M. E. (1996) Identification of neutrophil NADPH oxidase proteins gp91-phox, p22-phox, p67-phox, and p47-phox in mammalian species Am. J. Vet. Res. 57,672-676[Medline]
  26. Davis, A. R., Mascolo, P. L., Bunger, P. L., Sipes, K. M., Quinn, M. T. (1998) Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences J. Leukoc. Biol. 64,114-123[Abstract]
  27. Thomas, K. M., Pyun, H. Y., Navarro, J. (1990) Molecular cloning of the fMet-Leu-Phe receptor from neutrophils J. Biol. Chem. 265,20061-20064[Abstract/Free Full Text]
  28. Bunger, P. L., Swain, S. D., Clements, M. K., Siemsen, D. W., Davis, A. R., Gauss, K. A., Quinn, M. T. (2000) Cloning and expression of bovine p47-phox and p67-phox: comparison with the human and murine homologs J. Leukoc. Biol. 67,63-72[Abstract]
  29. Pagano, P. J., Chanock, S. J., Siwik, D. A., Colucci, W. S., Clark, J. K. (1998) Angiotensin II induces p67phox mRNA expression and NADPH oxidase superoxide generation in rabbit aortic adventitial fibroblasts Hypertension 32,331-337[Abstract/Free Full Text]
  30. DeLeo, F. R., Yu, L., Burritt, J. B., Loetterle, L. R., Bond, C. W., Jesaitis, A. J., Quinn, M. T. (1995) Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries Proc. Natl. Acad. Sci. USA 92,7110-7114[Abstract/Free Full Text]
  31. Sugawara, T., Miyamoto, M., Takayama, S., Kato, M. (1995) Separation of neutrophils from blood in human and laboratory animals and comparison of the chemotaxis J. Pharmacol. Toxicol. Methods 33,91-100[Medline]
  32. DeLeo, F. R., Ulman, K. V., Davis, A. R., Jutila, K. L., Quinn, M. T. (1996) Assembly of the human neutrophil NADPH oxidase involves binding of p67phox and flavocytochrome b to a common functional domain in p47phox J. Biol. Chem. 271,17013-17020[Abstract/Free Full Text]
  33. Burritt, J. B., Quinn, M. T., Jutila, M. A., Bond, C. W., Jesaitis, A. J. (1995) Topological mapping of neutrophil cytochrome b epitopes with phage-display libraries J. Biol. Chem. 270,16974-16980[Abstract/Free Full Text]
  34. Finkel, T. (2000) Redox-dependent signal transduction FEBS Lett 476,52-54[Medline]
  35. Dinauer, M. C., Pierce, E. A., Bruns, G. A. P., Curnutte, J. T., Orkin, S. H. (1990) Human neutrophil cytochrome b light chain (p22-phox): gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease J. Clin. Investig. 86,1729-1737
  36. Sumimoto, H., Hata, K., Mizuki, K., Ito, T., Kage, Y., Sakaki, Y., Fukumaki, Y., Nakamura, M., Takeshige, K. (1996) Assembly and activation of the phagocyte NADPH oxidase. Specific interaction of the N-terminal Src homology 3 domain of p47phox with p22phox is required for activation of the NADPH oxidase J. Biol. Chem. 271,22152-22158[Abstract/Free Full Text]
  37. Rotrosen, D., Yeung, C. L., Leto, T. L., Malech, H. L., Kwong, C. H. (1992) Cytochrome b558. The flavin-binding component of the phagocyte NADPH oxidase Science 256,1459-1462[Abstract/Free Full Text]
  38. Wientjes, F. B., Panayotou, G., Reeves, E., Segal, A. W. (1996) Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox Biochem. J. 317,919-924
  39. Cross, A. R. (2000) p40phox participates in the activation of NADPH oxidase by increasing the affinity of p47phox for flavocytochrome b558 Biochem. J. 349,113-117[Medline]
  40. Nakamura, R., Sumimoto, H., Mizuki, K., Hata, K., Ago, T., Kitajima, S., Takeshige, K., Sakaki, Y., Ito, T. (1998) The PC motif: a novel and evolutionarily conserved sequence involved in interaction between p40phox and p67phox, SH3 domain-containing cytosolic factors of the phagocyte NADPH oxidase Eur. J. Biochem. 251,583-589[Medline]
  41. Leto, T. L., Adams, A. G., de Mendez, I. (1994) Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to proline-rich targets Proc. Natl. Acad. Sci. USA 91,10650-10654[Abstract/Free Full Text]
  42. Swain, S. D., Helgerson, S. L., Davis, A. R., Nelson, L. K., Quinn, M. T. (1997) Analysis of activation-induced conformational changes in p47phox using tryptophan fluorescence spectroscopy J. Biol. Chem. 272,29502-29510[Abstract/Free Full Text]
  43. Faust, L. P., El Benna, J., Babior, B. M., Chanock, S. J. (1995) The phosphorylation targets of p47phox, a subunit of the respiratory burst oxidase. Functions of the individual target serines as evaluated by site-directed mutagenesis J. Clin. Investig. 96,1499-1505
  44. El Benna, J., Faust, L. P., Johnson, J. L., Babior, B. M. (1996) Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping. Phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase J. Biol. Chem. 271,6374-6378[Abstract/Free Full Text]
  45. Souza, H. P., Laurindo, F. R. M., Ziegelstein, R. C., Berlowitz, C. O., Zweier, J. L. (2001) Vascular NAD(P)H oxidase is distinct from the phagocytic enzyme and modulates vascular reactivity control Am. J. Physiol. Heart Circ. Physiol. 280,H658-H667[Abstract/Free Full Text]
  46. Meier, B., Jesaitis, A. J., Emmendörffer, A., Roesler, J., Quinn, M. T. (1993) The cytochrome b-558 molecules involved in the fibroblast and polymorphonuclear leucocyte superoxide-generating NADPH oxidase systems are structurally and genetically distinct Biochem. J. 289,481-486
  47. Lambeth, J. D., Cheng, G., Arnold, R. S., Edens, W. A. (2000) Novel homologs of gp91phox Trends Biochem. Sci. 25,459-461[Medline]
  48. Hsich, E., Segal, B. H., Pagano, P. J., Rey, F. E., Paigen, B., Deleonardis, J., Hoyt, R. F., Holland, S. M., Finkel, T. (2000) Vascular effects following homozygous disruption of p47phox An essential component of NADPH oxidase Circulation 101,1234-1236[Abstract/Free Full Text]
  49. Wang, H. D., Xu, S., Johns, D. G., Du, Y., Quinn, M. T., Cayatte, A. J., Cohen, R. A. (2001) Role of NADPH oxidase in the vascular hypertrophic and oxidative stress response to angiotensis II in mice Circ. Res. 88,947-953[Abstract/Free Full Text]
  50. Kirk, E. A., Dinauer, M. C., Rosen, H., Chait, A., Heinecke, J. W., LeBoeuf, R. C. (2000) Impaired superoxide production due to a deficiency in phagocyte NADPH oxidase fails to inhibit atherosclerosis in mice Arterioscler. Thromb. Vasc. Biol. 20,1529-1535[Abstract/Free Full Text]
  51. Meyer, J. W., Schmitt, M. E. (2000) A central role for the endothelial NADPH oxidase in atherosclerosis FEBS Lett 472,1-4[Medline]
  52. Cale, C. M., Jones, A. M., Goldblatt, D. (2000) Follow up of patients with chronic granulomatous disease diagnosed since 1990 Clin. Exp. Immunol. 120,351-355[Medline]
  53. Han, C-H., Freeman, J. L. R., Lee, T. H., Motalebi, S. A., Lambeth, J. D. (1998) Regulation of the neutrophil respiratory burst oxidase. Identification of an activation domain in p67phox J. Biol. Chem. 273,16663-16668[Abstract/Free Full Text]
  54. Koga, H., Terasawa, H., Nunoi, H., Takeshige, K., Inagaki, F., Sumimoto, H. (1999) Tetratricopeptide repeat (TPR) motifs of p67phox participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase J. Biol. Chem. 274,25051-25060[Abstract/Free Full Text]
  55. Ahmed, S., Prigmore, E., Govind, S., Veryard, C., Kozma, R., Wientjes, F. B., Segal, A. W., Lim, L. (1998) Cryptic Rac-binding and p21Cdc42HS/Rac-activated kinase phosphorylation sites of NADPH oxidase component p67phox J. Biol. Chem. 273,15693-15701[Abstract/Free Full Text]
  56. Aramburu, J., Rao, A., Klee, C. B. (2000) Calcineurin: from structure to function Curr. Top. Cell. Regul. 36,237-295[Medline]
  57. Sun, L., Youn, H. D., Loh, C., Stolow, M., He, W., Liu, J. O. (1998) Cabin 1, a negative regulator for calcineurin signaling in T lymphocytes Immunity 8,703-711[Medline]
  58. Serfling, E., Berberich-Siebelt, F., Chuvpilo, S., Jankevics, E., Klein-Hessling, S., Twardzik, T., Avots, A. (2000) The role of NF-AT transcription factors in T cell activation and differentiation Biochim. Biophys. Acta 1498,1-18[Medline]
  59. Krause, K. H., Campbell, K. P., Welsh, M. J., Lew, D. P. (1990) The calcium signal and neutrophil activation Clin. Biochem. 23,159-166[Medline]
  60. Hallett, M. B., Davies, E. V., Campbell, A. K. (1990) Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca2+ signal Cell Calcium 11,655-663[Medline]
  61. Stendahl, O., Krause, K-H., Krischer, J., Jerström, P., Theler, J-M., Clark, R. A., Carpentier, J-L., Lew, D. P. (1994) Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils Science 265,1439-1441[Abstract/Free Full Text]
  62. Lawson, M. A., Maxfield, F. R. (1995) Ca2+ and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils Nature 377,75-79[Medline]
  63. Nathan, C., Srimal, S., Farber, C., Sanchez, E., Kabbash, L., Asch, A., Gailit, J., Wright, S. D. (1989) Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins J. Cell Biol. 109,1341-1349[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
K. A. Gauss, L. K. Nelson-Overton, D. W. Siemsen, Y. Gao, F. R. DeLeo, and M. T. Quinn
Role of NF-{kappa}B in transcriptional regulation of the phagocyte NADPH oxidase by tumor necrosis factor-{alpha}
J. Leukoc. Biol., September 1, 2007; 82(3): 729 - 741.
[Abstract] [Full Text] [PDF]