Blood online
Home About Blood Authors Subscriptions Permission Advertising Public Access contact us
 

 
Advanced
Current Issue
First Edition
Future Articles
Archives
Submit to Blood
Search
American Society of Hematology
Meeting Abstracts
Email Alerts
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
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leusen, J. H. W.
Right arrow Articles by Roos, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leusen, J. H. W.
Right arrow Articles by Roos, D.
Related Collections
Right arrow Phagocytes
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

arrow to previous article Previous Article  |  Table of Contents  |  Next Article next article arrow

Blood, Vol. 95 No. 2 (January 15), 2000: pp. 666-673

PHAGOCYTES

Four novel mutations in the gene encoding gp91-phox of human NADPH oxidase: consequences for oxidase assembly

J. H. W. Leusen, C. Meischl, M. H. M. Eppink, P. M. Hilarius, M. de Boer, R. S. Weening, A. Åhlin, L. Sanders, D. Goldblatt, H. Skopczynska, E. Bernatowska, J. Palmblad, A. J. Verhoeven, W. J. H. van Berkel, and D. Roos

From the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, the Laboratory of Experimental and Clinical Immunology, and the Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; the Department of Biomolecular Sciences, Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands; Wilhelmina Children's Hospital, Utrecht, The Netherlands; the Department of Pediatrics, Sachs' Children's Hospital, Stockholm Söder Hospital, Stockholm, Sweden; the Department of Medicine, the Center for Inflammation and Hematology Research, Huddinge University Hospital, Huddinge, Sweden; the Department of Clinical Immunology, Children's Memorial Hospital, Warsaw, Poland; and the Institute of Child Health, University College London, London, Great Britain.


    Abstract
Top
Abstract
Introduction
Patients and methods
Results
Discussion
References

The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients with chronic granulomatous disease (CGD) are defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox or p22-phox, which constitute the membrane-bound cytochrome b558. In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal amounts of nonfunctional cytochrome b558 in the patients' neutrophils. In phorbol-myristate-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369right-arrowArg, Gly408right-arrowGlu, and Glu568right-arrow Lys substitutions was strongly disturbed. Only a Thr341right-arrowLys substitution, residing in a region of gp91-phox involved in flavin adenine dinucleotide (FAD) binding, supported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b558. Based on mutagenesis studies of structurally related flavin-dependent oxidoreductases, we propose that the Thr341right-arrowLys substitution results in impaired hydride transfer from NADPH to FAD. Because we found no electron transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical for electron transfer. Apparently, an intimate relation exists between domains of gp91-phox involved in electron transfer and in p47/p67-phox binding. (Blood. 2000;95:666-673)

© 2000 by The American Society of Hematology.


    Introduction
Top
Abstract
Introduction
Patients and methods
Results
Discussion
References

Phagocytic leukocytes use reactive oxygen metabolites to kill ingested microorganisms. The first step in the production of these compounds is the generation of superoxide by the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase enzyme in these cells. For an active NADPH oxidase, at least 5 different proteins are required: the membrane-bound cytochrome b558 (a flavocytochrome consisting of the subunits gp91-phox and p22-phox)1,2 and 3 cytosolic proteins, p47-phox,3 p67-phox,4 and a low-molecular-weight guanosine 5'-triphosphate-binding (GTP-binding) protein rac.5,6 A fourth cytosolic oxidase component, namely, p40-phox,7,8 has been described. However, this latter protein is not essential for oxidase activity because complete reconstitution of oxidase activity in a cell-free assay is achieved by purified cytochrome b558 with recombinant p47-phox, p67-phox, and rac protein.9

In resting neutrophils, most of the cytochrome b558 resides in the membrane of specific granules or secretory vesicles.10,11 Upon cell activation, these organelles fuse with the plasma membrane, which results in expression of cytochrome b558 in the plasma membrane. At the same time, a complex of cytosolic phox proteins translocates to the plasma membrane and forms an enzymatically active complex with cytochrome b558.12-14

Patients suffering from chronic granulomatous disease (CGD) typically lack p47-phox, p67-phox, p22-phox, or gp91-phox (reviewed elsewhere15,16). X-linked CGD patients bear mutations in CYBB, the gene encoding gp91-phox. In some of these patients, normal amounts of nonfunctional gp91-phox are found; such patients are designated X 91+ CGD patients.

Thus far, 6 X91+ CGD patients have been described (reviewed elsewhere17). Two of these patients have mutations leading to substitutions in the N-terminal part of gp91-phox (R54S and A57E), which affect heme binding and/or stable interaction with p22-phox.18,19 The remaining 4 X91+ patients bear mutations in the cytosolic C-terminal part of gp91-phox. This region of gp91-phox is important for FAD and NADPH binding and is also involved in recruitment of cytosolic phox proteins. From sequence comparison between the C-terminal half of gp91-phox and the ferredoxin-NADP+ reductase flavoenzyme family, the putative location of these FAD-binding and NADPH-binding domains within gp91-phox have been deduced.20-22 In 2 X91+ patients, there are substitutions in regions of gp91-phox predicted to be involved in NADPH binding: P415H and a replacement of residues 507-509 by HisIleTrpAla.23-24

The last 2 X91+ patients thus far described carry mutations in the cytosolic part of gp91-phox outside the putative binding regions for FAD or NADPH: a deletion of amino acids 488-497 and a D500G substitution.25-26 These mutations reside in a surface loop exposed to the cytosol as predicted by a structural model of gp91-phox.27 This suggests a role for this region of gp91-phox in the recruitment of cytosolic phox proteins. Indeed, the translocation of p47-phox and p67-phox to the plasma membranes of the neutrophils of the patient with the D500G substitution was abrogated in intact neutrophils as well as in the cell-free system.26 The other patient has not been studied in this respect. Residue 500 of gp91-phox is the only residue to date that has been proven to be critical for interaction with the cytosolic oxidase components.

In the present report, we characterize an additional 4 X91+ CGD patients. One of these patients has a substitution (T341K) in an FAD-binding region, the other patients have substitutions C369R, G408E, and E568K, which are predicted to be involved in NADPH binding or interaction with the cytosolic proteins p47-phox and p67-phox. We have studied the translocation of cytosolic proteins in these 4 patients. Indeed, the 3 latter patients have a strongly disturbed translocation of cytosolic phox proteins to the plasma membrane. Only the membranes harboring the T341K substitution supported a normal translocation, which indicates another cause of the oxidase defect in this patient, probably decreased FAD binding. With the help of these unique CGD patients we can verify predictions for the different binding regions derived from the structural model of gp91-phox and gain more insight into the complex interactions within the NADPH oxidase.


    Patients and methods
Top
Abstract
Introduction
Patients and methods
Results
Discussion
References

Clinical history

All 4 patients suffer from the classical form of CGD, with frequent bacterialand fungal infections of the airways, skin, and lymph nodes. Two patients (MK, DG) presented with clinical manifestations at 8 months of life, the other 2 (JW, CE) at 3 years of age. All patients have been hospitalized at least 3 times. Surgical drainage of lymph nodes was performed in 2 patients (JW, MK). Microorganisms cultured were Salmonella from blood (DG), Staphylococcus aureus from blood (DG), Aspergillus fumigatus from the lungs (CE), and Mycobacterium bovis from liquor (MK). Granulomatous lesions were found in the colon (CE) and in the cerebrum (MK). Treatment consisted of intravenous antibiotics during infectious episodes and prophylactic treatment with intracellular antibiotics (and itraconazole in MK and DG). Patient MK received granulocyte transfusions and interferon-gamma treatment during acute M bovis infection in the brain. In the family of JW, 2 maternal cousins with CGD have died. DG's mother has signs of lupus erythematosus.

Experimental procedures

Materials. We used the following materials: guanosine 5'-3-O-(thio)triphosphate (GTP-gamma -S) and NADPH (Boehringer Mannheim, Mannheim, Germany); reagents and molecular-weight markers for sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) (BioRad Laboratories, Richmond, CA); and nitrocellulose sheets BA84 for Western blotting (Schleicher & Schull, Dassel, Germany). Antibodies used in this study were mAb 449 and 48, directed against p22-phox and gp91-phox, respectively.20 Rabbit antisera specific for either p47-phox or p67-phox were raised against synthetic peptides identical to the last 12 residues of the C-termini. Goat antibody against rabbit immunoglobulin (Ig), conjugated to horseradish peroxidase, was produced within our institute (CLB, Amsterdam, The Netherlands). We also used a chemiluminescence kit with luminol (ECL; Amersham International, Uppsala, Sweden); diagnostic films (X-Omat AR; Kodak, Rochester, NY); and the following lipids (Sigma; product numbers appear in brackets): L-alpha -phosphatidylcholine type 2-S from soybean, 14% (P5638), and L-alpha -phosphatidic acid, sodium salt, from egg yolk lecithin, 98% (P9511).

Classification of CGD patients. Nitroblue tetrazolium (NBT) slide tests with PMA were performed on the neutrophils of the 4 patients,29 and respiratory burst activity after stimulation with opsonized yeast particles or PMA was determined by the rate of oxygen consumption30 and chemiluminescence with lucigenin.31 Cytochrome b558 contents were determined by absorption spectroscopy32 and immunodetection on Western blot.28 For immunodetection, 2 µg of protein from a neutrophil membrane fraction were dissolved in SDS sample buffer (125 mmol/L Tris, pH 6.8; 20% (w/v) SDS and 10% (v/v) beta -mercaptoethanol), loaded on a 10% polyacrylamide gel according to Laemmli,33 and then loaded in a gel apparatus (Mini-Protean II, BioRad). Western blotting was performed (Mini Trans-Blot cell, BioRad) according to the manufacturer's recommendations. The nitrocellulose was stained for gp91-phox and p22-phox with mAbs 48 and 449 and subsequently with goat-antimouse-Ig conjugated to horseradish peroxidase. Detection was performed by a chemiluminescence kit (ECL, Amersham International).

Preparation of RNA and DNA. Total RNA was purified from mononuclear leukocytes as described,34 and cDNA was synthesized with reverse transcriptase. The cDNA of the coding region of gp91-phox was amplified with PCR in 3 overlapping fragments as described,35,36 and it was subsequently sequenced (Sequenase Version 2.0 kit; US Biochemical, Cleveland, OH).37 Genomic DNA was isolated from circulating blood leukocytes37 of the 4 CGD patients.

Isolation and fractionation of leukocytes. Human neutrophils were prepared on 4 different occasions from 20 to 50 mL of citrated blood from the 4 CGD patients after obtaining informed consent. Neutrophils from a healthy donor were isolated in parallel on each occasion as previously described.38 Subsequently, sonicated neutrophils were fractionated over a sucrose gradient as described.26

Translocation of cytosolic proteins in intact neutrophils. Cells (20 × 106) of a patient and a healthy donor were incubated with PMA (100 ng/mL) or without PMA for 10 minutes at 37°C. The cells were then resuspended and sonicated in 1 mL of ice-cold oxidase buffer containing: sodium chloride (NaCl, 75 mmol/L); 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10 mmol/L); sucrose (170 mmol/L); magnesium dichloride (MgCl2, 1 mmol/L); ethyleneglycoltetraacetic acid (EGTA, 0.5 mmol/L); adenosine triphosphate (ATP, 10 µmol/L); and azide (2 mmol/L, pH 7.0) with GTPgamma S (5 µmol/L) and PMSF (100 µg/mL). After centrifugation (10 minutes, ×800 g), the sonicate was layered on a 15% sucrose gradient, as described previously,26 with MgCl2 (1 mmol/L); NaCl (40 mmol/L); EGTA, (0.5 mmol/L); and GTPgamma S (5 µmol/L). After centrifugation (45 minutes, ×100 000 g), 500 µL of plasma membranes were harvested. For immunodetection, 25 µL of membrane fraction were dissolved in SDS sample buffer (125 mmol/L Tris, pH 6.8; 20% (w/v) SDS and 10% (v/v) beta -mercaptoethanol) and were loaded on a 10% polyacrylamide gel, according to Laemmli,33 in a gel apparatus (Mini-Protean II, BioRad). Western blotting was performed (Mini Trans-Blot cell, BioRad) according to the manufacturer's recommendations. Detection of proteins was performed as described previously.26 When the blot was stained for 2 sets of proteins, the nitrocellulose was stripped, after the first staining, in 62.5 mmol/L Tris, pH 6.7; 2% SDS; and 100 mmol/L beta -mercaptoethanol.

Superoxide assay. NADPH oxidase activity with neutrophil membranes and cytosol was measured as the SOD-sensitive reduction of cytochrome c in a spectrophotometer (Lambda 2, Perkin Elmer, Norwalk, CT). The contents of 6 cuvettes measured in parallel were stirred continuously and were thermostatted at 28°C. Plasma membranes (10 µg of protein) and cytosol (200 µg of protein) were incubated in oxidase buffer (0.8 mL) and cytochrome c (60 µmol/L). After 2 minutes of incubation, oxidase assembly was induced by addition of SDS (100 µmol/L) and GTP-gamma -S (10 µmol/L). After 5 minutes, NADPH (250 µmol/L) was added, and the rate of cytochrome c reduction was measured at 550 nm.

Translocation of cytosolic proteins in the cell-free system. Neutrophil plasma membranes (20 µg protein) were mixed with neutrophil cytosol (400 µg of protein) in oxidase buffer (1 mL). Subsequently, SDS (100 µmol/L) and GTP-gamma -S (10 µmol/L) were added. After 10 minutes at room temperature, the mixture was loaded on a discontinuous sucrose gradient as previously described.26,39,40 NADPH oxidase activity of one-fifth part of the reisolated membranes was measured without cytosol, in the presence of SDS (100 µmol/L) and GTP-gamma -S (10 µmol/L). After 2 minutes, NADPH (250 µmol/L) was added and the rate of cytochrome c reduction was measured at 550 nm. In addition, the supernatant (50 µL) and the remaining four fifths of the reisolated membranes were analyzed by immunoblot for the presence of p47-phox, p67-phox, and cytochrome b558 subunits.

Oxygen consumption in plasma membranes without cytosol according to Koshkin and Pick. 41

Membrane protein (6 µg in 30 µL) was solubilized with 1-O-Octyl-beta -D-glucopyranoside (80 mmol/L) and buffer A (10 µL), which comprised: sodium phosphate (50 mmol/L, pH 7.4); EGTA (1 mmol/L); MgCl2 (1 mmol/L); sodium azide (NaN3, 2 mmol/L); dithiothreitol (1 mmol/L); and glycerol (20%).

Subsequently, the membranes were reconstituted with 4 µg each of L-alpha -phosphatidyl-choline and L-alpha -phosphatidic acid and diluted 8-fold in assay buffer: potassium (65 mmol/L), sodium phosphate buffer (pH 7.0), MgCl2 (1 mmol/L), EGTA (1 mmol/L), NaN3 (2 mmol/L), FAD (1 µmol/L), and SDS (30 µmol/L). The solution was allowed to stand on ice for 30 minutes. Oxygen consumption was measured with an oxygen electrode after the addition of NADPH (250 µmol/L).

Sequence alignment. A multiple sequence alignment of the structurally known members of the ferredoxin-NADP+ reductase (FNR) family was performed by superimposing the structures with the program TOP.42 The sequence incorporation of the cytosolic C-terminal half of gp91-phox into the alignment profile of the FNR family was based on a previous sequence alignment of gp91-phox with FNR.27


    Results
Top
Abstract
Introduction
Patients and methods
Results
Discussion
References

Diagnosis of CGD patients

The neutrophils of 4 patients (JW, CE, MK, and DG), stimulated with PMA or opsonized yeast particles, showed no respiratory burst as measured by oxygen consumption and chemiluminescence, which indicates a severe form of CGD. The cells of the mothers and/or sisters of patient JW, MK, and DG showed a mosaic pattern in the NBT slide test. These findings are compatible with an X-linked defect in these families. The cells of the only female patient (CE) showed a very low fraction of positive cells (4%) in the NBT slide test, which indicates an extreme lyonisation causing the CGD phenotype.

Because cytochrome b558 is typically absent in neutrophils of most patients with X-linked CGD, we measured the cytochrome b558 content in the patients' neutrophils by spectral and Western blot analysis. Interestingly, the optical spectrum of the patients' neutrophils showed an (almost) normal heme content (data not shown). On the immunoblot of the neutrophil membranes, both subunits of cytochrome b558 appeared to be present (see Figure 1).


View larger version (44K):
[in this window]
[in a new window]
  		Fig 1. Western blot of neutrophil plasma membranes. Plasma membranes (2 µg) were run on a 10% polyacrylamide minigel and blotted onto nitrocellulose. The blot was stained for gp91-phox and p22-phox with mAbs 48 and 449, respectively, as previously described. The patients are indicated above the lanes. For each patient, material of a control donor was processed in parallel.

Superoxide production in the cell-free assay

To localize the cellular defect in NADPH oxidase activity, membrane and cytosolic fractions were prepared from the patients' neutrophils and studied in a cell-free oxidase assay. As shown in Table 1, the membrane fraction of a control donor mixed with cytosol of the patients showed a normal rate of cytochrome c reduction, whereas the membrane fractions of the patients mixed with control cytosol showed almost no superoxide production. Only the membranes of patient CE supported a low rate of superoxide production, probably caused by the small amount of unaffected cytochrome b558 present in 4% of the neutrophils in this extremely lyonized patient. This demonstrates that the defect in all patients is localized in a membrane-bound component of the NADPH oxidase, ie, in cytochrome b558.

                              
View this table:
[in this window]
[in a new window]
  		Table 1. Superoxide production in the cell-free system

Genetic analysis of CGD patients

Because the defect in these patients was located in the membranes and was X-linked, we searched for mutations in gp91-phox. For this purpose, we amplified the coding region of the gp91-phox cDNA in 3 overlapping fragments and subsequently sequenced these fragments. We found 4 different point mutations in the 4 patients: JW (C1034A), CE (T1117C), MK (G1235A), and DG (G1714A), predicting amino acid substitutions T341K, C369R, G408E and E568K, respectively. These mutations were confirmed in the patients' genomic DNA after PCR amplification and subsequent sequencing (Figure 2).


View larger version (47K):
[in this window]
[in a new window]
  		Fig 2. Analysis of genomic DNA from both the patients and a healthy control. PCR products from each exon with intron boundaries and from the promoter region of CYBB were generated from genomic DNA obtained from circulating leukocytes and were analyzed by dye primer cycle sequencing. The figure shows the areas in which mutations were found in the patients. Patients are indicated by initials, nucleotide substitutions, and amino acid substitutions. Above each patient sequence, the normal sequence from the control is shown. Arrows indicate mutant sequence.

Translocation of p47-phox and p67-phox in neutrophils of CGD patients

To investigate the functional effect of these substitutions in the cytoplasmic tail of gp91-phox, we studied the association of the cytosolic proteins with the plasma membranes of PMA-activated neutrophils. For this purpose, neutrophils of the patients and those of a healthy donor were incubated in parallel in the absence or presence of PMA, and subcellular fractions were isolated. Figure 3 shows the Western blot of the supernatants and the plasma membranes of these neutrophils, stained for p47-phox and p67-phox. The figure clearly shows a strongly reduced translocation of both cytosolic oxidase components to the neutrophil membranes of the patients CE, MK, and DG upon stimulation with PMA. However, the neutrophils of patient JW showed a normal translocation.


View larger version (22K):
[in this window]
[in a new window]
  		Fig 3. Western blot analysis of plasma membranes of activated neutrophils. Cells (20 × 106) were incubated with PMA (100 ng/mL) or without PMA for 10 minutes at 37°C. Fractionation of the cells was performed as previously described. PAGE (10%) was performed for the plasma membranes with comparable amounts of cytochrome b558. The blot was stained with rabbit antisera against p47-phox and p67-phox. The patients are indicated above the lanes. For each patient, material of a control donor was processed in parallel. The treatment (-PMA or +PMA) is also indicated above the lanes. From patient MK, we did not obtain enough neutrophils to investigate the unstimulated cells; therefore, only the +PMA lane is shown.

Reprobing of the blots with antibodies against p22-phox and gp91-phox showed comparable amounts of these cytochrome b558 subunits between patients and controls (data not shown).

Effect of point mutations on oxide assembly

We also studied the neutrophil membranes of the 4 CGD patients for their ability to bind p47-phox and p67-phox in a cell-free translocation assay. The supernatant of the sucrose gradient and the reisolated membranes were analyzed on an immunoblot to determine the translocation of cytosolic oxidase components. Control membranes and the membranes of patient JW showed a significant association with p47-phox and p67-phox (Figure 4). In contrast, the neutrophil membranes of patients MK and DG did not contain appreciable amounts of cytosolic oxidase proteins. Only patient CE exhibited a very small amount of cytosolic proteins in the reisolated plasma membranes (6.5% of normal p67-phox translocation and 7.7% of normal p47-phox translocation, as determined by densitometry). This can be explained by the 4% of unaffected neutrophils in this extremely lyonized patient. The oxidase activity measured in the reisolated membranes is shown in Table 2. Similar to the cell-free system, only the membranes of patient CE generated a small amount of superoxide.


View larger version (36K):
[in this window]
[in a new window]
  		Fig 4. Western blot analysis of neutrophil membranes reisolated from the cell-free activation system. The reisolated membranes were prepared as described in the legend of Table 2. Four fifths of these membrane samples were precipitated with 10% (w/v) trichloric acid and resuspended in sample buffer as previously described. After 10% PAGE, the proteins were blotted and stained for p47-phox and p67-phox.


                              
View this table:
[in this window]
[in a new window]
  		Table 2. Superoxide production of reisolated membranes in the cell-free system

Oxygen consumption in neutrophil plasma membranes without cytosol

It has been described that relipidation of solubilized cytochrome b558 can elicit NADPH-dependent superoxide (O2-) production in the absence of cytosolic oxidase proteins,41 which suggests that cytochrome b558 contains the complete electron-transporting apparatus of the NADPH oxidase and that the cytosolic components merely function as activators. Therefore, we tested the neutrophil membranes of the patients for oxygen consumption in the absence of cytosol. For this purpose, membranes obtained from control donors or CGD patients were solubilized and activated with phospholipids in the presence of FAD and NADPH41 (Table 3). The neutrophil membranes of control donors exhibited significant oxygen consumption, whereas the neutrophil membranes of the CGD patients showed very low or no oxygen consumption.

                              
View this table:
[in this window]
[in a new window]
  		Table 3. Oxygen consumption of membranes without cytosol

Location of amino acid substitutions in gp91-phox

Figure 5 shows a sequence alignment of the cytosolic C-terminal domains of gp91-phox with members of the FNR family. From this alignment and the structural model of the globular portion of gp91-phox27 (Figure 6), the putative location of the amino acid substitutions in the X-linked CGD patients can be deduced.


View larger version (63K):
[in this window]
[in a new window]
  		Fig 5. Multiple sequence alignment of the cytosolic C-terminal nucleotide binding domains of gp91-phox with members of the FNR family with known 3-dimensional structure. The deduced amino acid sequence of gp91-phox is aligned with the amino acid sequences of ferredoxin NADP+ reductase from spinach (1fnb),43 Azotobacter vinelandii (1a8p),59 and Anabaena (1quf);60 phthalate dioxygenase reductase from Pseudomonas cepacia (2pia);46 nitrate reductase from corn (1cnf);61 cytochrome b5 reductase from pig liver (1ndh);62 flavohemoglobin from Alcaligenes eutrophus (1fhp);63 and flavodoxin reductase from Escherichia coli (1fdr).64 Secondary structure elements are underlined, conserved residues shown in bold, and mutated residues indicated by an asterisk.



View larger version (44K):
[in this window]
[in a new window]
  		Fig 6. Ribbon diagram of the 3-dimensional model of the cytosolic part of gp91-phox.27 The schematic diagram was generated with Ribbons.65 The alpha -helices are depicted as cylinders and the beta -sheets as arrows. The FAD-binding domain is drawn in orange, the NADPH-binding domain in blue, FAD in yellow, NADPH in red, and the mutated residues in white.

The T341K substitution in patient JW is located in the FAD-binding domain at the si-face of the flavin ring. Thr341 is part of a short sequence (HPFT, Figure 5) involved in binding the isoalloxazine moiety of FAD.20 Interestingly, this sequence is conserved in the plasma membrane iron reductase (FRE1) from yeast.43 However, in bacterial flavin reductase FRE proteins, the HPFT sequence is replaced by an RXYS motif,44 which is also omnipresent in the FNR family (Figure 5). In FNR, the equivalent serine residue interacts with the N5 atom of the flavin ring45,46 and is directly involved in the reduction of NADP+.47

The C369R substitution in patient CE is also situated in the FAD-binding domain (Figure 6). Cys369 resides in a surface loop and is next to the strictly conserved Gly370 (Figure 5). This glycine is at the beginning of a helix, proposed to be important for interaction with the pyrophosphate moiety of FAD.48

The G408E substitution in patient MK is localized in the NADPH-binding domain (Figure 6). Gly408 is predicted to be buried and close to the GXGXXP fingerprint sequence (Figure 5) for binding the pyrophosphate moiety of NADPH.48

Finally, the E568K substitution in patient DG is near the C-terminus (Figure 5), at the interface of the FAD-binding and NADPH-binding domain. This part of the protein has been implicated in interaction with p47-phox during assembly of the active oxidase complex.49


    Discussion
Top
Abstract
Introduction
Patients and methods
Results
Discussion
References

Cytochrome b558 of human phagocytes is a membrane-bound heterodimer of p22-phox and gp91-phox. The cytochrome has an NADPH-binding site and bears a flavin group that acts as the initial acceptor of a pair of electrons from NADPH.20-22 Two heme groups embedded in the flavocytochrome mediate the 1-electron transfer from FAD to molecular oxygen, thus generating superoxide (O2-).50 Sequence-homology studies between the C-terminal half of gp91-phox and the ferredoxin-NADP+ reductase flavoenzyme family suggest that both FAD and NADPH are bound by specific domains within this part of gp91-phox.20-22

CGD patients with X-linked, cytochrome b558-positive CGD (X91+) can be very helpful in verifying the various binding domains in gp91-phox. In this study we have characterized the defects in 4 X91+ CGD patients. We found 4 different point mutations in the gene encoding gp91-phox, leading to amino acid substitutions in the cytosolic C-terminal part of gp91-phox.

Patient JW carries a T341K substitution in the FAD-binding site of gp91-phox. This mutation does not result in a lower amount of cytochrome b558, which points to a proper folding of the gp91-phox polypeptide chain. In PMA-stimulated neutrophils of this patient, the translocation of p47-phox and p67-phox was unaffected (Figure 3). Also in the cell-free system the translocation appeared to be normal. These data, and the fact that the NADPH oxidase is inactive (Table 1 and 2), suggest that the defect in this patient is not due to an impaired regulation of cytochrome b558 activity by the cytosolic oxidase subunits, but is restricted to a subsequent step. Based on mutagenesis studies of FNR47 and flavin reductase,44 it is obvious that introduction of a positive charge at position 341 in gp91-phox impairs the efficient hydride transfer between NADPH and FAD. The T341K substitution probably influences the correct stacking of nicotinamide and isoalloxazine rings and most likely also the redox properties of the flavin. The lack of NADPH oxidase activity in this patient is probably not due to weak binding of the FAD. This may be derived from the fact that proper binding of FAD is essential for normal gp91-phox expression.51

The other 3 X91+ CGD patients studied in this report carry C369R, G408E, and E568K substitutions. In contrast to the first patient, translocation of p47-phox and p67-phox to the plasma membranes upon activation of the neutrophils of these patients was strongly disturbed (Figure 3). Also, in the cell-free system no appreciable amounts of p47-phox and p67-phox were detected in the membrane fractions (Figure 4). Thus far, only 1 other X91+ CGD patient has been described with a disturbed translocation of both cytosolic proteins. This patient is carrying a D500G substitution, situated in an exposed protein loop, as predicted by the structural model of the C-terminal half of gp91-phox. As can be seen from the sequence alignment in Figure 5, this surface loop is absent in other members of the FNR family. The disturbed translocation of cytosolic oxidase components in this patient is indicative for a role of the NADPH-binding domain of gp91-phox in p47-phox and/or p67-phox recognition. This idea corresponds with the notion of an additional NADPH-binding domain in p67-phox.52

The C369R substitution in patient CE described here also resides in a surface loop (Figure 6). However, this loop is located in the FAD-binding domain, which suggests that this domain is also involved in the translocation process. Based on the structural properties of the FNR family,48 the loop comprising residue 369 is supposed to be flexible. Therefore, defective translocation of cytosolic proteins in patient CE is most simply explained by an electrostatic repulsion mechanism. On the other hand, the C369R substitution is also close to helix F1, which is involved in binding the pyrophosphate moiety of FAD. This might explain the lack of electron transfer in solubilized plasma membranes of this patient.

In patient MK, the G408E substitution is localized between beta N1 and alpha N1, close to the GXGXXP fingerprint sequence (Figure 5) for binding the pyrophosphate moiety of NADPH.20,48 Although Gly408 is predicted to be buried and not likely a binding site for 1 of the cytosolic proteins, introduction of a more bulky acidic residue at this position could perturb normal NADPH binding through electrostatic repulsion of the pyrophosphate group and/or introduce local structural changes in the NADPH-binding domain that are transmitted to the protein surface. Such structural changes might also hamper NADPH binding, leading to impaired oxygen consumption of solubilized plasma membranes, but apparently also disturb the binding sites for p47-phox and/or p67-phox.

The substitution E568K in patient DG resides in the extreme C-terminal region of gp91-phox, implicated as a contact point for p47-phox.53-55 These published studies were performed with (inhibitory) peptides in the cell-free system. However, recently Dinauer et al56 used a different approach: In a human myeloid leukemia cell-line, the gp91-phox gene was knocked out by targeted disruption. Then site-directed mutagenesis and transfection of gp91-phox was used to probe the role of the C-terminus of gp91-phox in NADPH oxidase activity.49 Despite the expectations, none of the substitutions introduced in the region 560-570 of gp91-phox had a dramatic effect on oxidase activity in intact cells, but several substitutions were associated with reduced expression of gp91-phox.

In patient DG, the E568K substitution was associated with normal amounts of both subunits of cytochrome b558 (Figure 1). This contrasts with the in vitro mutagenesis studies,49 where C-terminal replacements resulted in poor protein expression. Nonetheless, in patient DG almost no oxidase activity was measured. Also, the translocation of p47-phox and p67-phox in PMA-stimulated neutrophils, as well as in the cell-free system, was abrogated. In addition, electron transfer in the solubilized neutrophil membranes of this patient was also disturbed. These results imply that Glu568 of gp91-phox is indeed required for oxidase activity and for the binding of the cytosolic phox proteins. In relation to this observation, it is interesting to note that in spinach FNR57 and Anabaena FNR,58 the equivalent C-terminal glutamic acid is critical for rapid electron transfer with the iron-sulfur cluster of ferredoxin, without appreciably affecting complex formation.

In conclusion, this paper reports on 4 X91+ CGD patients with unique single amino acid substitutions. These substitutions are localized in different regions of the FAD-binding and NADPH-binding domains of gp91-phox and cause strongly impaired superoxide production without affecting gp91-phox expression. Because 3 of these amino acid substitutions disturb the binding of cytosolic NADPH oxidase components to the membrane-bound gp91-phox, this points at an intimate relation between domains of gp91-phox involved in electron transfer and those involved in p47-phox/p67phox binding.


    Acknowledgments

We thank Dr U. Ermler for providing us with the coordinates of the flavohemoglobin structure (1fhp) and Dr A.W. Segal for the coordinates of the structural model of the globular portion of gp91-phox.


    Footnotes

Submitted April 28, 1999; accepted September 15, 1999.

Supported by grants from The Netherlands Fund for Preventive Medicine (Praeventiefonds) and from the German Research Foundation (Deutsche Forschungsgesellschaft) to C.M..

Reprints: D Roos, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; e-mail: d_roos{at}clb.nl.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.


    References
Top
Abstract
Introduction
Patients and methods
Results
Discussion
References

1. Parkos CA, Allen RA, Cochrane CG, Jesaitis AJ. Purified cytochrome b from human plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000. J Clin Invest. 1987;80:732-742.

2. Dinauer MC, Pierce EA, Bruns GA, Curnutte JT, Orkin SH. Human neutrophil cytochrome b light chain (p22-phox): gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. J Clin Invest. 1990;86:1729-1737.

3. Volpp BD, Nauseef WM, Donelson JE, Moser DR, Clark RA. Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase [erratum in Proc Natl Acad Sci U S A. 1989;86:9563.]. Proc Natl Acad Sci U S A. 1989;86:7195-7199[Abstract/Free Full Text].

4. Leto TL, Lomax KJ, Volpp BD, et al. Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. Science. 1990;248:727-730[Abstract/Free Full Text].

5. Knaus UG, Heyworth PG, Evans T, Curnutte JT, Bokoch GM. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science. 1991;254:1512-1515[Abstract/Free Full Text].

6. Abo A, Pick E, Hall A, Totty N, Teahan CG, Segal AW. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 1991;353:668-670[Medline] [Order article via Infotrieve].

7. Wientjes FB, Hsuan JJ, Totty NF, Segal AW. p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. Biochem J. 1993;296:557-561.

8. Tsunawaki S, Mizunari H, Nagata M, Tatsuzawa O, Kuratsuji T. A novel cytosolic component, p40phox, of respiratory burst oxidase associates with p67phox and is absent in patients with chronic granulomatous disease who lack p67phox. Biochem Biophys Res Commun. 1994;199:1378-1387[Medline] [Order article via Infotrieve].

9. Abo A, Boyhan A, West I, Thrasher A, Segal AW. Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1 and cytochrome b-245. J Biol Chem. 1992;267:16,767-16,770[Abstract/Free Full Text].

10. Bjerrum OW, Borregaard N. Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils [erratum in Eur J Haematol. 1989;8:270]. Eur J Haematol. 1989;43:67-77[Medline] [Order article via Infotrieve].

11. Johansson A, Jesaitis AJ, Lundqvist H, et al. Different subcellular localization of cytochrome b and the dormant NADPH oxidase in neutrophils and macrophages: effects on the production of reactive oxygen species during phagocytosis. Cell Immunol. 1995;161:61-71[Medline] [Order article via Infotrieve].

12. Park JW, Ma M, Ruedi JM, Smith RM, Babior BM. The cytosolic components of the respiratory burst oxidase exist as a M(r) approximately 240,000 complex that acquires a membrane-binding site during activation of the oxidase in a cell-free system. J Biol Chem. 1992;267:17,327-17,332[Abstract/Free Full Text].

13. Clark RA, Volpp BD, Leidal KG, Nauseef WM. Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J Clin Invest. 1990;85:714-721.

14. Ambruso DR, Bolscher BGJM, Stokman PM, Verhoeven AJ, Roos D. Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate [erratum in J Biol Chem. 1990;265:19,370.] J Biol Chem. 1990;265:924-930[Abstract/Free Full Text].

15. Roos D, de Boer M, Kuribayashi F, et al. Mutations in the X-linked and autosomal recessive forms of chronic granulomatous disease. Blood. 1996;87:1663-1681[Free Full Text].

16. Meischl C, Roos D. The molecular basis of chronic granulomatous disease. Springer Semin Immunopathol. 1998;19:417-434[Medline] [Order article via Infotrieve].

17. Leusen JH, Verhoeven AJ, Roos D. Interactions between the components of the human NADPH oxidase: intrigues in the phox family. J Lab Clin Med. 1996;128:461-476[Medline] [Order article via Infotrieve].

18. Cross AR, Heyworth PG, Rae J, Curnutte JT. A variant X-linked chronic granulomatous disease patient (X91+) with partially functional cytochrome b. J Biol Chem. 1995;270:8194-8200[Abstract/Free Full Text].

19. Ariga T, Sakiyama Y, Tomizawa K, Imajoh-Ohmi S, Kanegasaki S, Matsumoto S. A newly recognized point mutation in the cytochrome b558 heavy chain gene replacing alanine57 by glutamic acid, in a patient with cytochrome b positive X-linked chronic granulomatous disease. Eur J Pediatr. 1993;152:469-472[Medline] [Order article via Infotrieve].

20. Segal AW, West I, Wientjes F, et al. Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. Biochem J. 1992;284:781-788.

21. Rotrosen D, Yeung CL, Leto TL, Malech HL, Kwong CH. Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. Science. 1992;256:1459-1462[Abstract/Free Full Text].

22. Sumimoto H, Sakamoto N, Nozaki M, Sakaki Y, Takeshige K, Minakami S. Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein. Biochem Biophys Res Commun. 1992;186:1368-1375[Medline] [Order article via Infotrieve].

23. Dinauer MC, Curnutte JT, Rosen H, Orkin SH. A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease. J Clin Invest. 1989;84:2012-2016.

24. Azuma H, Oomi H, Sasaki K, et al. A new mutation in exon 12 of the gp91-phox gene leading to cytochrome b-positive X-linked chronic granulomatous disease. Blood. 1995;85:3274-3277[Abstract/Free Full Text].

25. Schapiro BL, Newburger PE, Klempner MS, Dinauer MC. Chronic granulomatous disease presenting in a 69-year-old man. N Engl J Med. 1991;325:1786-1790[Medline] [Order article via Infotrieve].

26. Leusen JHW, de Boer M, Bolscher BGJM, et al. A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox. J Clin Invest. 1994;93:2120-2126.

27. Taylor WR, Jones DT, Segal AW. A structural model for the nucleotide binding domains of the flavocytochrome b-245 beta-chain. Protein Sci. 1993;2:1675-1685[Medline] [Order article via Infotrieve].

28. Verhoeven AJ, Bolscher BGJM, Meerhof LJ, et al. Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils. Blood. 1989;73:1686-1694[Abstract/Free Full Text].

29. Meerhof LJ, Roos D. Heterogeneity in chronic granulomatous disease detected with an improved nitroblue tetrazolium slide test. J Leukoc Biol. 1986;39:699-711[Abstract].

30. Weening RS, Roos D, Loos J. Oxygen consumption of phagocytizing cells in human leukocyte and granulocyte preparations: a comparative study. J Lab Clin Med. 1974;83:570-577[Medline] [Order article via Infotrieve].

31. Weening RS, Corbeel L, De Boer M, et al. Cytochrome b deficiency in an autosomal form of chronic granulomatous disease: a third form of chronic granulomatous disease recognized by monocyte hybridization. J Clin Invest. 1985;75:915-920.

32. Lutter R, van Schaik ML, van Zwieten R, Wever R, Roos D, Hamers MN. Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes. J Biol Chem. 1985;260:2237-2244[Abstract/Free Full Text].

33. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685[Medline] [Order article via Infotrieve].

34. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biological active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;18:5294-5299[Medline] [Order article via Infotrieve].

35. Bolscher BGJM, de Boer M, de Klein A, Weening RS, Roos D. Point mutations in the beta-subunit of cytochrome b558 leading to X-linked chronic granulomatous disease. Blood. 1991;77:2482-2487[Abstract/Free Full Text].

36. de Boer M, Bolscher BGJM, Sijmons RH, Scheffer H, Weening RS, Roos D. Prenatal diagnosis in a family with X-linked chronic granulomatous disease with the use of the polymerase chain reaction. Prenat Diagn. 1992;12:773-777[Medline] [Order article via Infotrieve].

37. de Boer M, Bolscher BGJM, Dinauer MC, et al. Splice site mutations are a common cause of X-linked chronic granulomatous disease. Blood. 1992;80:1553-1558[Abstract/Free Full Text].

38. Roos D, de Boer M. Purification and cryopreservation of phagocytes from human blood. In: Di Sabato G,Everse J, eds. Immunochemical Techniques Part J Phagocytosis and Cell-Mediated Cytotoxicity. Methods Enzymol. Vol 132. New York, NY: Academic Press; 1986:225-243.

39. Leusen JHW, Bolscher BGJM, Hilarius PM, et al. 156Pro--->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22-phox) leads to defective translocation of the cytosolic proteins p47-phox and p67-phox. J Exp Med. 1994;180:2329-2334[Abstract/Free Full Text].

40. Leusen JHW, de Klein A, Hilarius PM, et al. Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease. J Exp Med 1996;184:1243-1249[Abstract/Free Full Text].

41. Koshkin V, Pick E. Generation of superoxide by purified and relipidated cytochrome b559 in the absence of cytosolic activators. FEBS Lett. 1993;327:57-62[Medline] [Order article via Infotrieve].

42. Lu G. A WWW service system for automatic comparison of protein structures. Protein Data Bank Newsletter. 1996;78:10-11.

43. Finegold AA, Shatwell KP, Segal AW, Klausner RD, Dancis A. Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase. J Biol Chem. 1996;271:31,021-31,024[Abstract/Free Full Text].

44. Nivière V, Fieschi F, Frier C, Décout J-L, Fontecave M. Is the NAD(P)H: flavin oxidoreductase from Escherichia coli a member of the ferredoxin: NADP+ reductase family? J Biol Chem. 1996;271:16,656-16,661[Abstract/Free Full Text].

45. Karplus PA, Daniels MJ, Herriot JR. Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family. Science. 1991;251:60-66[Abstract/Free Full Text].

46. Bruns CM, Karplus PA. Refined crystal structure of spinach ferredoxin reductase at 1.7 Å resolution: oxidized, reduced and 2'-phospho-5'-AMP bound states. J Mol Biol. 1995;247:125-145[Medline] [Order article via Infotrieve].

47. Aliverti A, Bruns CM, Pandini VE, et al. Involvement of serine 96 in the catalytic mechanism of ferredoxin-NADP+ reductase: structure-function relationship as studied by site-directed mutagenesis and X-ray crystallography. Biochemistry. 1995;34:8371-8379[Medline] [Order article via Infotrieve].

48. Correll CC, Ludwig ML, Bruns CM, Karplus PA. Structural prototypes for an extended family of flavoprotein reductases: comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin. Protein Sci. 1993;2:2112-2133[Medline] [Order article via Infotrieve].

49. Zhen L, Yu L, Dinauer MC. Probing the role of the carboxyl terminus of the gp91phox subunit of neutrophil flavocytochrome b558 using site-directed mutagenesis. J Biol Chem. 1998;273:6575-6581[Abstract/Free Full Text].

50. Quinn MT, Mullen ML, Jesaitis AJ. Human neutrophil cytochrome b contains multiple hemes: evidence for heme associated with both subunits. J Biol Chem. 1992;267:7303-7309[Abstract/Free Full Text].

51. Yoshida LS, Saruta F, Yoshikawa K, Tatsuzawa O, Tsunawaki S. Mutation of histidine 338 of gp91-phox depletes FAD and affects expression of cytochrome b558 of the human NADPH oxidase. J Biol Chem. 1998;273:27,879-27,886[Abstract/Free Full Text].

52. Smith RM, Connor JA, Chen LM, Babior BM. The cytosolic subunit p67phox contains an NADPH binding site that participates in catalysis by the leukocyte NADPH oxidase. J Clin Invest. 1996;98:977-983[Medline] [Order article via Infotrieve].

53. Rotrosen D, Kleinberg ME, Nunoi H, Leto T, Gallin JI, Malech HL. Evidence for a functional domain of phagocyte oxidase cytochrome b558. J Biol Chem. 1990;265:8745-8750[Abstract/Free Full Text].

54. Kleinberg ME, Mital D, Rotrosen D, Malech HL. Characterization of a phagocyte cytochrome b558 91-kilodalton subunit functional domain: identification of peptide sequence and amino acids essential for activity. Biochemistry. 1992;31:2686-2690[Medline] [Order article via Infotrieve].

55. DeLeo FR, Nauseef WM, Jesaitis AJ, Burritt JB, Clark RA, Quinn MT. A domain of p47phox that interacts with human neutrophil flavocytochrome b558. J Biol Chem. 1995;270:26,246-26,251[Abstract/Free Full Text].

56. Zhen L, King AA, Xiao Y, Chanock SJ, Orkin SH, Dinauer MC. Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox. Proc Natl Acad Sci U S A. 1993;90:9832-9836[Abstract/Free Full Text].

57. Zanetti G, Aliverti A, Ravashi D, Curti B, Deng Z, Karplus PA. On the role of glutamate 312 of spinach ferredoxin-NADP+ reductase. In: Stevenson KJ,Massey V,Williams CH Jr, eds. Flavins and Flavoproteins XII. Calgary, Canada: University Press; 1997:509-512.

58. Medina M, Martínez-Júlvez M, Hurley JK, Tollin G, Gómez-Moreno C. Involvement of glutamic acid 301 in the catalytic mechanism of ferredoxin-NADP+ reductase from Anabaena PCC 7119. Biochemistry. 1998;37:2715-2728[Medline] [Order article via Infotrieve].

59. Sridhar Prasad G, Kresge N, Muhlberg AB, et al. The crystal structure of NADPH: ferredoxin reductase from Azotobacter vinelandii. Protein Sci. 1998;7:2541-2549[Medline] [Order article via Infotrieve].

60. Serre L, Vellieux FMD, Medina M, Gomez-Moreno C, Fontecilla-Camps JC, Frey M. X-ray structure of the ferredoxin NADP reductase from the cyanobacterium Anabaena PCC 7119 at 1.8 Å resolution and crystallographic studies of NADP binding at 2.25 Å resolution. J Mol Biol. 1996;263:20-39[Medline] [Order article via Infotrieve].

61. Lu G, Campbell WH, Schneider G, Lindqvist Y. Crystal structure of the FAD-containing fragment of corn nitrate reductase at 2.5 Å resolution: relationship to other flavoprotein reductases. Structure. 1994;2:809-821[Medline] [Order article via Infotrieve].

62. Nishida H, Inaka K, Yamanaka M, Kaida S, Kobayashi K, Miki K. Crystal structure of NADH-cytochrome b5 reductase from pig liver at 2.4 Å resolution. Biochemistry. 1995;34:2763-2767[Medline] [Order article via Infotrieve].

63. Ermler U, Siddiqui RA, Cramm R, Friedrich B. Crystal structure of the flavohemoglobin from Alcaligenes eutrophus at 1.75 Å resolution. EMBO J. 1995;14:6067-6077[Medline] [Order article via Infotrieve].

64. Ingelman M, Bianchi V, Eklund H. The three-dimensional structure of flavodoxin reductase from Escherichia coli at 1.7 Å resolution. J Mol Biol. 1997;268:147-157[Medline] [Order article via Infotrieve].

65. Carson M. Ribbons 2.0. J Appl Crystallogr. 1991;24:958-961.

© 2000 by The American Society of Hematology.
 

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
X. J. Li, D. Grunwald, J. Mathieu, F. Morel, and M.-J. Stasia
Crucial Role of Two Potential Cytosolic Regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH Oxidase Activation
J. Biol. Chem., April 15, 2005; 280(15): 14962 - 14973.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
K. Shinozaki, K. Ayajiki, Y. Nishio, T. Sugaya, A. Kashiwagi, and T. Okamura
Evidence for a Causal Role of the Renin-Angiotensin System in Vascular Dysfunction Associated With Insulin Resistance
Hypertension, February 1, 2004; 43(2): 255 - 262.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. B. Burritt, T. R. Foubert, D. Baniulis, C. I. Lord, R. M. Taylor, J. S. Mills, T. D. Baughan, D. Roos, C. A. Parkos, and A. J. Jesaitis
Functional Epitope on Human Neutrophil Flavocytochrome b558
J. Immunol., June 15, 2003; 170(12): 6082 - 6089.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. Dekker, M. H. M. Eppink, R. van Zwieten, T. de Rijk, A. F. Remacha, L. K. Law, A. M. Li, K. L. Cheung, W. J. H. van Berkel, and D. Roos
Seven new mutations in the nicotinamide adenine dinucleotide reduced-cytochrome b5 reductase gene leading to methemoglobinemia type I
Blood, February 15, 2001; 97(4): 1106 - 1114.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. S. Wolin
How Could a Genetic Variant of the p22phox Component of NAD(P)H Oxidases Contribute to the Progression of Coronary Atherosclerosis?
Circ. Res., March 3, 2000; 86(4): 365 - 366.
[Full Text] [PDF]

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
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leusen, J. H. W.
Right arrow Articles by Roos, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leusen, J. H. W.
Right arrow Articles by Roos, D.
Related Collections
Right arrow Phagocytes
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

click for free articles
home about blood authors subscriptions permissions advertising public access contact us
  Copyright © 2000 by American Society of Hematology         Online ISSN: 1528-0020