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(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 p67^phox homolog

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

^* Department of Veterinary Molecular Biology, Montana State University, Bozeman; and
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

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 p67^phox, the rabbit phox proteins were highly conserved. In contrast, rabbit p67^phox had a very divergent C-terminus and was 17 amino acids longer than any other known p67^phox 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 p67^phox and a mutated rabbit p67^phox 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 p67^phox proteins were able to support oxidase activity, although the truncated form reproducibly supported a higher level of activity than full-length p67^phox. 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

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 (gp91^phox and p22^phox) [⁵ ]. The cytosolic NADPH oxidase proteins include p40^phox, p47^phox, p67^phox, as well as a low molecular-weight GTPase, Rac2 (reviewed in ref. [⁶ ]).

Through analysis of neutrophils from patients with CGD, p22^phox, p47^phox, p67^phox, and gp91^phox 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 p47^phox and p67^phox exist in a complex in the cytosol [⁷ ], however p47^phox 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 p67^phox [⁸ ]. During NADPH oxidase activation, p47^phox has been shown to undergo extensive phosphorylation [⁹ , ¹⁰ ], and inhibition of phosphorylation during neutrophil activation markedly decreases O₂^- generation and translocation of p47^phox and other cytosolic oxidase proteins [¹¹ ]. Less is understood regarding the role of p67^phox in NADPH oxidase. It has been shown that p67^phox is phosphorylated during neutrophil activation [¹² ], and recently Forbes et al. [¹³ ] mapped the major p67^phox phosphorylation site to Thr233. In addition, it has been proposed that p67^phox contains the NADPH-binding site of the NADPH oxidase [¹⁴ ] and that p67^phox 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 p47^phox and possibly flavocytochrome b, p67^phox has also been shown to bind to Rac (Rac1 and/or Rac2) [¹⁷ ] and p40^phox [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 p67^phox was much longer than any other p67^phox homolog sequenced to date, we also expressed rabbit p67^phox and a truncated rabbit p67^phox 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.

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 p22^phox, p40^phox, p47^phox, p67^phox, and gp91^phox were cloned from a rabbit neutrophil cDNA library [²⁷ ]. Briefly, cDNAs corresponding to the full-length open-reading frame (ORF) of bovine p22^phox (576 bp), p40^phox (1020 bp), and gp91^phox (1713 bp) and partial ORF of bovine p47^phox (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 p67^phox (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 p22^phox (three clones), p40^phox (three clones), p47^phox (one clone), p67^phox (one clone), and gp91^phox (three clones). Phage lysates from the positive clones were amplified by PCR using 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 p67^phox clone isolated by cDNA library screening was significantly longer than all other known p67^phox 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 p67^phox. 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 p67^phox
The full-length rabbit p67^phox clone was digested out of pT-Adv using EcoRI. Truncated rabbit p67^phox was prepared from the full-length rabbit p67^phox 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 p67^phox 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 p67^phox 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 p47^phox and p67^phox 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 GTPS. Each well contained 6 µg human neutrophil membrane protein, 8 µg human p47^phox, the indicated concentrations of rabbit or human p67^phox, 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 V_max rates, calculated using an = 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).

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 p22^phox, p40^phox, gp91^phox, a partial cDNA of bovine p47^phox, and a partial cDNA of rabbit fibroblast p67^phox were labeled with ³²P and used individually to screen the 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 p22^phox screen was 666 nucleotides long and contained an ORF of 582 nucleotides encoding 194 amino acids (GenBank accession #AF323787). Comparison of the rabbit p22^phox amino acid sequence with the other known p22^phox 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 ).

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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 gp91^phox screen was 1857 nucleotides long and contained an ORF of 1713 nucleotides encoding 570 amino acids (GenBank accession #AF323788). Comparison of the rabbit gp91^phox amino acid sequence with the other known gp91^phox 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 ).

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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 (). 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 p40^phox screen was 1270 nucleotides long and contained an ORF of 1020 nucleotides encoding 340 amino acids (GenBank accession #AF323790). Comparison of the rabbit p40^phox amino acid sequence with the other known p40^phox sequences demonstrated considerable similarity with the human (89.7%), murine (86.5%), and dolphin (85.3%) homologs (Fig. 3 ).

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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 p47^phox screen was 1458 nucleotides long and contained an ORF of 1173 nucleotides encoding 391 amino acids (GenBank accession #AF324409). Comparison of the rabbit p47^phox amino acid sequence with the other known p47^phox 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 p67^phox screen was 1757 nucleotides long and contained an ORF of 1629 nucleotides encoding 543 amino acids (GenBank accession #AF323789). Comparison of the rabbit p67^phox amino acid sequence with the other known p67^phox sequences demonstrated considerable similarity with the human (77.6%), bovine (78.0%), murine (75.1%), and dolphin (77.2%) proteins, however rabbit p67^phox was 16–17 amino acids longer than all other p67^phox homologs (Fig. 5 ). Consequently, the predicted molecular weight of rabbit p67^phox (60,302 Da) is also larger than that of the other homologs. This difference in p67^phox was surprising, considering the rabbit homologs for all other NADPH oxidase proteins were highly conserved. To verify that this difference in p67^phox 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 p67^phox. Sequence analysis of this fragment again confirmed that rabbit p67^phox contained these additional residues (unpublished results).

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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.

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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 p22^phox (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 p22^phox (R3179) showed little cross-reactivity with the rabbit homolog, suggesting it recognizes a region of divergence between the two homologs (unpublished results).

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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 gp91^phox (54.1) and polyclonal anti-human gp91^phox peptide (R2085; not shown) antibodies recognized gp91^phox 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 gp91^phox in human and rabbit samples is consistent with the heterogeneous glycosylation of this protein [⁵ ]. Note, however, the molecular weight of rabbit gp91^phox 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 p40^phox was recognized in samples of rabbit neutrophil cytosol by monoclonal anti-human p40^phox (1.9) and polyclonal anti-human p40^phox peptide (not shown) antibodies. Although rabbit p40^phox is one amino acid shorter than human p40^phox, 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 p47^phox was recognized in samples of rabbit neutrophil cytosol by mouse polyclonal anti-bovine p47^phox antibodies (MB47; Fig. 6 ), goat polyclonal anti-human p47^phox (not shown), and rabbit polyclonal (R360) anti-human p47^phox antibodies (data not shown). Rabbit p47^phox migrated as a band of 48 kDa, which is slightly larger than that of the human protein (47 kDa).

Rabbit p67^phox was recognized in samples of rabbit neutrophil cytosol by mouse polyclonal antibovine p67^phox antibodies (MB8; Fig. 6 ) and by rabbit polyclonal (R1497) anti-human p67^phox antibodies but with lower affinity (data not shown). Rabbit p67^phox migrated as a band of 73.1 kDa, which is larger than that of the human protein (66 kDa) and is consistent with rabbit p67^phox being 17 amino acids longer than the human counterpart.

Functional analysis of recombinant wild-type and truncated rabbit p67^phox
To investigate the functional consequences of the C-terminal 17 amino acid extension on rabbit p67^phox, we expressed and purified recombinant full-length rabbit p67^phox and a truncated rabbit p67^phox 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 p67^phox (see Fig. 6 ). As shown in Figure 8 , the full-length and truncated forms of rabbit p67^phox 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 p67^phox, further confirming the highly conserved nature of the proteins making up the NADPH oxidase [²⁶ ]. It is interesting that truncated rabbit p67^phox 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.

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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.

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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 GTPS-loaded Rac2, and the indicated concentrations of recombinant human p67phox (), full-length rabbit p67phox (), and truncated rabbit p67phox (). The results are expressed as mean ± SD; N = 3. Representative of three separate experiments.

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 (p22^phox, p40^phox, p47^phox, p67^phox, and gp91^phox) and provide evidence for the presence of a unique p67^phox 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 p22^phox sequence encodes a protein 194 amino acids long, which is one amino acid shorter than human p22^phox, two amino acids longer than pig, rat, mouse, and dolphin p22^phox, 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 p22^phox to be about 21.7 kDa and the human homolog to be 22 kDa. Comparison of rabbit p22^phox with other known p22^phox 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 p22^phox 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 p22^phox [³⁵ ] and is absent in p22^phox from all other species, provides further support for the conclusion that p22^phox 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 p47^phox [³⁶ ], is also present in rabbit p22^phox and is completely conserved across all species, further confirming the importance of this domain in p22^phox function.

The rabbit gp91^phox sequence encodes a protein 570 amino acids long, which is identical in length to human, bovine, and murine gp91^phox and one amino acid longer than the dolphin homolog. Overall, rabbit gp91^phox was the most highly conserved of all rabbit NADPH oxidase proteins (average 91.1±1.0% similarity). All gp91^phox homologs, including rabbit, contained the same number of histidines in identical locations, suggesting that transmembrane orientation and heme coordination in gp91^phox are very similar or identical regardless of the species of origin of gp91^phox. Further analysis of rabbit gp91^phox showed conserved regions of homology with the putative NADPH-binding domains [¹⁶ , ³⁷ ] and p47^phox-binding domains (reviewed in ref. [² ]) proposed for human gp91^phox. Analysis of rabbit gp91^phox for potential N-linked glycosylation sequences revealed unique differences in the putative glycosylation sites compared with other gp91^phox homologs. Rabbit gp91^phox contains consensus glycosylation motifs (N-X-S/T) at Asn97, Asn132, and Asn149, which are present in all gp91^phox sequences except mouse (missing Asn132) and dolphin (missing Asn149). Although human (Asn240), bovine (Asn247), dolphin (Asn247), and pig (Asn247) gp91^phox contain a third putative external glycosylation site, this site is absent in rabbit gp91^phox, which does contain a consensus glycosylation motif at Asn430, conserved in human, mouse, and dolphin gp91^phox. However, this region of the protein is adjacent to several p47^phox-binding domains (e.g., see ref. [³⁰ ]) and consequently, would be located in the cytosol. Our Western blot results show that rabbit gp91^phox migrates as a broad band of 73 kDa, which is smaller than that of human gp91^phox (91 kDa). Thus, based on the smaller size of the rabbit protein and the location of putative glycosylation sites, we conclude that rabbit gp91^phox is glycosylated only at Asn132 and Asn149.

The rabbit p40^phox 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 p40^phox to be about 40.4 kDa and the human homolog to be 41.2 kDa. Rabbit p40^phox is also well-conserved when compared with other known p40^phox homologs (average 86.9±2.0% similarity). Rabbit p40^phox 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 p40^phox is consistent with the functional role it plays in binding to p47^phox [³⁸ ], and recent studies by Cross [³⁹ ] suggest that this interaction may play a role in increasing the affinity of p47^phox for flavocytochrome b. Additionally, a region in the C-terminus of p40^phox has been shown to participate in association with p67^phox 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 p40^phox function, this motif is highly conserved in rabbit p40^phox.

The rabbit p47^phox sequence encodes a protein of 391 amino acids, which is one amino acid longer than human and murine p47^phox 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 p47^phox with other p47^phox sequences revealed a high degree of divergence among all species near the C-terminus (residues 339–361), with an average of 22% homology among p47^phox homologs in this region of the protein.

Rabbit p47^phox 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 p47^phox sequences. The C-terminal proline-rich region of p47^phox, corresponding to residues 361–371 of rabbit p47^phox, represents a consensus SH3 domain-binding site and is identical in all p47^phox homologs, which is consistent with the key role this domain plays in binding a p67^phox SH3 domain [⁴¹ ]. Previously, we found that p67^phox and flavocytochrome b used a common binding site in p47^phox (residues 323–332), presumably at distinct stages of the activation process [³² ]. These residues are located within a highly cationic p47^phox domain that also contains multiple, potential phosphorylation sites. During activation, phosphorylation and subsequent conformation changes in p47^phox appear to result in exposure of this domain for binding to other oxidase proteins [³² ]. The high level of conservation among p47^phox 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 p47^phox becomes multiply phosphorylated during NADPH oxidase activation [⁹ , ¹⁰ ]. This phosphorylation has been proposed to neutralize a highly cationic region of p47^phox 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 p47^phox, 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 p47^phox contains serines at all of these sites, except for the residues corresponding to human p47^phox Ser345 and Ser359 (where a conserved Thr is substituted). Consistent with previous studies showing that phosphorylation of Ser379 played an essential role in p47^phox translocation to the membrane and NADPH oxidase activity [⁴³ ], this serine and flanking consensus phosphorylation site are conserved in rabbit p47^phox. Previously, El Benna et al. [⁴⁴ ] demonstrated that the p47^phox Ser345 and Ser348 (human sequence) were located in consensus sequences (-PXSP-), which are recognized by mitogen-activated protein kinase (MAPK), and postulated that p47^phox was regulated by MAPK-mediated phosphorylation. However, this consensus sequence was not conserved in murine or bovine p47^phox. Furthermore, this consensus site is also not conserved in rabbit p47^phox, providing even greater support to the conclusion that p47^phox is probably not phosphorylated by a MAPK or another proline-directed kinase during NADPH oxidase activation.

The rabbit p67^phox sequence encodes a protein of 543 amino acids, which is 17 amino acids longer than human and dolphin p67^phox, 16 amino acids longer than bovine p67^phox, 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 p67^phox 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 p67^phox 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 p67^phox corresponding to amino acids 74–312 of the human sequence. It is interesting that comparison of rabbit leukocyte p67^phox with this fragment rabbit fibroblast p67^phox 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 p67^phox 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 gp91^phox 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 p47^{phox -/-} mice did not influence basal blood pressure significantly, yet recent studies by Wang et al. [⁴⁹ ] showed significantly lower basal blood pressure in gp91^{phox -/-} 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 p47^{phox -/-}/apoE^-/- [⁴⁸ ] or gp91^{phox -/-}/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 gp91^phox and p67^phox. Further sequencing of NADPH oxidase proteins from different tissues in the same species will be necessary to address this issue.

Overall, rabbit p67^phox was the least conserved of all rabbit NADPH oxidase proteins (average 77.0±1.3% similarity) when compared with other known p67^phox sequences. Furthermore, rabbit p67^phox 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 p67^phox homologs in this region of the protein. Rabbit p67^phox 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 p67^phox 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 p67^phox 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 p47^phox during NADPH oxidase assembly [¹⁹ ]. Comparison of p67^phox homologs shows that this region is essentially identical in rabbit p67^phox, supporting its putative role in NADPH oxidase function. An "activation domain" has also been identified in human p67^phox (residues 199–210) [⁵³ ], and comparison of this domain in rabbit p67^phox shows that this sequence is identical, supporting its putative role in oxidase activation. The interaction between p67^phox and Rac is also essential for NADPH oxidase activation and appears to be mediated primarily by binding Rac to the amino-terminal region of p67^phox (residues 1–199) [¹⁷ ]. It is interesting that residues 1–199 of rabbit p67^phox fall within the most conserved region of p67^phox, and it has been demonstrated that the tetratricopeptide repeat (TPR) motifs located in the N-terminus of p67^phox 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 p67^phox, a region that is almost identical between rabbit p67^phox and all other known p67^phox homologs.

The function of the 17 additional C-terminal amino acids found in rabbit p67^phox is still unclear. Comparative oxidase assays using full-length and a truncated form of rabbit p67^phox 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 p67^phox 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 p67^phox 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.

 
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 p47^phox.

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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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