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Activation factors of neutrophil NADPH oxidase complex
Life Sciences, Vol. 55, No. 1, pp. 1-13, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/94 $6.00 + .00
Pergamon 0024-3205(94) 00075-1
MINIREVIEW
ACTIVATION FACTORS OF NEUTROPHIL NADPH OXIDASE COMPLEX Shigenobu Umeki Department of Medicine Toshida-kai Kumeda Hospital 2944 Obu-Cho, Kishiwada, Osaka 596, Japan (Received in final form April 26, 1994)
Summary Professional phagocytes, neutrophils, possess a unique membraneassociated NADPH oxidase system, dormant in resting cells, which becomes activated upon exposure to the appropriate stimuli and catalyzes the one-electron reduction of molecular oxygen to superoxide, O;,. Oxidase activation involves the assembly, in the plasma membrane, ot~membrane-bound and cytosolic constituents of the oxidase system, which are disassembled in the resting state. The oxidase system consists of two plasma membrane-bound components; low-potential cytochrome b558, which is composed of two subunits of 22 kDa and 91 kDa, and a flavoprotein related to the electron transport between NADPH and heme-binding domains of the oxidase. Recent reports have indicated that FAD-binding sites of the oxidase are contained in cytochrome b~o~o(flavocytochrome b...). At least two cytosolic components, 67 kDa protein and a phosphory°l=,~°ted 47 kDa protein, are known to translocate to the plasma membrane, ensuring assembly of an active O2-generating NADPH oxJdase system. More recently, the membrane (Raps) and cytosolic (Racs) GTPbinding proteins have been established as essential to oxidase assembly. It is the purpose of this review to focus on recent data concerning the regulatory mechanisms which lead to organization and activation of the neutrophil NADPH oxidase system. Key Words: neutrophils, N A D P H oxidase, electron transfer system, chronic granulomatous disease
Human neutrophils serve a crucial role in the host defense through normal activation of a latent plasma membrane multicomponent NADPH oxidase (EC 1.6.99.6) system associated with the principal defense against a wide variety of bacteria (1-3). Activation of the neutrophil oxidative metabolism is characterized by strong cyanide-insensitive oxygen consumption (respiratory burst) (4,5) and by concomitant production of active oxygen derivatives such as superoxide (O;), H>O~, and .OH (6). Superoxide is produced primarily through the activation of plasma ?nemq3rAne-bound NADPH oxidase by stimulation with phagocytizable particles (7) or soluble agents (8). The major contribution of O2 and its dismutation products to the bactericidal capacity of the body is readily demonstrated by the susceptibility to infection of patients with chronic granulomatous disease, in which there is a defect in NADPH oxidase or its activating apparatus (6,9). As a result of many studies of cell-free systems, important insights into the sequence of activation steps required for a normal neutrophil response have been obtained. By sequencing of cDNA clones and tracing of exogenous agonist-induced stimulation of NADPH oxidase, the basic elements of the activation sequence have been now disclosed. The present review focuses on recent findings regarding the nature of the electron transport components of the neutrophil NADPH oxidase system and its activation/regulation mechanism in relation to specific pathological aspects of chronic granulomatous disease.
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1. NADPH oxidase components
1.1. Plasma membrane components 1.1.1. Cytochromeb558 Cytochrome bs.^ is localized both in the plasma membrane, which becomes the wall of the phagocytic vac~°ole by its invagination (10), and in the specific granule membrane, which transfers to the membrane of the phagocytic vacuole (11). The cytochrome is a heterodimer consisting of a small (a) subunit of 22 kDa (p22-phox; phox indicates phagocyte oxidase), which carries the heme and a large (/3) subunit of 91 kDa (gp91-phox), which is highly glycosylated. Studies of cytochrome b558 were begun in earnest after a correlation was found between the absence of cytochrome b~.. in neutrophils from patients with a classic form of chronic granulomatous disease (CGI~ ~ linked to the X chromosome and the inability of these deficient neutrophils to mount the respiratory burst. Cytochrome b.... which was first discovered in horse neutrophils, was identified from human neutrophlls ~y Segal and Jones (12). This cytochrome has an extremely low mid-point oxidation-reduction (redox) potential (Ern7. = -245 mv) (13), lower than that of any other mammalian cytochrome and sufficiently low to reduce oxygen to 02 (Era7 = -160 mv) (14). since the identification of this cytochrome, much evidence supporting participation of cytochrome bs58 in the respiratory burst has accumulated. With cloning of the X-linked defective genes responsible for a classic form of CGD and the discovery that these genes encode the protein moiety of p22-phox and gp91-phox of cytochrome b558 (15-18), cytochrome b~oo has been accepted as an important component of respiratory burst oxldase. This c~ochrome has now been purified and shown to be a heterodimeric protein consisting of two subunits; p22-phox with a molecular weight of 22,000 - 23,000 and gp91 -phox with a molecular weight of 76,000 - 92,000 as determined by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 2 (19-22). The p22-phox is a heme-bearing protein (22,23) which may be the terminal component of the respiratoryburst electron transport system. By comparing the binding characteristics of a monoclonal antibody reacting with p22-phox to intact and permeabilized neutrophils, it was established that p22-phox is a transmembrane protein (24,25). The gp91 -phox of cytochrome b558 is a heavily glycosylated integral membrane protein with several membrane-spanning regions predicted by hydrophobicity analysis (15,16,19). The glycosylated portion of the gp91-phox is not essential for oxidase activity, but may be required for optimum production or stability of the cytochrome b~58 and could modulate the activity. This has been shown by the fact that, when N-glyc~sylation in differentiating HL60 cells was inhibited by tunicamycin, the gp91-phox was synthesized with a mass close to 58 kDa, similar to that found after cleavage with endoglycosidase F (26). Based on an analysis with antibodies raised against synthetic peptides corresponding to various regions in gp91-phox, it was concluded that this subunit is a transmembrane protein (27). In experiments using the synthetic peptide-containing gp91-phox and antibody-binding assays of the cytoplasmic carboxyl-terminal tail of the gp91-phox, the subunit was found .to mediate interactions with other cytosolic proteins essential to activation of the respiratory burst, such as p47-phox and p67-phox (28). Therefore, gp91-phox may play a central role in regulating the cellular assembly of the heterodimeric cytochrome bs58. The two subunits are very tightly associated and any maneuvers designed to separate them also displace the heme. Recently, it has been argued that gp91-phox might play a structural role in the maintenance of the heme-bearing p22-phox in the plasma membrane (3). Using electron paramagnetic resonance spectroscopy, it was determined that cytochrome bs.o is a hexa-coordinated ferric hemoprotein, and that the heme iron is ligated in its °f~fth and sixth coordination positions by two imidazole nitrogens of histidine residues (29). As only one histidine residue exists in the p22-phox (30). This evidence suggests that gp91-phox may also participate in heme binding. Quinn et al. (31), using lithium dodecyl sulfate-PAGE followed by tetramethylbenzidine heme 1CGD, chronic granulomatous disease; 2SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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NADPH Oxidase Activation Factors
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staining, reported that a multi-heme structure residing in both the p22-phox and gp91phox subunits would be consistent with an electron transfer function for cytochrome b558 by providing an efficient mechanism for transferring electrons across the plasma membrane to the extracellular surface where oxygen could be reduced to create 0 2. The genes for both subunits have been cloned and sequenced (15-18). Transcription of the genes for the two subunits, p22-phox and gp91-phox, each of which has close physical interaction, is independently regulated (1,3). Although mRNA for p22-phox exists in a wide variety of cell lines derived from non-phagocytic cells, synthesis of gp91-phox mRNA transcript is limited to phagocytic cells and the myeloid cell lineage. This suggests that regulation of cytochrome b55. assembly may occur through the expression of gp91-phox. Transcription of gp91-~hox is increased in parallel with expression of the cytochrome after the induction of oxidase activity by interferon-y in the cells of patients with variant CGD (1). Although the oxidase activity and special activity of cytochrome b558 are enhanced by interferon-y, expression of p22-phox is not (32). Recently, Knoller et al. (33) hypothesized that cytochrome b558 might be the only redox component associated with activated NADPH oxidase. This Is supported by three proposals: 1) that the NADPH-binding site is located on the cytochrome b558 dimer itself, 2) that it is present on one of the cytosolic components, and 3) that it is a complex structure to which both cytochrome bs58 and a cytosolic component contribute. According to Rotrosen et al. (34), NADPH oxidase activity was reconstituted in a cell-free system with only purified oxidase proteins, such as cytosolic proteins (p47-phox and p67-phox), ras-related guanosine triphosphate-(GTP-) 1 binding proteins and membrane-bound cytochrome b.... requiring exogenous flavin adenine dinucleotide (FAD) 2. The FAD binding site was~°~ocated on cytochrome b558. Based on evidence that purified cytochrome b.58 was completely dependent on exogenous FAD to obtain its high 02 production, S~mimoto et al. (35) aligned the amino acid sequence of gp91-phox with previously characterized flavoproteins, and concluded that the middle and carboxylterminal portions of gp91-phox are probably FAD- and NADPH-binding domains, respectively. Similar results were obtained by Segal et al. (36). These results strongly indicate that cytochrome b558 is a fiavoprotein (flavoc.ytochrome b558) bearing the complete electron-transporting apparatus of the NADPH oxldase system. By measunng FAD and heme contents in membranes from resting and activated neutrophils, Segal et al. (36) found that flavocytochrome b~58 bound to both FAD and heme at a molar ratio of 1:2. Structural and functional data frorfi several laboratories in the United States, Europe and Japan established flavocytochrome b558 as both the NADPH- and FAD-binding component of the oxidase. Quinn et al. (37)coisolated a protein with a relative molecular mass of 22 kDa with heterodimeric flavocytochrome b.... This protein was considered to be identical to the sequence of a ras-related GTF~2~inding protein identified as Rap-1 on the basis of its primary structure deduced from cDNA. Immunoaffinity purification of flavocytochrome b#~8 using a monoclonal antibody reactive to the highly conserved GTP-binding domains oTnuman H-ras, N-ras and K-ras proteins showed an association between flavocytochrome b..o and the ras-related protein (37), indicating a role for such a protein in the transductlon, regulation or structure of the NADPH oxidase system. In immunoelectron microscopic studies with Rap-l-specific antibodies, furthermore, Quinn et al. (38) found that Rap-1 is colocalized with flavocytochrome b... in both plasma membranes and specific granules of human neutrophils and is cotr,~n%located with flavocytochrome /9558 to the phagolysosomal membranes during the course of oxidase activation. Rap-~ binding to flavocytochrome bs. 8 was inhibited by phosphorylation of Rap-1 with cAMP-dependent protein kinas~e (39). These results suggest a functional association of these two molecules in the resting state, a role for Rap-1 in the structure or function of the activated NADPH oxidase, and a phosphorylation-coupled regulation of Rap-1 function in the respiratory burst system. A GTPase-activating protein (GAP) 3 involved 1GTP, guanosine triphosphate; 2FAD, flavin adenine dinucleotide; 3GAP, GTPase-activating protein.
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NADPH Oxidase Activation Factors
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with Ras proteins (40) possesses a significant sequence similar to the cytosolic component of p47-phox required for oxidase activation (41), suggesting a possible association of Rap-1 with the sensitivity of the respiratory burst to GTP in a cell-free system. A recent study of a cell-free system by Eklund et al. (42) showed that purified human recombinant Rap-1 could completely reconstitute cytosol from which ras-related proteins were immunologically depleted. This may be consistent with Rap-1 as a necessary cofactor. However, the data of Rotrosen et al. (34) and Abo et al. (43) using purified flavocytochrome b devoid of Rap-1 suggest that Rap-1 is not an essential component of the oxidase, but it may still play a regulatory role in the whole cell. In this regard, further detailed studies should be done. .
558
1.1.2. NADPH-dehydrogenase Although the involvement of cytochrome b..~ as a terminal electron transporter of the NADPH oxidase system is evident, it is st~°unclear which protein is the flavoprotein transferring electrons from NADPH to the heme as the prosthetic group in the oxidase system. The association of a flavoprotein in this oxidase was postulated by Cagan and Karnovsky (43). There is much evidence indicating that the cofactor is FAD (44-47) and that a FAD-containing flavoprotein is one of the electron carriers in the NADPH oxidase system (2,3,45,48-54). A definite conclusion, however, must await the identification of this flavoprotein, reports concerning its kinetic competency in cell-free reconstituted studies, and its DNA sequence analysis. An NADPH-binding protein focusing at pl 5.0 on gel (51) with a molecular weight of 65,000 - 67,000 (45, 49, 51), which appears to be a fiavoprotein, has been purified from activated neutrophils. The capacity to produce O~ in membranes prepared from activated neutrophils was inhibited by the addition of ant~odies raised against this possible flavoprotein (53). On the other hand, Yea et al. (54)purified a 45 kDa flavoprotein shown to have a pl of 4.0 which bound FAD with a molar stoichiometry of 1:1, from human neutrophils by affinity chromatography. The flavoprotein was a target for diphenylene iodonium, a potent inhibitor of O; generation by the activated oxidase system (50). Antibodies raised against the 45 kDa protein inhibited 02 production of oxidase in a cell-free reconstituted system (54), suggesting that this protein has a dual localization in plasma membranes and cytosol. These antibodies precipitated the 45 kDa protein together with a heme-containing 23 kDa protein thought to be the p22-phox of cytochrome b558. These findings were criticized, however, in a recent report in which cytochrome b.58 was said to also be a target for diphenylene iodonlum (55). Using oxtdase preparations derived from activated cells (56) and ones activated in a cell-free system with crude membrane fractions (57), it was demonstrated that FAD is not obligatory but stimulatory for the O 3 production. Recently, Sumimoto et al. (35) found that exogenous FAD was required for oxidase activation with purified cytochrome b558 in a cell-free system, and suggested that FAD acts through cytochrome b558. .
.
.
.
.
o
Following the partial purification of a cytosolic component containing a possible NADPH-binding site from guinea pig macrophages by Sha'ag and Pick (58), studies have recently started to determine whether an NADPH-binding component is present in dormant neutrophils in a soluble form in cytosol and thence is transferred to the plasma membrane during activation to form an active oxidase complex by interaction with cytochrome b .... Many substantial studies indicate that, in activated neutrophils, the NADPH-bindi°r~ component of the oxidase system resides in the plasma membrane. Using affinity labelling with NADPH dialdehyde as an inhibitor of and an affinity ligand to the binding site of dehydrogenase, however, a cytosolic component with a molecular weight of 66 kDa (59) or 32 kDa (60) was identified in resting neutrophils. These results suggest that an NADPH-binding component, present in the cytosol of dormant cells, is translocated to the plasma membrane during activation. However, this NADPH analogue has been known to induce rather nonspecific labelling (36). This is also supported by the report of a labelled protein that was membrane-bound with a molecular mass of 66 kDa (52). Segal et al. (36) showed that the heme/FAD ratio in the membranes from neutrophils does not change significantly on activation of the NADPH oxidase, indicating that the FAD is present in the plasma membrane from the outset and not recruited from the cytosol.
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NADPH Oxidase Activation Factors
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More recently, much interest has turned to the question of whether the NADPH-binding site is independent of membrane-bound cytochrome b558 or exists in cytochrome b55a. (also see the cytochrome b55a section) The NADPH oxlaase enzyme is very unstable when the plasma membranes are exposed to some detergents (61-63). This instability has obstructed most attempts to purify FAD-containing flavoprotein and has resulted in its low yield (45). If the multiple components comprising the membrane-bound oxidase system cause their dissociation in relation to inactivation or are labile due to denaturation, covalent cross-linking may be expected to stabilize the activity. According to Tamura et al. (64), cross-linking with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide, which has been known to stabilize oxidase, failed to stabilize proteinbound FAD. Knoller et al. (33)showed that cytochrome b558which was free of FAD could alone replace whole membranes in a cell-free system, But only if it was relipidated before use. Therefore, Segal et al. (36) attempted to reflavinate cytochrome b..~ after purification by incubation with FAD and detergent-solubilized lipid followed ~ removal of the detergent by dialysis. This led to its separation from free FAD on sucrose gradients. They found that cytochrome b. ~ is a flavocytochrome b..o binding both FAD and the heme, at a molar ratio of 1:2. ~otoaffinity labelling for th~°~NADPHbinding site with 32p-labelled 2-azido-NADP has been observed in the region of the gp91-phox of cytochrome b~.~, indicating that cytochrome b558 is a flavocytochrome containing FAD and the NADPH-binding site and bearing the complete electron-transporting apparatus of the NADPH oxidase. Recently, several investigators have demonstrated strong homologies in flavin- and NADPH-binding domains in the nitrite reductase family of proteins (65,66). Also with flavocytochrome b .... amino acid sequence homology has been detected between its gp91-phoxand the ~rredoxln-NADP reductase family of reductases in the putative NADPH- and FAD-binding sites (34-36), suggesting that the gp91-phox of flavocytochrome b..o may originate through a fusion of the ancestral genes for these two distinct typ~°of flavoproteins (65). Furthermore, the alignment of the amino acid sequence of the gp91-phox with previously characterized flavoproteins has raised the possibility that the middle and carboxyl-terminal portions of the gp91-phox are FAD- and NADPH-binding domains, respectively (35). It seems inevitable that details of the FAD-binding site in flavocytochrome b558 will be answered in the near future. OO
,
+
1.2.Cytosofic components Following the development of cell-free activation system of the oxidase enzyme, it has been reported that NADPH-dependent O; production can be activated in subcellular fractions (membranes and specific granules) 5y SDS (57,67), protein kinase C (68), arachidonic acid (69), and other fatty acids (70). All these cell-free activation systems exhibit an absolute requirement for cytosolic fractions (56,67,71-74). Later reports regarding an obligatory translocation of cytosolic components to the plasma membrane during the activation process have led to several studies directed toward the identification, purification and characterization of the cytosolic components and then analysis of their involvement in the oxidase activation mechanism. Through the genetic approach and complementation studies, at least two variants of autosomal CGD were initially recognized. These were characterized by deficiencies in two distinct cytosolic proteins of 47 - 48 kDa and 65 - 67 kDa (75-78), which are referred to as p47-phox and p67-phox, respectively. Many substantial studies have disclosed that the p47-phox takes up phosphate when intact neutrophils are activated, and loses it again when the cells return to the resting state (79). This has also been observed in cell-free systems (80,81). The evidence that the phosphorylation of p47-phox is abnormal in both X-linked (79) and autosomal recessive (82,83) forms of CGD strongly suggests an association between the phosphorylation of p47-phox and the activation of the NADPH oxidase. Phosphorylation of isolated p47-phox has been observed in the presence of purified protein kinase C (84), suggesting that p47-phox is a direct substrate for protein kinase C. It has been proven that SDS induces the phosphorylation of many cytosolic and membrane-associated proteins in cell-free reconstituted systems. Caldwell et al. (77) demonstrated that SDS can activate protein kinase C in cytosolic fractions from human neutrophils as measured by exogenous histone phosphorylation. Uhlinger et
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al. (81) recently reported that diacylglycerol synergizes with SDS in activating both O~generation and p47-phox phosphorylation in a cell-free activation system and that its action is independent of protein kinase C or other protein kinase activity, and suggested a novel role for diacylglycerol in cell regulation. However, Badwey et al. (85)demonstrated oxidase activation in the absence of p47-phox phosphorylation in a guinea pig (intact cell) system. This indicates that phosphorylation of p47-phox in cell-free systems is not a sufficient stimulus for oxidase activation. In any case, phosphorylation of p47phox does not seem to be obligatory for activation, either in intact cells (86) or in cellfree systems (80,81). Using immunoprecipitation from neutrophil cytosolic and membrane fractions followed by two-dimensional gel electrophoresis and autoradiography, it has been shown that phosphorylation of p47-phox may be associated with its translocation to the plasma membrane during activation (87). In contrast to the strict cytosolic localization of p47-phox and p67-phox in unstimulated neutrophils, both proteins have been detected in plasma membrane fractions of cells activated by phorbol myristate acetate and other stimuli (88), indicating that this translocation event is important in oxidase activation. Studies with cytosols of variant forms of CGD suggest that cytochrome b558 or a closely linked factor provides an essential docking site for the cytosolic oxi~ase components and that p47-phox mediates the assembly of these components on the plasma membrane (89). In a cell-free translocation with an in vitro translated radio-labelled recombinant p47-phox, Tyagi et al. (90) proposed the possibility that SDS and diacylglycerol act on the cytosolic components to alter protein-protein and/or protein-membrane associations, which are necessary for activation, and that these altered associations are likely to function by increasing the local concentrations of p47-phox and other components in the plasma membrane. Furthermore, they suggested that the translocation mechanism of the cytosolic components in a cell-free system differs from that seen in intact neutrophils. In the neutrophil cytosol, p67-phox is considered to be at least partly complexed to p47-phox and to be in the form of the complex participating in oxidase activation (91). Although p47-phox is translocated from the cytosol to the plasma membrane during oxidase activation, neither its complexation to p67-phox nor concomitance of other protein phosphorylation is necessary for this translocation (91). Similarity in the rates of oxidase activation and p67-phox inactivation induced by SDS has been observed (92), suggesting that the activation of the NADPH oxidase in a cell-free system could involve an SDS-mediated alteration in p67-phox. Babior et al. (74) reported that p47-phox and active NADPH oxidase were recovered in the cytoskeleton-rich pellet of activated neutrophils solubilized in Triton X-100. Priming of neutrophils with phorbol ester and subsequent activation with an agonist have given rise to oscillations both in oxidase activity and in the cell shape due to periodic changes in the content of filamentous actin (93,94). These results suggest a functional interaction between NADPH oxidase and the cytoskeleton. Likewise, in the translocation associated with phorbol ester-induced oxidase activation, the protein kinase-dependent phosphorylation of p47-phox has been found to correlate with association of p47-phoxwith the cytoskeleton and with translocation of both p47-phox and p67-phox to the plasma membrane (95). Based on the results of a study using fluorescence microscopy with the NADPH-associated autofluorescence imaging technique, it has also been suggested that NADPH, including its sources and/or carriers, accumulates near phagosomes prior to the oxidation of antibody-coated target erythrocytes, and that local NADPH molecules are consumed during the target oxidation (96). At present, both cytosolic components, p47-phox and p67-phox, have been purified to homogeneity. The former was purified from the cytosol of human neutrophils by chromatography on ion exchange and hydroxyapatite resins (97). Antibodies to purified p47-phoxdemonstrated that this protein is indeed the protein defective in the most common autosomal form of CGD. This was fully supported by the fact that the phosphorylated p47-phox was recognized by the polyclonal antiserum B - l , which recognizes both p47-phox and p67-phox (95). As for the latter component, a 63 kDa polypeptide, corresponding to human neutrophil cytosolic p67-phox, was recently purified from porcine (98) and bovine (99) neutrophils. Antibodies to the two proteins derived from porcine and bovine neutrophils could immunoreact with human p67-phox. Immunoblotting with
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the 63 kDa bovine protein antiserum demonstrated that activation of oxidase by phorbol esters induced translocation of the 63 kDa protein from cytosol to the plasma membrane. The peptide (RGVHFIF), corresponding to a cytoplasmic carboxyl-terminal domain of the gp91-phox of cytochrome b~o, failed to inhibit an arachidonate-related cell-free activation of the NADPH oxidas~°~Nhen added after initial incubation if p47-phox was present during the incubation, but it markedly inhibited oxidase activity if p47-phox was absent during the initial incubation (100). These results suggest that p47-phox, but not p67-phox, is a participant with membrane and/or other cytosolic components in early arachidonate-dependent reactions. The cDNAs for both p47-phox (41,101,102) and p67-phox (103) have been already cloned. The predicted amino acid sequence for p47-phox shows a number of potential protein kinase C-related phosphorylation sites characterized by serine residues flanked by arginine residues clustered within the highly basic carboxyl-terminal of the protein (41,101). These findings are sufficiently supported by phosphorylation studies of 32p-labelled neutrophils activated by exposure to phorbol esters, fMet-Leu-Phe, opsonized latex beads, calcium ionophore A23187 and fluoride (83,104). In addition, it is of particular interest that p47-phox has an NH>-terminal glycine residue which could potentially serve as a site for myristoylation-and thereby provide a mechanism by which this component could translocate to the plasma membrane during oxidase activation (101). The two p47-phoxand p67-phox components possess two peptide domains similar to a sequence motif found in the noncatalytic domain of src-related protein kinases and to some domains of other proteins which do not exhibit kinase activity, including nonerythroid a-spectrin, GAP, and phospholipase C-~ (101,103). Some functional relationships between p47-phox and GAP are considered to exist, based on results showing that a 33 amino acid segment shared 49% identity with the corresponding segment of GAP (41). Rodaway et al. (102) demonstrated the possibility that two src homology domains of p47-phox may interact with the Rap-1 protein associated with flavocytochrome b~.~ (37). In addition, using nuclear run-on transcription assays, they showed that the p47-phox gene responds to induction by retinoic acid at the transcription level in a cycloheximide-insensitive manner, and they suggested that p47-phox transcription is likely to be directly activated by a nuclear retinoic acid receptor. In addition to the cytosolic components of p47-phox and p67-phox, other cytosolic components are considered to be associated with NADPH oxidase activation. Some of them have been identified as members of a ras-related superfamily of GTP-binding proteins, a group of 20 - 28 kDa proteins whose members are thought to play an important role in cell regulation (105). Two cytosolic GTP-binding proteins, Rac-1 and Rac-2, which can stimulate NADPH oxidase activity in cell-free systems, have been independently purified from guinea pig and human neutrophils, with a molecular weight of 22,000 on SDS-PAGE (106,107). Didsbury et al. (108) isolated the cDNA clones for Rac-1 and Rac-2. The two cDNAs contain open reading frames encoding proteins with a length of 192 amino acids with calculated M_ values of 21,450 and 21,429, respectively. Both Racs have been reported to be pos~ranslationally modified by addition of an isoprenoid group, the likely site of which is the carboxyl-terminal cystein (109). This modification is essential for membrane association and biological activity of ras-related proteins. Isoprenylation was found only in Racs association with particulate cell fractions (109), suggesting that this modification may be associated with membrane localization of the proteins. In a cell-free system containing macrophage membranes, p47-phoxand p67-phox, partially purified Rac-1 increased oxidase activity by threefold (106), suggesting a possible involvement of Rac-1 in activating or promoting the formation of an active oxidase complex at the membrane. On the other hand, Knaus et al. (110) demonstrated that a specific Rac-2 antibody inhibited by up to 70% the generation of O; in a fully active cell-free system, and suggested that Rac-2 is likely to be subject to regulatory cofactors in vivo. In addition to Racs, their regulatory proteins, such as GAPs, GDP dissociation inhibitors and GDP/GTP dissociation stimulators, would be expected to exert significant control over the activity of the oxidase system. It will be important to direct future studies to identifying and understanding the interaction of these other components with Racs and the NADPH oxidase.
Phosphorylated during activation At least six potential serine phosphorylation sites, cytoskeleton binding sites
Heme-binding domains
Carboxyl-terminal binds cytosolic components FAD-binding domains
Functional domains
ISDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis 2NCF, neutrophil cytosol factor
Cytoskeletonbinding sites
NCF2 Iq25
NCFI 2 7qli.23
Phosphorylated
Chromosome localization
=26 kDa NADPHbinding domainlinked carbohydrate
6
60.9 67
526
p67-phox
i0
44.6 47
390
p47-phox
Posttranslational modification
10
20.9 22
195
p22-phox
CYBA 16q24
9.7
65.0 91
569
gp91-phox
CYBB Xp21.1
pI
Molecular weight (kDa) Predicted On SDS-PAGE 1
Amino acids
Factors
TABLE I Properties of NADPH Oxidase Components
Z 9
Ln
O
<
o
>
©
O0
Function
Posttranslational modification
GTPTS 1 binding
regulatory cofactor
phosphorylated
activating (promoting) cofactor
isoprenylated
regulatory cofactor
isoprenylated
pendent
pendent
(+)
myeloid
7.56
21.4 22
192
cytosol
Rac-2
Mg 2+ de-
(+)
mRNA in a large number of cells
8.53
21.5 22
192
cytosol
Rac-i
Mg 2+ de-
(+)
mRNA in a large number of cells
6.5
20.8 21
184
membrane
Rap-i
IGTPTS, guanosine 5 ' - 3 - O - ( t h i o ) - t r i p h o s p h a t e
(kDa)
Tissue specificity
pl
Molecular weight Predicted On SDS-PAGE
Amino acids
Localization
Factors
TABLE I I Properties of G T P - B i n d i n g Proteins in Human Neutrophils
5"
2"
O
Z P
~a
o_
<
10
NADPH Oxidase Activation Factors
Vol. 55, No. 1, 1994
2. Conclusions
Among the probably numerous protein components which are assembled to make an active oxidase complex, at least two cytosolic components, p47-phox and p67-phox, are essential. Recently, it has been proposed that the cytosolic components of the oxidase exist in the form of a M r 240,000 complex in SDS-dependent cell-free system, which suggests that the exposure of this complex to SDS induces a structural change that may or may not be associated with the loss of an inhibitory subunit too small to cause a detectable change in the size of the complex (111). Current evidence has disclosed many properties of the NADPH oxidase system, including membrane components (gp91phox, p22-phox and Rap-l) and cytosolic components (p47-phox, p67-phox and Rac1/2) (Tables I and II). However, the nature of the interactions between the different pieces of this amazing puzzle and the structural modifications of the components' assembly which lead to the respiratory burst remain to be determined. References
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Pergamon 0024-3205(94) 00075-1
MINIREVIEW
ACTIVATION FACTORS OF NEUTROPHIL NADPH OXIDASE COMPLEX Shigenobu Umeki Department of Medicine Toshida-kai Kumeda Hospital 2944 Obu-Cho, Kishiwada, Osaka 596, Japan (Received in final form April 26, 1994)
Summary Professional phagocytes, neutrophils, possess a unique membraneassociated NADPH oxidase system, dormant in resting cells, which becomes activated upon exposure to the appropriate stimuli and catalyzes the one-electron reduction of molecular oxygen to superoxide, O;,. Oxidase activation involves the assembly, in the plasma membrane, ot~membrane-bound and cytosolic constituents of the oxidase system, which are disassembled in the resting state. The oxidase system consists of two plasma membrane-bound components; low-potential cytochrome b558, which is composed of two subunits of 22 kDa and 91 kDa, and a flavoprotein related to the electron transport between NADPH and heme-binding domains of the oxidase. Recent reports have indicated that FAD-binding sites of the oxidase are contained in cytochrome b~o~o(flavocytochrome b...). At least two cytosolic components, 67 kDa protein and a phosphory°l=,~°ted 47 kDa protein, are known to translocate to the plasma membrane, ensuring assembly of an active O2-generating NADPH oxJdase system. More recently, the membrane (Raps) and cytosolic (Racs) GTPbinding proteins have been established as essential to oxidase assembly. It is the purpose of this review to focus on recent data concerning the regulatory mechanisms which lead to organization and activation of the neutrophil NADPH oxidase system. Key Words: neutrophils, N A D P H oxidase, electron transfer system, chronic granulomatous disease
Human neutrophils serve a crucial role in the host defense through normal activation of a latent plasma membrane multicomponent NADPH oxidase (EC 1.6.99.6) system associated with the principal defense against a wide variety of bacteria (1-3). Activation of the neutrophil oxidative metabolism is characterized by strong cyanide-insensitive oxygen consumption (respiratory burst) (4,5) and by concomitant production of active oxygen derivatives such as superoxide (O;), H>O~, and .OH (6). Superoxide is produced primarily through the activation of plasma ?nemq3rAne-bound NADPH oxidase by stimulation with phagocytizable particles (7) or soluble agents (8). The major contribution of O2 and its dismutation products to the bactericidal capacity of the body is readily demonstrated by the susceptibility to infection of patients with chronic granulomatous disease, in which there is a defect in NADPH oxidase or its activating apparatus (6,9). As a result of many studies of cell-free systems, important insights into the sequence of activation steps required for a normal neutrophil response have been obtained. By sequencing of cDNA clones and tracing of exogenous agonist-induced stimulation of NADPH oxidase, the basic elements of the activation sequence have been now disclosed. The present review focuses on recent findings regarding the nature of the electron transport components of the neutrophil NADPH oxidase system and its activation/regulation mechanism in relation to specific pathological aspects of chronic granulomatous disease.
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1. NADPH oxidase components
1.1. Plasma membrane components 1.1.1. Cytochromeb558 Cytochrome bs.^ is localized both in the plasma membrane, which becomes the wall of the phagocytic vac~°ole by its invagination (10), and in the specific granule membrane, which transfers to the membrane of the phagocytic vacuole (11). The cytochrome is a heterodimer consisting of a small (a) subunit of 22 kDa (p22-phox; phox indicates phagocyte oxidase), which carries the heme and a large (/3) subunit of 91 kDa (gp91-phox), which is highly glycosylated. Studies of cytochrome b558 were begun in earnest after a correlation was found between the absence of cytochrome b~.. in neutrophils from patients with a classic form of chronic granulomatous disease (CGI~ ~ linked to the X chromosome and the inability of these deficient neutrophils to mount the respiratory burst. Cytochrome b.... which was first discovered in horse neutrophils, was identified from human neutrophlls ~y Segal and Jones (12). This cytochrome has an extremely low mid-point oxidation-reduction (redox) potential (Ern7. = -245 mv) (13), lower than that of any other mammalian cytochrome and sufficiently low to reduce oxygen to 02 (Era7 = -160 mv) (14). since the identification of this cytochrome, much evidence supporting participation of cytochrome bs58 in the respiratory burst has accumulated. With cloning of the X-linked defective genes responsible for a classic form of CGD and the discovery that these genes encode the protein moiety of p22-phox and gp91-phox of cytochrome b558 (15-18), cytochrome b~oo has been accepted as an important component of respiratory burst oxldase. This c~ochrome has now been purified and shown to be a heterodimeric protein consisting of two subunits; p22-phox with a molecular weight of 22,000 - 23,000 and gp91 -phox with a molecular weight of 76,000 - 92,000 as determined by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 2 (19-22). The p22-phox is a heme-bearing protein (22,23) which may be the terminal component of the respiratoryburst electron transport system. By comparing the binding characteristics of a monoclonal antibody reacting with p22-phox to intact and permeabilized neutrophils, it was established that p22-phox is a transmembrane protein (24,25). The gp91 -phox of cytochrome b558 is a heavily glycosylated integral membrane protein with several membrane-spanning regions predicted by hydrophobicity analysis (15,16,19). The glycosylated portion of the gp91-phox is not essential for oxidase activity, but may be required for optimum production or stability of the cytochrome b~58 and could modulate the activity. This has been shown by the fact that, when N-glyc~sylation in differentiating HL60 cells was inhibited by tunicamycin, the gp91-phox was synthesized with a mass close to 58 kDa, similar to that found after cleavage with endoglycosidase F (26). Based on an analysis with antibodies raised against synthetic peptides corresponding to various regions in gp91-phox, it was concluded that this subunit is a transmembrane protein (27). In experiments using the synthetic peptide-containing gp91-phox and antibody-binding assays of the cytoplasmic carboxyl-terminal tail of the gp91-phox, the subunit was found .to mediate interactions with other cytosolic proteins essential to activation of the respiratory burst, such as p47-phox and p67-phox (28). Therefore, gp91-phox may play a central role in regulating the cellular assembly of the heterodimeric cytochrome bs58. The two subunits are very tightly associated and any maneuvers designed to separate them also displace the heme. Recently, it has been argued that gp91-phox might play a structural role in the maintenance of the heme-bearing p22-phox in the plasma membrane (3). Using electron paramagnetic resonance spectroscopy, it was determined that cytochrome bs.o is a hexa-coordinated ferric hemoprotein, and that the heme iron is ligated in its °f~fth and sixth coordination positions by two imidazole nitrogens of histidine residues (29). As only one histidine residue exists in the p22-phox (30). This evidence suggests that gp91-phox may also participate in heme binding. Quinn et al. (31), using lithium dodecyl sulfate-PAGE followed by tetramethylbenzidine heme 1CGD, chronic granulomatous disease; 2SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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staining, reported that a multi-heme structure residing in both the p22-phox and gp91phox subunits would be consistent with an electron transfer function for cytochrome b558 by providing an efficient mechanism for transferring electrons across the plasma membrane to the extracellular surface where oxygen could be reduced to create 0 2. The genes for both subunits have been cloned and sequenced (15-18). Transcription of the genes for the two subunits, p22-phox and gp91-phox, each of which has close physical interaction, is independently regulated (1,3). Although mRNA for p22-phox exists in a wide variety of cell lines derived from non-phagocytic cells, synthesis of gp91-phox mRNA transcript is limited to phagocytic cells and the myeloid cell lineage. This suggests that regulation of cytochrome b55. assembly may occur through the expression of gp91-phox. Transcription of gp91-~hox is increased in parallel with expression of the cytochrome after the induction of oxidase activity by interferon-y in the cells of patients with variant CGD (1). Although the oxidase activity and special activity of cytochrome b558 are enhanced by interferon-y, expression of p22-phox is not (32). Recently, Knoller et al. (33) hypothesized that cytochrome b558 might be the only redox component associated with activated NADPH oxidase. This Is supported by three proposals: 1) that the NADPH-binding site is located on the cytochrome b558 dimer itself, 2) that it is present on one of the cytosolic components, and 3) that it is a complex structure to which both cytochrome bs58 and a cytosolic component contribute. According to Rotrosen et al. (34), NADPH oxidase activity was reconstituted in a cell-free system with only purified oxidase proteins, such as cytosolic proteins (p47-phox and p67-phox), ras-related guanosine triphosphate-(GTP-) 1 binding proteins and membrane-bound cytochrome b.... requiring exogenous flavin adenine dinucleotide (FAD) 2. The FAD binding site was~°~ocated on cytochrome b558. Based on evidence that purified cytochrome b.58 was completely dependent on exogenous FAD to obtain its high 02 production, S~mimoto et al. (35) aligned the amino acid sequence of gp91-phox with previously characterized flavoproteins, and concluded that the middle and carboxylterminal portions of gp91-phox are probably FAD- and NADPH-binding domains, respectively. Similar results were obtained by Segal et al. (36). These results strongly indicate that cytochrome b558 is a fiavoprotein (flavoc.ytochrome b558) bearing the complete electron-transporting apparatus of the NADPH oxldase system. By measunng FAD and heme contents in membranes from resting and activated neutrophils, Segal et al. (36) found that flavocytochrome b~58 bound to both FAD and heme at a molar ratio of 1:2. Structural and functional data frorfi several laboratories in the United States, Europe and Japan established flavocytochrome b558 as both the NADPH- and FAD-binding component of the oxidase. Quinn et al. (37)coisolated a protein with a relative molecular mass of 22 kDa with heterodimeric flavocytochrome b.... This protein was considered to be identical to the sequence of a ras-related GTF~2~inding protein identified as Rap-1 on the basis of its primary structure deduced from cDNA. Immunoaffinity purification of flavocytochrome b#~8 using a monoclonal antibody reactive to the highly conserved GTP-binding domains oTnuman H-ras, N-ras and K-ras proteins showed an association between flavocytochrome b..o and the ras-related protein (37), indicating a role for such a protein in the transductlon, regulation or structure of the NADPH oxidase system. In immunoelectron microscopic studies with Rap-l-specific antibodies, furthermore, Quinn et al. (38) found that Rap-1 is colocalized with flavocytochrome b... in both plasma membranes and specific granules of human neutrophils and is cotr,~n%located with flavocytochrome /9558 to the phagolysosomal membranes during the course of oxidase activation. Rap-~ binding to flavocytochrome bs. 8 was inhibited by phosphorylation of Rap-1 with cAMP-dependent protein kinas~e (39). These results suggest a functional association of these two molecules in the resting state, a role for Rap-1 in the structure or function of the activated NADPH oxidase, and a phosphorylation-coupled regulation of Rap-1 function in the respiratory burst system. A GTPase-activating protein (GAP) 3 involved 1GTP, guanosine triphosphate; 2FAD, flavin adenine dinucleotide; 3GAP, GTPase-activating protein.
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with Ras proteins (40) possesses a significant sequence similar to the cytosolic component of p47-phox required for oxidase activation (41), suggesting a possible association of Rap-1 with the sensitivity of the respiratory burst to GTP in a cell-free system. A recent study of a cell-free system by Eklund et al. (42) showed that purified human recombinant Rap-1 could completely reconstitute cytosol from which ras-related proteins were immunologically depleted. This may be consistent with Rap-1 as a necessary cofactor. However, the data of Rotrosen et al. (34) and Abo et al. (43) using purified flavocytochrome b devoid of Rap-1 suggest that Rap-1 is not an essential component of the oxidase, but it may still play a regulatory role in the whole cell. In this regard, further detailed studies should be done. .
558
1.1.2. NADPH-dehydrogenase Although the involvement of cytochrome b..~ as a terminal electron transporter of the NADPH oxidase system is evident, it is st~°unclear which protein is the flavoprotein transferring electrons from NADPH to the heme as the prosthetic group in the oxidase system. The association of a flavoprotein in this oxidase was postulated by Cagan and Karnovsky (43). There is much evidence indicating that the cofactor is FAD (44-47) and that a FAD-containing flavoprotein is one of the electron carriers in the NADPH oxidase system (2,3,45,48-54). A definite conclusion, however, must await the identification of this flavoprotein, reports concerning its kinetic competency in cell-free reconstituted studies, and its DNA sequence analysis. An NADPH-binding protein focusing at pl 5.0 on gel (51) with a molecular weight of 65,000 - 67,000 (45, 49, 51), which appears to be a fiavoprotein, has been purified from activated neutrophils. The capacity to produce O~ in membranes prepared from activated neutrophils was inhibited by the addition of ant~odies raised against this possible flavoprotein (53). On the other hand, Yea et al. (54)purified a 45 kDa flavoprotein shown to have a pl of 4.0 which bound FAD with a molar stoichiometry of 1:1, from human neutrophils by affinity chromatography. The flavoprotein was a target for diphenylene iodonium, a potent inhibitor of O; generation by the activated oxidase system (50). Antibodies raised against the 45 kDa protein inhibited 02 production of oxidase in a cell-free reconstituted system (54), suggesting that this protein has a dual localization in plasma membranes and cytosol. These antibodies precipitated the 45 kDa protein together with a heme-containing 23 kDa protein thought to be the p22-phox of cytochrome b558. These findings were criticized, however, in a recent report in which cytochrome b.58 was said to also be a target for diphenylene iodonlum (55). Using oxtdase preparations derived from activated cells (56) and ones activated in a cell-free system with crude membrane fractions (57), it was demonstrated that FAD is not obligatory but stimulatory for the O 3 production. Recently, Sumimoto et al. (35) found that exogenous FAD was required for oxidase activation with purified cytochrome b558 in a cell-free system, and suggested that FAD acts through cytochrome b558. .
.
.
.
.
o
Following the partial purification of a cytosolic component containing a possible NADPH-binding site from guinea pig macrophages by Sha'ag and Pick (58), studies have recently started to determine whether an NADPH-binding component is present in dormant neutrophils in a soluble form in cytosol and thence is transferred to the plasma membrane during activation to form an active oxidase complex by interaction with cytochrome b .... Many substantial studies indicate that, in activated neutrophils, the NADPH-bindi°r~ component of the oxidase system resides in the plasma membrane. Using affinity labelling with NADPH dialdehyde as an inhibitor of and an affinity ligand to the binding site of dehydrogenase, however, a cytosolic component with a molecular weight of 66 kDa (59) or 32 kDa (60) was identified in resting neutrophils. These results suggest that an NADPH-binding component, present in the cytosol of dormant cells, is translocated to the plasma membrane during activation. However, this NADPH analogue has been known to induce rather nonspecific labelling (36). This is also supported by the report of a labelled protein that was membrane-bound with a molecular mass of 66 kDa (52). Segal et al. (36) showed that the heme/FAD ratio in the membranes from neutrophils does not change significantly on activation of the NADPH oxidase, indicating that the FAD is present in the plasma membrane from the outset and not recruited from the cytosol.
Vol. 55, No. 1, 1994
NADPH Oxidase Activation Factors
5
More recently, much interest has turned to the question of whether the NADPH-binding site is independent of membrane-bound cytochrome b558 or exists in cytochrome b55a. (also see the cytochrome b55a section) The NADPH oxlaase enzyme is very unstable when the plasma membranes are exposed to some detergents (61-63). This instability has obstructed most attempts to purify FAD-containing flavoprotein and has resulted in its low yield (45). If the multiple components comprising the membrane-bound oxidase system cause their dissociation in relation to inactivation or are labile due to denaturation, covalent cross-linking may be expected to stabilize the activity. According to Tamura et al. (64), cross-linking with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide, which has been known to stabilize oxidase, failed to stabilize proteinbound FAD. Knoller et al. (33)showed that cytochrome b558which was free of FAD could alone replace whole membranes in a cell-free system, But only if it was relipidated before use. Therefore, Segal et al. (36) attempted to reflavinate cytochrome b..~ after purification by incubation with FAD and detergent-solubilized lipid followed ~ removal of the detergent by dialysis. This led to its separation from free FAD on sucrose gradients. They found that cytochrome b. ~ is a flavocytochrome b..o binding both FAD and the heme, at a molar ratio of 1:2. ~otoaffinity labelling for th~°~NADPHbinding site with 32p-labelled 2-azido-NADP has been observed in the region of the gp91-phox of cytochrome b~.~, indicating that cytochrome b558 is a flavocytochrome containing FAD and the NADPH-binding site and bearing the complete electron-transporting apparatus of the NADPH oxidase. Recently, several investigators have demonstrated strong homologies in flavin- and NADPH-binding domains in the nitrite reductase family of proteins (65,66). Also with flavocytochrome b .... amino acid sequence homology has been detected between its gp91-phoxand the ~rredoxln-NADP reductase family of reductases in the putative NADPH- and FAD-binding sites (34-36), suggesting that the gp91-phox of flavocytochrome b..o may originate through a fusion of the ancestral genes for these two distinct typ~°of flavoproteins (65). Furthermore, the alignment of the amino acid sequence of the gp91-phox with previously characterized flavoproteins has raised the possibility that the middle and carboxyl-terminal portions of the gp91-phox are FAD- and NADPH-binding domains, respectively (35). It seems inevitable that details of the FAD-binding site in flavocytochrome b558 will be answered in the near future. OO
,
+
1.2.Cytosofic components Following the development of cell-free activation system of the oxidase enzyme, it has been reported that NADPH-dependent O; production can be activated in subcellular fractions (membranes and specific granules) 5y SDS (57,67), protein kinase C (68), arachidonic acid (69), and other fatty acids (70). All these cell-free activation systems exhibit an absolute requirement for cytosolic fractions (56,67,71-74). Later reports regarding an obligatory translocation of cytosolic components to the plasma membrane during the activation process have led to several studies directed toward the identification, purification and characterization of the cytosolic components and then analysis of their involvement in the oxidase activation mechanism. Through the genetic approach and complementation studies, at least two variants of autosomal CGD were initially recognized. These were characterized by deficiencies in two distinct cytosolic proteins of 47 - 48 kDa and 65 - 67 kDa (75-78), which are referred to as p47-phox and p67-phox, respectively. Many substantial studies have disclosed that the p47-phox takes up phosphate when intact neutrophils are activated, and loses it again when the cells return to the resting state (79). This has also been observed in cell-free systems (80,81). The evidence that the phosphorylation of p47-phox is abnormal in both X-linked (79) and autosomal recessive (82,83) forms of CGD strongly suggests an association between the phosphorylation of p47-phox and the activation of the NADPH oxidase. Phosphorylation of isolated p47-phox has been observed in the presence of purified protein kinase C (84), suggesting that p47-phox is a direct substrate for protein kinase C. It has been proven that SDS induces the phosphorylation of many cytosolic and membrane-associated proteins in cell-free reconstituted systems. Caldwell et al. (77) demonstrated that SDS can activate protein kinase C in cytosolic fractions from human neutrophils as measured by exogenous histone phosphorylation. Uhlinger et
6
NADPH Oxidase Activation Factors
Vol. 55, No. 1, 1994
al. (81) recently reported that diacylglycerol synergizes with SDS in activating both O~generation and p47-phox phosphorylation in a cell-free activation system and that its action is independent of protein kinase C or other protein kinase activity, and suggested a novel role for diacylglycerol in cell regulation. However, Badwey et al. (85)demonstrated oxidase activation in the absence of p47-phox phosphorylation in a guinea pig (intact cell) system. This indicates that phosphorylation of p47-phox in cell-free systems is not a sufficient stimulus for oxidase activation. In any case, phosphorylation of p47phox does not seem to be obligatory for activation, either in intact cells (86) or in cellfree systems (80,81). Using immunoprecipitation from neutrophil cytosolic and membrane fractions followed by two-dimensional gel electrophoresis and autoradiography, it has been shown that phosphorylation of p47-phox may be associated with its translocation to the plasma membrane during activation (87). In contrast to the strict cytosolic localization of p47-phox and p67-phox in unstimulated neutrophils, both proteins have been detected in plasma membrane fractions of cells activated by phorbol myristate acetate and other stimuli (88), indicating that this translocation event is important in oxidase activation. Studies with cytosols of variant forms of CGD suggest that cytochrome b558 or a closely linked factor provides an essential docking site for the cytosolic oxi~ase components and that p47-phox mediates the assembly of these components on the plasma membrane (89). In a cell-free translocation with an in vitro translated radio-labelled recombinant p47-phox, Tyagi et al. (90) proposed the possibility that SDS and diacylglycerol act on the cytosolic components to alter protein-protein and/or protein-membrane associations, which are necessary for activation, and that these altered associations are likely to function by increasing the local concentrations of p47-phox and other components in the plasma membrane. Furthermore, they suggested that the translocation mechanism of the cytosolic components in a cell-free system differs from that seen in intact neutrophils. In the neutrophil cytosol, p67-phox is considered to be at least partly complexed to p47-phox and to be in the form of the complex participating in oxidase activation (91). Although p47-phox is translocated from the cytosol to the plasma membrane during oxidase activation, neither its complexation to p67-phox nor concomitance of other protein phosphorylation is necessary for this translocation (91). Similarity in the rates of oxidase activation and p67-phox inactivation induced by SDS has been observed (92), suggesting that the activation of the NADPH oxidase in a cell-free system could involve an SDS-mediated alteration in p67-phox. Babior et al. (74) reported that p47-phox and active NADPH oxidase were recovered in the cytoskeleton-rich pellet of activated neutrophils solubilized in Triton X-100. Priming of neutrophils with phorbol ester and subsequent activation with an agonist have given rise to oscillations both in oxidase activity and in the cell shape due to periodic changes in the content of filamentous actin (93,94). These results suggest a functional interaction between NADPH oxidase and the cytoskeleton. Likewise, in the translocation associated with phorbol ester-induced oxidase activation, the protein kinase-dependent phosphorylation of p47-phox has been found to correlate with association of p47-phoxwith the cytoskeleton and with translocation of both p47-phox and p67-phox to the plasma membrane (95). Based on the results of a study using fluorescence microscopy with the NADPH-associated autofluorescence imaging technique, it has also been suggested that NADPH, including its sources and/or carriers, accumulates near phagosomes prior to the oxidation of antibody-coated target erythrocytes, and that local NADPH molecules are consumed during the target oxidation (96). At present, both cytosolic components, p47-phox and p67-phox, have been purified to homogeneity. The former was purified from the cytosol of human neutrophils by chromatography on ion exchange and hydroxyapatite resins (97). Antibodies to purified p47-phoxdemonstrated that this protein is indeed the protein defective in the most common autosomal form of CGD. This was fully supported by the fact that the phosphorylated p47-phox was recognized by the polyclonal antiserum B - l , which recognizes both p47-phox and p67-phox (95). As for the latter component, a 63 kDa polypeptide, corresponding to human neutrophil cytosolic p67-phox, was recently purified from porcine (98) and bovine (99) neutrophils. Antibodies to the two proteins derived from porcine and bovine neutrophils could immunoreact with human p67-phox. Immunoblotting with
Vol. 55, No. 1, 1994
NADPH Oxidase Activation Factors
7
the 63 kDa bovine protein antiserum demonstrated that activation of oxidase by phorbol esters induced translocation of the 63 kDa protein from cytosol to the plasma membrane. The peptide (RGVHFIF), corresponding to a cytoplasmic carboxyl-terminal domain of the gp91-phox of cytochrome b~o, failed to inhibit an arachidonate-related cell-free activation of the NADPH oxidas~°~Nhen added after initial incubation if p47-phox was present during the incubation, but it markedly inhibited oxidase activity if p47-phox was absent during the initial incubation (100). These results suggest that p47-phox, but not p67-phox, is a participant with membrane and/or other cytosolic components in early arachidonate-dependent reactions. The cDNAs for both p47-phox (41,101,102) and p67-phox (103) have been already cloned. The predicted amino acid sequence for p47-phox shows a number of potential protein kinase C-related phosphorylation sites characterized by serine residues flanked by arginine residues clustered within the highly basic carboxyl-terminal of the protein (41,101). These findings are sufficiently supported by phosphorylation studies of 32p-labelled neutrophils activated by exposure to phorbol esters, fMet-Leu-Phe, opsonized latex beads, calcium ionophore A23187 and fluoride (83,104). In addition, it is of particular interest that p47-phox has an NH>-terminal glycine residue which could potentially serve as a site for myristoylation-and thereby provide a mechanism by which this component could translocate to the plasma membrane during oxidase activation (101). The two p47-phoxand p67-phox components possess two peptide domains similar to a sequence motif found in the noncatalytic domain of src-related protein kinases and to some domains of other proteins which do not exhibit kinase activity, including nonerythroid a-spectrin, GAP, and phospholipase C-~ (101,103). Some functional relationships between p47-phox and GAP are considered to exist, based on results showing that a 33 amino acid segment shared 49% identity with the corresponding segment of GAP (41). Rodaway et al. (102) demonstrated the possibility that two src homology domains of p47-phox may interact with the Rap-1 protein associated with flavocytochrome b~.~ (37). In addition, using nuclear run-on transcription assays, they showed that the p47-phox gene responds to induction by retinoic acid at the transcription level in a cycloheximide-insensitive manner, and they suggested that p47-phox transcription is likely to be directly activated by a nuclear retinoic acid receptor. In addition to the cytosolic components of p47-phox and p67-phox, other cytosolic components are considered to be associated with NADPH oxidase activation. Some of them have been identified as members of a ras-related superfamily of GTP-binding proteins, a group of 20 - 28 kDa proteins whose members are thought to play an important role in cell regulation (105). Two cytosolic GTP-binding proteins, Rac-1 and Rac-2, which can stimulate NADPH oxidase activity in cell-free systems, have been independently purified from guinea pig and human neutrophils, with a molecular weight of 22,000 on SDS-PAGE (106,107). Didsbury et al. (108) isolated the cDNA clones for Rac-1 and Rac-2. The two cDNAs contain open reading frames encoding proteins with a length of 192 amino acids with calculated M_ values of 21,450 and 21,429, respectively. Both Racs have been reported to be pos~ranslationally modified by addition of an isoprenoid group, the likely site of which is the carboxyl-terminal cystein (109). This modification is essential for membrane association and biological activity of ras-related proteins. Isoprenylation was found only in Racs association with particulate cell fractions (109), suggesting that this modification may be associated with membrane localization of the proteins. In a cell-free system containing macrophage membranes, p47-phoxand p67-phox, partially purified Rac-1 increased oxidase activity by threefold (106), suggesting a possible involvement of Rac-1 in activating or promoting the formation of an active oxidase complex at the membrane. On the other hand, Knaus et al. (110) demonstrated that a specific Rac-2 antibody inhibited by up to 70% the generation of O; in a fully active cell-free system, and suggested that Rac-2 is likely to be subject to regulatory cofactors in vivo. In addition to Racs, their regulatory proteins, such as GAPs, GDP dissociation inhibitors and GDP/GTP dissociation stimulators, would be expected to exert significant control over the activity of the oxidase system. It will be important to direct future studies to identifying and understanding the interaction of these other components with Racs and the NADPH oxidase.
Phosphorylated during activation At least six potential serine phosphorylation sites, cytoskeleton binding sites
Heme-binding domains
Carboxyl-terminal binds cytosolic components FAD-binding domains
Functional domains
ISDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis 2NCF, neutrophil cytosol factor
Cytoskeletonbinding sites
NCF2 Iq25
NCFI 2 7qli.23
Phosphorylated
Chromosome localization
=26 kDa NADPHbinding domainlinked carbohydrate
6
60.9 67
526
p67-phox
i0
44.6 47
390
p47-phox
Posttranslational modification
10
20.9 22
195
p22-phox
CYBA 16q24
9.7
65.0 91
569
gp91-phox
CYBB Xp21.1
pI
Molecular weight (kDa) Predicted On SDS-PAGE 1
Amino acids
Factors
TABLE I Properties of NADPH Oxidase Components
Z 9
Ln
O
<
o
>
©
O0
Function
Posttranslational modification
GTPTS 1 binding
regulatory cofactor
phosphorylated
activating (promoting) cofactor
isoprenylated
regulatory cofactor
isoprenylated
pendent
pendent
(+)
myeloid
7.56
21.4 22
192
cytosol
Rac-2
Mg 2+ de-
(+)
mRNA in a large number of cells
8.53
21.5 22
192
cytosol
Rac-i
Mg 2+ de-
(+)
mRNA in a large number of cells
6.5
20.8 21
184
membrane
Rap-i
IGTPTS, guanosine 5 ' - 3 - O - ( t h i o ) - t r i p h o s p h a t e
(kDa)
Tissue specificity
pl
Molecular weight Predicted On SDS-PAGE
Amino acids
Localization
Factors
TABLE I I Properties of G T P - B i n d i n g Proteins in Human Neutrophils
5"
2"
O
Z P
~a
o_
<
10
NADPH Oxidase Activation Factors
Vol. 55, No. 1, 1994
2. Conclusions
Among the probably numerous protein components which are assembled to make an active oxidase complex, at least two cytosolic components, p47-phox and p67-phox, are essential. Recently, it has been proposed that the cytosolic components of the oxidase exist in the form of a M r 240,000 complex in SDS-dependent cell-free system, which suggests that the exposure of this complex to SDS induces a structural change that may or may not be associated with the loss of an inhibitory subunit too small to cause a detectable change in the size of the complex (111). Current evidence has disclosed many properties of the NADPH oxidase system, including membrane components (gp91phox, p22-phox and Rap-l) and cytosolic components (p47-phox, p67-phox and Rac1/2) (Tables I and II). However, the nature of the interactions between the different pieces of this amazing puzzle and the structural modifications of the components' assembly which lead to the respiratory burst remain to be determined. References
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