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The biochemical basis of the NADPH oxidase of phagocytes
TIBS 1 8 - FEBRUARY 1993
VIEWS THE PRESENCE OF OXYGEN is a vital
condition for the effective killing and digestion of pathogens by phagocytes, but it is not necessary for phagocytosis itself. Oxygen is consumed in the 'respiratory burst', a process that has fascinated scientists for the past three decades. This review focuses on our current understanding of the underlying mechanisms of this oxidase and the regulatory proteins that are required for activity (for more detailed reviews see Refs I--4).
What is the respiratory burst? While investigating the phagocytosis of tubercle bacilli by neutrophils and monocytes in 1959, Sbarra and Karnovsky made the novel obserJation that the sudden increase in the consumption of oxygen by the phagocytes was resistant to inhibitors like azide and cyanide. This distinguished it from mitochondrial respiration and suggested that it was involved in some process other than the production of the energy that was required for engulfment of the microorganism, most of which is provided by glycolysis. The natural substrate of this oxidase was the subject of considerable speculation. It was eventually clearly shown to be NADPH4, a continuous supply of which is generated from glucose via the hexose monophosphate shunt, leading to the somewhat unimaginative title of 'the NADPH oxidase' for this system. The most dramatic example of the importance of this NADPH oxidase is Chronic Granulomatous Disease (CGD), characterized by the absence of respiratory burst activity from the phagocytes of these patients 5. This rare condition manifests as a profound predisposition to severe and protracted, often fatal, infection. The responsible organisms include a wide variety of bacteria and fungi including many, such as Serracia marsescens, that are generally not pathogenic in normal subjects. The cells from these patients have proved to be an invaluable model sys-
The NADPH oxidase is an electron transport chain found in lymphocytes and in the wall of the endocytic vacuole of 'professional' phagocytic cells. It is so called because NADPH is used as an electron donor to reduce oxygen to superoxide and hydrogen peroxide. The redox components are provided by a very unusual flavocytochrome b from the membrane, which is dependent upon cytosolic factors (including two specialized proteins, p47 "°" and p67 ."°") for activation. The small GTP-binding protein, p21 rac, is also implicated in this system, possibly as the switch that triggers electron transport. This system provides a key to our understanding of the way in which these GTP-binding proteins function.
granule constituents, can use H202 to oxidize halides to reactive, toxic compounds like HOCI3 and chloramines 7. However, neither in myeloperoxidase The function of the NADPHoxidase How does the respiratory burst in- deficiency nor in ovine neutrophils, duce killing in normal cells? The prod- which lack this enzyme, is microbial uct of this reaction was initially ident- killing overtly defective, therefore other ified as H202, because it was found to factors must be involved in the effect of oxidize formate, which was subsequently the respiratory burst on killing by granshown to be generated by the dis- ule proteins. The explanation appears mutation of superoxide (O~), the one- to be remarkably simpleS: the pumping electron reduction product of oxygen. of millimolar concentrations of elecWhen the neutrophils phagocytose par- trons, unaccompanied by protons, into ticles the superoxide is only produced the vacuole results in the consumption in the region of the plasma membrane of hydrogen ions within the lumen, thus in direct contact with the particle, and increasing the pH. The granule contents the lag period provides the time re- are maintained in an inactive state at a quired for closure of the vacuole, thereby pH of about 5.0, and the neutral prolimiting the release of the reduced teinases are activated when exposed to the relatively alkaline environment oxygen species to the exterior. The discovery that free radicals were within the vacuole. The phagocytic vacuoles in CGD are produced in large amounts within the phagocytic vacuole (Fig. 1) implicated abnormally small, and the tissues of the reduced oxygen species themselves these subjects are infiltrated by granuin the microbial killing. However, both lomata composed of macrophages and superoxide and hydrogen peroxide are lymphocytes. These abnormalities apfairly long-lived and relatively unreactive, pear to result from defective digestion of endocytosed microbes and autoloso other explanations were needed. The contents of the cytoplasmic gous debris, an indigestion resulting granules are clearly important. Cyto- from excessively acid vacuoles! There is a growing realization that plasts (enucleated, granule-free bodies) phagocytose bacteria normally and pro- this oxidase system is not confined to A. W. Segaland A. Abo are at the duce a normal respiratory burst, but phagocytes, and that its component Departmentof Medicine,UniversityCollege kill the bacteria much less efficiently~. proteins are also found in T and B lymLondon,RayneInstitute,UniversityStreet, Myeloperoxidase, one of the major phocytes (see Ref. 9). It has been London,UKWCIE6JJ. 43 © 1993,ElsevierSciencePublishers,(UK) 0968-0004/93/$06.00 tem for the study of the biochemistry and cell biology of this oxidase.
TIBS 1 8 -
Plasma
e
NADP*+H*
202"-.-I~H202
Rgure 1 Schematic representation of a phagocyte engulfing a microbe into a phagocytic vacuole. The NADPH oxidase is selectively activated in the wall of the vacuole, generating O~ and hydrogen peroxide in the vacuolar lumen. Enzymes are also released into the vacuole by degranutation of cytoplasmic granules.
suggested that the superoxide it produces might play a role in regulating the expression of genes involved in inflammatory and immune responses. This oxidase could also play a role in antigen digestion and processing in the endosomes of antigen-processing cells by similar mechanisms to those responsible for optimizing conditions for the degradation of phagocytosed microbes in the endocytlc vacuole of phagocytes.
Activation of the oxldase The oxldase is normally dormant but can be rapidly activated by a number of stimulF "4. These include agonists that interact with surface receptors like opsonized particles and fMetLeuPhe (an example of immature peptide chains from microorganisms), and protein kinase C agonists like phorbol myristate acetate (PMA). Different stimuli activate different proximal pathways. The response to PMA is seen after about 25 s and lasts for many minutes, is calcium independent and predictably, is blocked by kinase inhibitors. The fMetLeuPhe response occurs after a lag of 5--10s, is of lower intensity and duration, is calcium dependent and resistant to inhibitors of PKC. Paradoxically, the phosphatase inhibitor okadaic acid almost completely inhibits the PMA response, whereas it greatly prolongs that induced by fMetLeuPhe~°, indicating that at least after activation with this agonist, termination of electron 44
transport might result from dephosphorylation of one or more of the specialized components. A 'primed' state can also be induced by subactirating levels of various stimuli. It reduces the lag and amplifies the response to a subsequent stimulus. Whatever the differences in the proximal activation pathways, the downstream processes converge in their requirements for the cytosolic components of the activation complex described below, no activators being effective in cells from patients with CGD in whom these are lacking.
A flavocytochromeb is responsiblefor electron transport The electron transporting mechanism of this system is very unusual. It contains a cytochrome b (Ref. 1I) with a very low mid-point potential which, at -245mV (hence the name b_245,also known as cytochrome bs~ because of the absorption maximum of its a-band) is lower than that of any such molecule in mammalian cells, and capable of directly reducing oxygen to superoxide. This cytochrome b is most abundant in neutrophils, monocytes, macrophages and eosinophils, but has been reported in a number of other cells including fibroblasts, T and B lymphocytes and renal mesangial cells3,9. It is located in the plasma membrane, and in neutrophils it is also distributed in the specific granules which fuse with the plasma
FEBRUARY1993
membrane, replenishing stocks of this and various other molecules when required. This cytochrome was also unusual in having two subunits 12,~3, an a-subunit (also called p21Ph~, and a heavily glycosylated [3-subunit (or gp91Ph~, with apparent molecular weights of approximately 21 and 76-92kDa respectively. Most mammalian cytochromes b are composed of a single polypeptide about the size of the a-subunit. The involvement of this cytochrome b in the NADPH oxidase was confirmed by its absence from neutrophils of patients with the X-linked type of CGD, with approximately half the normal concentrations in heterozygote carriers 14.This study also clearly differentiated the site of the lesion in X-linked patients from those with the autosomal recessive inheritance in most of whom the cytochrome was normal. The genes for both subunits have been cloned and sequenced, that for the ~-subunit by the then novel approach of 'reverse genetics 'Is. It was shown to code for the [5-subunit of this cytochrome when the amino acid sequence obtained from this protein was found to correspond with that derived from the nucleotide sequence of the gene responsible for X-linked CGD16.
The ~subunlt contains the FAD.and NADPH.blndlngsites For over three decades attempts have been made to identify the involvement of a flavoprotein in this oxidase. It has recently been shown that the ~-subunit of cytochrome b is in fact this FADcontaining flavoprotein dehydrogenase ~7. A number of lines of evidence support this conclusion. Membranes of neutrophils from X-CGDpatients, which selectively lack the two subunits of this cytochrome b, contain grossly reduced levels of both FAD and haem. The purified cytochrome b could be reflavinated, but only after relipidation, and the subunit was shown to bind the photoaffinity ligand 2-azido--NADP. The most convincing evidence was the identification of strong homology between the amino acid sequence of the ~-subunit of the cytochrome b and members of the ferredoxin-NADP÷ reductase (FNR) family of reductases, particularly in the conserved nucleotidebinding regions of these modular proteins (Fig. 2). Finally, as described below, we~8and others ~9have successfully reconstituted the oxidase from purified proteins, of
TIBS 18 - FEBRUARY1993
NADH reductases Nitrate reductase
(a) FAD-isoalloxazine (b) NAD/P-dbose
(c) NAD/P-adenine
712
880
Tomato
784 RAYTPSS
ARIAG---GTGITPV
66
Cytochmme-b5 reductase
Human
RPYTPIS
Saccharomyces cerevisiae
NADPH reductases Cytochrome b_us, ~-chain
337
Escherichia
HPFTLTS
coil
RLYSIAS
93 Fcrredoxin-NADP÷ reductasc
RLYSIA.
Tomato
S
454 Cytochmme P-450 reductase
RYYSIAS
Rat 1173
Nitric-oxide synthase
Rat
649 RNLVGVAAGLGVAA!
386 Sulphite reductase
VLNCGP
5~ HPFTVLS
Human
245 6NIAG---GTGITPN
462 Ferric-chelate reductase
LAACGP
150
RYYSISS
403 VNLVG--AGIGVTPF 453 VIRI6--P6T6IAPF 166 IINLG--TGTGIAPF 5~ VINVG--P6T6IAPF 1242 CILVG--PGT61APF
FYSCGP
~
533 VFLCGP
549 IY~C6D
269 VYNCGL
627 IYVCGD
1345 IYVCGD
Rgure2 ~subunit of flavocytochrome b is a memberof the FNRfarr,ily of reductasest7. Sequencealignmentof reductases and the ~-subunitof the flavocytochrome b in the proposedconserved(a) FADand (b), (c) NAD(P)H-bindingsites identified from the crystal structure of NAL)PHferredoxin reductase38. The site of the Pro/His mutation in a patient with X-linkedCGD,unusual because His membranescontain a cytochromeb with a normal spectrum, and content of FADand haem2° is shown(arrow).
which the flavocytochrome b is the only one in which there is any evidence for a redox function. The electron-transporting apparatus of this NADPH oxidase is thus very unusual in being entirely contained within a membrane-bound flavocytochrome, the only such molecule described to date in higher eukaryotic cells.
The 10oatl0nof the ham remainsto be determined Having sited the FAD in the ~subunit, finding the location of the two possible haems within this molecule proved to be equally as elusive, since they are displaced when the two subunlts are separated. Electron paramagnetic resonance (EPR) and Raman spectroscopy 2° indicated that the haems were of the low-spin, six-coordinate type with axial imidazole or imidazolate ligands. The cx-subunit has a region of limited sequence similarity with the haem-binding region of polypeptide 1 of cytochrome c oxidase, suggesting that a haem might be attached to this portion of the cx-subunit. However, the (x-subunit only has one invariant His residue 2], indicating an unusual ligand or attachment of the haems between more than one protein, either between two (x-, or the (x- and ~subunits. The []-subunit has five His residues in hydrophobic, possibly membrane-spanning, regions of the carboxy-terminal third of the molecule. This region of the molecule might act purely as a membrane anchor, or might have been further adapted for electron transport.
Two pieces of evidence suggest that the haem could be located exclusively on the wsubunit. It was claimed that this composite protein could be purified with the haem attached, although the amino acid composition did not correspond very closely with that derived from the nucleotide sequence of the gene 22. Radiation inactivation analysis of the haem spectrum revealed a vulnerable structure with a molecular mass of about 21 kDa, representing the wsubunit, the amino-terminal third of the ~-subunit, or both 23.
p47~ and p67~ are requiredfor activity The first indication that factors other than cytochrome b are required for activation was the discovery that the majority of patients with autosomal recessive CGD lack a 47 kDa cytosolic phosphoprotein, p47ph°x (Ref. 24). The phosphorylation of p47p~°~ coincides with the respiratory burst and it is translocated to the membranes upon activation. This movement of p47p~°~ was not observed in X-CGD neutrophils, indicating that the cytochrome b in the membranes was required for binding of p47ph°x to the membranes in this translocation event. The cloning and sequencing of the genes encoding both p47p~°~and p67Ph% another cytosolic protein component of this oxidase, resulted from a serendipitous experiment. Since a G protein was implicated in the activation of this system, proteins were fractionated on a GTP affinity column and the eluate, composed of a complex mixture of pro-
teins, was injected into rabbits. One rabbit produced antibodies to two main proteins, identified as p47p~ by its absence in most patients with autosomal recessive CGD, and p67Ph% which was missing in a few other individuals with the same inheritance. The predicted sequence of these proteins 2s,28indicated that they both contained two SH3 domains (SH, s r c homology region), which are present in many signalling proteins and are important for interactions between proteins, possibly including ,mall G proteins 27. Apart from these SH3 domains, which are likely to be involved in the association of the proteins of the activation complex described below, the amino acid sequences failed to provide any other clues as to their possible function.
Involvementof small GTP-bindlngproteins Cytosolic small GTP-binding proteins were found to be involved thro~gh use of a cell-free system 28 in which oxidase activity is induced in a mixture of cytosoi and membranes by the addition of arachidonic acid or sodium dodecyl sulphate. Separation of functional components showed that the flavocytochrome b was the only molecule in the membranes that was required for activity29. Fractionation of cytosol indicated that factor(s) other than p47p~°x and p67ph°~were required to activate the respiratory burst. Upon purification, these additional factors resolved into a heterodimeric complex of proteins with molecular weights of 21kDa and 26kDa3°; these were subsequently
45
TIBS 1 8 -
FEBRUARY1993
tern, is associated with movement of Rac, unaccompanied by GDI, to the membranes. The stimulus-induced dissociation of these two molecules, poss! kDa ibly by phosphorylation of the com-93 2o ponents or separation by liberated lipids such as arachidonic acid, might _ '67 be the switch that initiates activation of the oxidase. Our current data favours the formation of an activation complex involving Rac, p47p'°x and p67ph°~. This complex docks with the flavocytochrome, inducing a conformational -30 i lo . change favourable to electron transport. The mechanisms responsible for the formation of this complex and the way in which it induces electron transport . o . , .o.o o.o o., o., o.,o remain to be established. The continued -14 requirement for the association of these complexed cytosolic proteins 0 25 nM 50 75 with the flavocytochrome in the membranes while the oxidase is active37and Rgure 3 the roughly equimolar ratios of the speProteins required and sufficient for activity of the NADPH oxidase in the 'cell free' assayTM. cialized component proteins ~8 suggest (a) SDS/PAGE of p67p'°x, p47ph°", p21rac and the two subunits of purified flavocytochrome b that they induce a conformational (lanes 1-4 respectively), which together reconstitute an active oxidase upon the addition of change in the flavocytochrome favourNADPH and activation by SDS. (b) The effect of varying the concentration of the recombinant able for NADPH binding or electron cytosolic proteins, p67 ph°x(ll), p47 ph°x([:3) and p21 rac (Q), and NADPH(insert) in the cell-free assay are shown. transport, rather than by enzymic modification. It is also probable that the converidentified '~= as the ms-related small G dase system in the ceil.free system TMare sion of bound GTP to GDP on Rac is inprotein, p21'~'~ (Ref. 32) and rhoGDl shown in Fig. 3. volved in the regulation of its activity. (GDP-dissociation inhibition factor)33, On the basis of preliminary exper- As described in Fig. 4, the GTPase acrespectively. Pure, recombinant p21"'~ iments on the biology of p21"" in neu- tivity itself might act as a timing device coulC replace the complex of these two trophils, both in intact cells and in the that can regulate the duration of acproteins in the cell.free assay, but only cell-free assay, a schematic represen- tivity of the oxidase. This GTPase activity after exchange of the nucleotide into tation of the possible role that this mol- could be the unifying property of the the GTP form. The p21"~'~ was initially ecule plays in activation of the NADPH small GTP-binding proteins that convey purified from the cytosol of guinea-pig oxidase has been constructed (Fig. 4). their regulatory role to diverse protein macrophages. Similar results were sub- As described above, p21~d was purified interactions, each with a different applisequently obtained from human neutro- in a complex with rhoGDl, and chroma- cation depending upon the duration of phil cytosol, in which the GTP-binding tography of neutrophil cytosol indicates required activation. The NADPH oxiprotein was shown to be p21~c2 (Ref. that the bulk of the Rac is naturally dase, within both the intact cell and in 34). These two proteins, which share complexed to GDI. the cell-free system, provides a model 92% amino acid sequence homolo~, Small GTP-binding proteins are gen- system with which we can begin to probably play similar roles in these dif- erally active in the GTP-bound form; the answer some of these questions. ferent cells. The involvement of Rac in same was found to be true in the ceilthe oxidase is also supported by the free assay in which the Rac was active Acknowledgements parallel reduction of Rac content and in the GTP and non-hydrolysable GTP-TS We thank the Wellcome Trust for NADPH oxidase activity in Epstein-Barr forms, as was the Vail2 mutant that support and S. Blackwell for the figures. virus-transformed B-cells cultured in lacks inherent GTPase activity. The the presence of antisense oligonucleo- Rac-GD! complex purified from cytosol References tides 9. was active in the absence of exogenous Due to TIBS policy of short reference lists we have The p21~p~protein has been reported GTP, suggesting that some of the Rac is been unable to cite all the original work, most of which may be accessed through the reviews in to copurify and cross-immunoprecipi- naturally present in the GTP-bound Refs 1-4. tare with flavocytochrome b (Ref. 35). form in the GDI complex. This is consist1 Segal, A. W. (1989) J. C/in. Invest. 83, However, further purification separated ent with the recent observation that 1785-1793 these two molecules (see Ref. 18), and GDI inhibited the hydrolysis of GTP 2 Cross, A. R. and Jones, O. T. G. (1991) Biochim. the involvement of this small GTP-bind- bound to CDC42H, a Ras-related small G Biophys. Acta 1057, 281-298 ing protein in the regulation of the oxi- proteir with 70?/0amino acid sequence 3 Morel, F., Doussiere, J. and Vignais, P. V. (1991) Eur. J. Biochem. 201, 523-546 dase remains to be established. The identity to Rac~. 4 Rossi, F. (1986) Biochim. Biophys. Acta 853, five proteins that are both required and Activation of the oxidase, both in the 65-89 sufficient for activity of the NADPH oxi- intact cell and the cell-free assay sys- 5 Smith, R. M. and Cumutte, J. T. (1991) Blood 46
W
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-
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.
.
.
.
.
.
TIBS 1 8 - F E B R U A R Y 1 9 9 3
Active
202
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NADPH NADP +GDP-GTP excha~""~
(i)
(4) o, os,
SH3 domains ":~!i:p~
\
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Flavocytochrome b
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Figure 4 Model of the role p21= might play in the activation complex of the NADPH oxidase. (1) The specialized components of the NADPH oxidase include the e~-and ~subunits of tim flavocytochrome b and the two cytosolic proteins, p47 ph°x and p67 ph°x. (2) The p21 'ac protein is present largely in the GDP-bound form in a complex with GDI. This association requires modification by isoprenylation near its carboxyl terminus ( ~ )~, which could also be required for attachment to the membrane. (3) Activation is associated with separation of p21 'acfrom GDI and its movement with p47 ~h°xand p67ph°xin an activation complex into association with the flavocytochrome in the membranes. (4) At some stage in this process GDP is exchanged for GTP and the complex becomes activated. The bound GTP is then hydrolysed to GDP as a consequence of endogenous Rac GTPase activity, which may be modulated by mediators that accelerate or retard GTPase activity, including association with the effector molecules, as has been demonstrated with phospholipase C and elongation factor 4°. (5) The Rac is inactive in the GDP-bound form, so the rate of GTP hydrolysis provides a possible timing mechanism (~) for regulating the duration of the oxidase. Deactivation of the oxidase complex probably also involves dephosphorylation.
77, 673-686 6 Roos, D., Voetman,A. A. and Meerhof,L. J. (1983) J. Cell Biol. 97, 368-377 7 Weiss, S. J. (1989) New EngL J. Med. 320, 365-376 8 Segal,A. W. et al. (1981) Nature 290, 406-409 9 Dorseuil,O. et al. (1992) J. BioL Chem. 267, 20540-20542 10 Garcia,R. C., Whitaker, M., Heyworth,P. G. and Segal,A. W. (1992) Biochem. J. 286, 687-692 11 5egal,A. W. and Jones, O. T. G. (1978) Nature 276, 515-517 12 Segal,A. W. (1987) Nature 326, 88-91 13 Parkos,C. A., Allen, R. A., Cochrane,C. G. and Jesaitis,A. J. (1987)J. Clin. Invest. 80, 732-742 14 Segal,A. W. et aL (1983) New Engl. J. Med. 308, 245-251 15 RoyerPokora,B. et al. (1986) Nature 322, 32-38 16 Teahan,C. et al. (1987) Nature 327, 720-721 17 Segal,A. W. et al. (1992) Biochem. J. 284, 781-788
18 Abo, A. et al. (1992) J. BioL Chem. 267,
16767-16770 19 Rotrosen, D. et aL (1992) Science 256, 1459-1462 20 Hurst, J. K., Loehr,T. M., Cumutte,J. T. and Rosen,H. (1991)J. BioL Chem. 266,1627-1634 21 Dinauer, M. C. et al. (1990) J. Clin. Invest. 86, 1729-1737 22 Yamaguchi,T. et aL (1989) J. Biol. Chem. 264, 112-118 23 Nugent,J. H. A., Gratzer,W. and Segal,A. W. (1989) Biochem. J. 264, 921-924 24 Segal,A. W., Heyworth,P. G., Cockcroff,S. and Barrowman,M. M. (1985) Nature 316, 547-549 25 Volpp, B. D. et al. (1989) Proc. Natl Acad. Sci. USA 86, 7195-7199 26 Leto,T. L. et el. (1990) Science 248, 727-730 27 Pawson,T. and Gish,G. D. (1992) Cell 71, 359-362 28 Bmmberg,Y. and Pick, E. (1984) Cell Immunol.
88, 213-221 29 Knoller,S., Shpungin,S. and Pick, E. (1991) J. Biol. Chem. 266, 2795-2804 30 Abo, A. and Pick, E. (1991) J. Biol. Chem. 266, 23577-23585 31 Abo,A. et aL (1991) Nature 353, 668-670 32 Didsbufy,J. et el. (1989) J. BioL Chem. 264, 16378-16382 33 Fukumoto,Y. et el. (1990) Oncogene 5, 1321-1328 34 Knaus, U. G. et el. (1992) Science 254, 1512-1515 35 Quinn, M. T. et al. (1989) Nature 342, 198--200 36 Hart, M. J. et aL (1992) Science 258, 812-815 37 Tamura,M. et al. (1989) Arch. Biochem. Biophys. 275, 23-32 38 Karplus,P. A., Daniels,M. J. and Herriott,J. R. (1991) Science 251, 60-66 39 Hori, Y. et al. (1991) Oncogene6, 515-522 40 Boume,H. R. and Stryer,L. (1992) Nature 358, 541-543
47
VIEWS THE PRESENCE OF OXYGEN is a vital
condition for the effective killing and digestion of pathogens by phagocytes, but it is not necessary for phagocytosis itself. Oxygen is consumed in the 'respiratory burst', a process that has fascinated scientists for the past three decades. This review focuses on our current understanding of the underlying mechanisms of this oxidase and the regulatory proteins that are required for activity (for more detailed reviews see Refs I--4).
What is the respiratory burst? While investigating the phagocytosis of tubercle bacilli by neutrophils and monocytes in 1959, Sbarra and Karnovsky made the novel obserJation that the sudden increase in the consumption of oxygen by the phagocytes was resistant to inhibitors like azide and cyanide. This distinguished it from mitochondrial respiration and suggested that it was involved in some process other than the production of the energy that was required for engulfment of the microorganism, most of which is provided by glycolysis. The natural substrate of this oxidase was the subject of considerable speculation. It was eventually clearly shown to be NADPH4, a continuous supply of which is generated from glucose via the hexose monophosphate shunt, leading to the somewhat unimaginative title of 'the NADPH oxidase' for this system. The most dramatic example of the importance of this NADPH oxidase is Chronic Granulomatous Disease (CGD), characterized by the absence of respiratory burst activity from the phagocytes of these patients 5. This rare condition manifests as a profound predisposition to severe and protracted, often fatal, infection. The responsible organisms include a wide variety of bacteria and fungi including many, such as Serracia marsescens, that are generally not pathogenic in normal subjects. The cells from these patients have proved to be an invaluable model sys-
The NADPH oxidase is an electron transport chain found in lymphocytes and in the wall of the endocytic vacuole of 'professional' phagocytic cells. It is so called because NADPH is used as an electron donor to reduce oxygen to superoxide and hydrogen peroxide. The redox components are provided by a very unusual flavocytochrome b from the membrane, which is dependent upon cytosolic factors (including two specialized proteins, p47 "°" and p67 ."°") for activation. The small GTP-binding protein, p21 rac, is also implicated in this system, possibly as the switch that triggers electron transport. This system provides a key to our understanding of the way in which these GTP-binding proteins function.
granule constituents, can use H202 to oxidize halides to reactive, toxic compounds like HOCI3 and chloramines 7. However, neither in myeloperoxidase The function of the NADPHoxidase How does the respiratory burst in- deficiency nor in ovine neutrophils, duce killing in normal cells? The prod- which lack this enzyme, is microbial uct of this reaction was initially ident- killing overtly defective, therefore other ified as H202, because it was found to factors must be involved in the effect of oxidize formate, which was subsequently the respiratory burst on killing by granshown to be generated by the dis- ule proteins. The explanation appears mutation of superoxide (O~), the one- to be remarkably simpleS: the pumping electron reduction product of oxygen. of millimolar concentrations of elecWhen the neutrophils phagocytose par- trons, unaccompanied by protons, into ticles the superoxide is only produced the vacuole results in the consumption in the region of the plasma membrane of hydrogen ions within the lumen, thus in direct contact with the particle, and increasing the pH. The granule contents the lag period provides the time re- are maintained in an inactive state at a quired for closure of the vacuole, thereby pH of about 5.0, and the neutral prolimiting the release of the reduced teinases are activated when exposed to the relatively alkaline environment oxygen species to the exterior. The discovery that free radicals were within the vacuole. The phagocytic vacuoles in CGD are produced in large amounts within the phagocytic vacuole (Fig. 1) implicated abnormally small, and the tissues of the reduced oxygen species themselves these subjects are infiltrated by granuin the microbial killing. However, both lomata composed of macrophages and superoxide and hydrogen peroxide are lymphocytes. These abnormalities apfairly long-lived and relatively unreactive, pear to result from defective digestion of endocytosed microbes and autoloso other explanations were needed. The contents of the cytoplasmic gous debris, an indigestion resulting granules are clearly important. Cyto- from excessively acid vacuoles! There is a growing realization that plasts (enucleated, granule-free bodies) phagocytose bacteria normally and pro- this oxidase system is not confined to A. W. Segaland A. Abo are at the duce a normal respiratory burst, but phagocytes, and that its component Departmentof Medicine,UniversityCollege kill the bacteria much less efficiently~. proteins are also found in T and B lymLondon,RayneInstitute,UniversityStreet, Myeloperoxidase, one of the major phocytes (see Ref. 9). It has been London,UKWCIE6JJ. 43 © 1993,ElsevierSciencePublishers,(UK) 0968-0004/93/$06.00 tem for the study of the biochemistry and cell biology of this oxidase.
TIBS 1 8 -
Plasma
e
NADP*+H*
202"-.-I~H202
Rgure 1 Schematic representation of a phagocyte engulfing a microbe into a phagocytic vacuole. The NADPH oxidase is selectively activated in the wall of the vacuole, generating O~ and hydrogen peroxide in the vacuolar lumen. Enzymes are also released into the vacuole by degranutation of cytoplasmic granules.
suggested that the superoxide it produces might play a role in regulating the expression of genes involved in inflammatory and immune responses. This oxidase could also play a role in antigen digestion and processing in the endosomes of antigen-processing cells by similar mechanisms to those responsible for optimizing conditions for the degradation of phagocytosed microbes in the endocytlc vacuole of phagocytes.
Activation of the oxldase The oxldase is normally dormant but can be rapidly activated by a number of stimulF "4. These include agonists that interact with surface receptors like opsonized particles and fMetLeuPhe (an example of immature peptide chains from microorganisms), and protein kinase C agonists like phorbol myristate acetate (PMA). Different stimuli activate different proximal pathways. The response to PMA is seen after about 25 s and lasts for many minutes, is calcium independent and predictably, is blocked by kinase inhibitors. The fMetLeuPhe response occurs after a lag of 5--10s, is of lower intensity and duration, is calcium dependent and resistant to inhibitors of PKC. Paradoxically, the phosphatase inhibitor okadaic acid almost completely inhibits the PMA response, whereas it greatly prolongs that induced by fMetLeuPhe~°, indicating that at least after activation with this agonist, termination of electron 44
transport might result from dephosphorylation of one or more of the specialized components. A 'primed' state can also be induced by subactirating levels of various stimuli. It reduces the lag and amplifies the response to a subsequent stimulus. Whatever the differences in the proximal activation pathways, the downstream processes converge in their requirements for the cytosolic components of the activation complex described below, no activators being effective in cells from patients with CGD in whom these are lacking.
A flavocytochromeb is responsiblefor electron transport The electron transporting mechanism of this system is very unusual. It contains a cytochrome b (Ref. 1I) with a very low mid-point potential which, at -245mV (hence the name b_245,also known as cytochrome bs~ because of the absorption maximum of its a-band) is lower than that of any such molecule in mammalian cells, and capable of directly reducing oxygen to superoxide. This cytochrome b is most abundant in neutrophils, monocytes, macrophages and eosinophils, but has been reported in a number of other cells including fibroblasts, T and B lymphocytes and renal mesangial cells3,9. It is located in the plasma membrane, and in neutrophils it is also distributed in the specific granules which fuse with the plasma
FEBRUARY1993
membrane, replenishing stocks of this and various other molecules when required. This cytochrome was also unusual in having two subunits 12,~3, an a-subunit (also called p21Ph~, and a heavily glycosylated [3-subunit (or gp91Ph~, with apparent molecular weights of approximately 21 and 76-92kDa respectively. Most mammalian cytochromes b are composed of a single polypeptide about the size of the a-subunit. The involvement of this cytochrome b in the NADPH oxidase was confirmed by its absence from neutrophils of patients with the X-linked type of CGD, with approximately half the normal concentrations in heterozygote carriers 14.This study also clearly differentiated the site of the lesion in X-linked patients from those with the autosomal recessive inheritance in most of whom the cytochrome was normal. The genes for both subunits have been cloned and sequenced, that for the ~-subunit by the then novel approach of 'reverse genetics 'Is. It was shown to code for the [5-subunit of this cytochrome when the amino acid sequence obtained from this protein was found to correspond with that derived from the nucleotide sequence of the gene responsible for X-linked CGD16.
The ~subunlt contains the FAD.and NADPH.blndlngsites For over three decades attempts have been made to identify the involvement of a flavoprotein in this oxidase. It has recently been shown that the ~-subunit of cytochrome b is in fact this FADcontaining flavoprotein dehydrogenase ~7. A number of lines of evidence support this conclusion. Membranes of neutrophils from X-CGDpatients, which selectively lack the two subunits of this cytochrome b, contain grossly reduced levels of both FAD and haem. The purified cytochrome b could be reflavinated, but only after relipidation, and the subunit was shown to bind the photoaffinity ligand 2-azido--NADP. The most convincing evidence was the identification of strong homology between the amino acid sequence of the ~-subunit of the cytochrome b and members of the ferredoxin-NADP÷ reductase (FNR) family of reductases, particularly in the conserved nucleotidebinding regions of these modular proteins (Fig. 2). Finally, as described below, we~8and others ~9have successfully reconstituted the oxidase from purified proteins, of
TIBS 18 - FEBRUARY1993
NADH reductases Nitrate reductase
(a) FAD-isoalloxazine (b) NAD/P-dbose
(c) NAD/P-adenine
712
880
Tomato
784 RAYTPSS
ARIAG---GTGITPV
66
Cytochmme-b5 reductase
Human
RPYTPIS
Saccharomyces cerevisiae
NADPH reductases Cytochrome b_us, ~-chain
337
Escherichia
HPFTLTS
coil
RLYSIAS
93 Fcrredoxin-NADP÷ reductasc
RLYSIA.
Tomato
S
454 Cytochmme P-450 reductase
RYYSIAS
Rat 1173
Nitric-oxide synthase
Rat
649 RNLVGVAAGLGVAA!
386 Sulphite reductase
VLNCGP
5~ HPFTVLS
Human
245 6NIAG---GTGITPN
462 Ferric-chelate reductase
LAACGP
150
RYYSISS
403 VNLVG--AGIGVTPF 453 VIRI6--P6T6IAPF 166 IINLG--TGTGIAPF 5~ VINVG--P6T6IAPF 1242 CILVG--PGT61APF
FYSCGP
~
533 VFLCGP
549 IY~C6D
269 VYNCGL
627 IYVCGD
1345 IYVCGD
Rgure2 ~subunit of flavocytochrome b is a memberof the FNRfarr,ily of reductasest7. Sequencealignmentof reductases and the ~-subunitof the flavocytochrome b in the proposedconserved(a) FADand (b), (c) NAD(P)H-bindingsites identified from the crystal structure of NAL)PHferredoxin reductase38. The site of the Pro/His mutation in a patient with X-linkedCGD,unusual because His membranescontain a cytochromeb with a normal spectrum, and content of FADand haem2° is shown(arrow).
which the flavocytochrome b is the only one in which there is any evidence for a redox function. The electron-transporting apparatus of this NADPH oxidase is thus very unusual in being entirely contained within a membrane-bound flavocytochrome, the only such molecule described to date in higher eukaryotic cells.
The 10oatl0nof the ham remainsto be determined Having sited the FAD in the ~subunit, finding the location of the two possible haems within this molecule proved to be equally as elusive, since they are displaced when the two subunlts are separated. Electron paramagnetic resonance (EPR) and Raman spectroscopy 2° indicated that the haems were of the low-spin, six-coordinate type with axial imidazole or imidazolate ligands. The cx-subunit has a region of limited sequence similarity with the haem-binding region of polypeptide 1 of cytochrome c oxidase, suggesting that a haem might be attached to this portion of the cx-subunit. However, the (x-subunit only has one invariant His residue 2], indicating an unusual ligand or attachment of the haems between more than one protein, either between two (x-, or the (x- and ~subunits. The []-subunit has five His residues in hydrophobic, possibly membrane-spanning, regions of the carboxy-terminal third of the molecule. This region of the molecule might act purely as a membrane anchor, or might have been further adapted for electron transport.
Two pieces of evidence suggest that the haem could be located exclusively on the wsubunit. It was claimed that this composite protein could be purified with the haem attached, although the amino acid composition did not correspond very closely with that derived from the nucleotide sequence of the gene 22. Radiation inactivation analysis of the haem spectrum revealed a vulnerable structure with a molecular mass of about 21 kDa, representing the wsubunit, the amino-terminal third of the ~-subunit, or both 23.
p47~ and p67~ are requiredfor activity The first indication that factors other than cytochrome b are required for activation was the discovery that the majority of patients with autosomal recessive CGD lack a 47 kDa cytosolic phosphoprotein, p47ph°x (Ref. 24). The phosphorylation of p47p~°~ coincides with the respiratory burst and it is translocated to the membranes upon activation. This movement of p47p~°~ was not observed in X-CGD neutrophils, indicating that the cytochrome b in the membranes was required for binding of p47ph°x to the membranes in this translocation event. The cloning and sequencing of the genes encoding both p47p~°~and p67Ph% another cytosolic protein component of this oxidase, resulted from a serendipitous experiment. Since a G protein was implicated in the activation of this system, proteins were fractionated on a GTP affinity column and the eluate, composed of a complex mixture of pro-
teins, was injected into rabbits. One rabbit produced antibodies to two main proteins, identified as p47p~ by its absence in most patients with autosomal recessive CGD, and p67Ph% which was missing in a few other individuals with the same inheritance. The predicted sequence of these proteins 2s,28indicated that they both contained two SH3 domains (SH, s r c homology region), which are present in many signalling proteins and are important for interactions between proteins, possibly including ,mall G proteins 27. Apart from these SH3 domains, which are likely to be involved in the association of the proteins of the activation complex described below, the amino acid sequences failed to provide any other clues as to their possible function.
Involvementof small GTP-bindlngproteins Cytosolic small GTP-binding proteins were found to be involved thro~gh use of a cell-free system 28 in which oxidase activity is induced in a mixture of cytosoi and membranes by the addition of arachidonic acid or sodium dodecyl sulphate. Separation of functional components showed that the flavocytochrome b was the only molecule in the membranes that was required for activity29. Fractionation of cytosol indicated that factor(s) other than p47p~°x and p67ph°~were required to activate the respiratory burst. Upon purification, these additional factors resolved into a heterodimeric complex of proteins with molecular weights of 21kDa and 26kDa3°; these were subsequently
45
TIBS 1 8 -
FEBRUARY1993
tern, is associated with movement of Rac, unaccompanied by GDI, to the membranes. The stimulus-induced dissociation of these two molecules, poss! kDa ibly by phosphorylation of the com-93 2o ponents or separation by liberated lipids such as arachidonic acid, might _ '67 be the switch that initiates activation of the oxidase. Our current data favours the formation of an activation complex involving Rac, p47p'°x and p67ph°~. This complex docks with the flavocytochrome, inducing a conformational -30 i lo . change favourable to electron transport. The mechanisms responsible for the formation of this complex and the way in which it induces electron transport . o . , .o.o o.o o., o., o.,o remain to be established. The continued -14 requirement for the association of these complexed cytosolic proteins 0 25 nM 50 75 with the flavocytochrome in the membranes while the oxidase is active37and Rgure 3 the roughly equimolar ratios of the speProteins required and sufficient for activity of the NADPH oxidase in the 'cell free' assayTM. cialized component proteins ~8 suggest (a) SDS/PAGE of p67p'°x, p47ph°", p21rac and the two subunits of purified flavocytochrome b that they induce a conformational (lanes 1-4 respectively), which together reconstitute an active oxidase upon the addition of change in the flavocytochrome favourNADPH and activation by SDS. (b) The effect of varying the concentration of the recombinant able for NADPH binding or electron cytosolic proteins, p67 ph°x(ll), p47 ph°x([:3) and p21 rac (Q), and NADPH(insert) in the cell-free assay are shown. transport, rather than by enzymic modification. It is also probable that the converidentified '~= as the ms-related small G dase system in the ceil.free system TMare sion of bound GTP to GDP on Rac is inprotein, p21'~'~ (Ref. 32) and rhoGDl shown in Fig. 3. volved in the regulation of its activity. (GDP-dissociation inhibition factor)33, On the basis of preliminary exper- As described in Fig. 4, the GTPase acrespectively. Pure, recombinant p21"'~ iments on the biology of p21"" in neu- tivity itself might act as a timing device coulC replace the complex of these two trophils, both in intact cells and in the that can regulate the duration of acproteins in the cell.free assay, but only cell-free assay, a schematic represen- tivity of the oxidase. This GTPase activity after exchange of the nucleotide into tation of the possible role that this mol- could be the unifying property of the the GTP form. The p21"~'~ was initially ecule plays in activation of the NADPH small GTP-binding proteins that convey purified from the cytosol of guinea-pig oxidase has been constructed (Fig. 4). their regulatory role to diverse protein macrophages. Similar results were sub- As described above, p21~d was purified interactions, each with a different applisequently obtained from human neutro- in a complex with rhoGDl, and chroma- cation depending upon the duration of phil cytosol, in which the GTP-binding tography of neutrophil cytosol indicates required activation. The NADPH oxiprotein was shown to be p21~c2 (Ref. that the bulk of the Rac is naturally dase, within both the intact cell and in 34). These two proteins, which share complexed to GDI. the cell-free system, provides a model 92% amino acid sequence homolo~, Small GTP-binding proteins are gen- system with which we can begin to probably play similar roles in these dif- erally active in the GTP-bound form; the answer some of these questions. ferent cells. The involvement of Rac in same was found to be true in the ceilthe oxidase is also supported by the free assay in which the Rac was active Acknowledgements parallel reduction of Rac content and in the GTP and non-hydrolysable GTP-TS We thank the Wellcome Trust for NADPH oxidase activity in Epstein-Barr forms, as was the Vail2 mutant that support and S. Blackwell for the figures. virus-transformed B-cells cultured in lacks inherent GTPase activity. The the presence of antisense oligonucleo- Rac-GD! complex purified from cytosol References tides 9. was active in the absence of exogenous Due to TIBS policy of short reference lists we have The p21~p~protein has been reported GTP, suggesting that some of the Rac is been unable to cite all the original work, most of which may be accessed through the reviews in to copurify and cross-immunoprecipi- naturally present in the GTP-bound Refs 1-4. tare with flavocytochrome b (Ref. 35). form in the GDI complex. This is consist1 Segal, A. W. (1989) J. C/in. Invest. 83, However, further purification separated ent with the recent observation that 1785-1793 these two molecules (see Ref. 18), and GDI inhibited the hydrolysis of GTP 2 Cross, A. R. and Jones, O. T. G. (1991) Biochim. the involvement of this small GTP-bind- bound to CDC42H, a Ras-related small G Biophys. Acta 1057, 281-298 ing protein in the regulation of the oxi- proteir with 70?/0amino acid sequence 3 Morel, F., Doussiere, J. and Vignais, P. V. (1991) Eur. J. Biochem. 201, 523-546 dase remains to be established. The identity to Rac~. 4 Rossi, F. (1986) Biochim. Biophys. Acta 853, five proteins that are both required and Activation of the oxidase, both in the 65-89 sufficient for activity of the NADPH oxi- intact cell and the cell-free assay sys- 5 Smith, R. M. and Cumutte, J. T. (1991) Blood 46
W
t..
-
E
.
.
.
.
.
.
TIBS 1 8 - F E B R U A R Y 1 9 9 3
Active
202
20~
NADPH NADP +GDP-GTP excha~""~
(i)
(4) o, os,
SH3 domains ":~!i:p~
\
~eassociation
Flavocytochrome b
Is)
Figure 4 Model of the role p21= might play in the activation complex of the NADPH oxidase. (1) The specialized components of the NADPH oxidase include the e~-and ~subunits of tim flavocytochrome b and the two cytosolic proteins, p47 ph°x and p67 ph°x. (2) The p21 'ac protein is present largely in the GDP-bound form in a complex with GDI. This association requires modification by isoprenylation near its carboxyl terminus ( ~ )~, which could also be required for attachment to the membrane. (3) Activation is associated with separation of p21 'acfrom GDI and its movement with p47 ~h°xand p67ph°xin an activation complex into association with the flavocytochrome in the membranes. (4) At some stage in this process GDP is exchanged for GTP and the complex becomes activated. The bound GTP is then hydrolysed to GDP as a consequence of endogenous Rac GTPase activity, which may be modulated by mediators that accelerate or retard GTPase activity, including association with the effector molecules, as has been demonstrated with phospholipase C and elongation factor 4°. (5) The Rac is inactive in the GDP-bound form, so the rate of GTP hydrolysis provides a possible timing mechanism (~) for regulating the duration of the oxidase. Deactivation of the oxidase complex probably also involves dephosphorylation.
77, 673-686 6 Roos, D., Voetman,A. A. and Meerhof,L. J. (1983) J. Cell Biol. 97, 368-377 7 Weiss, S. J. (1989) New EngL J. Med. 320, 365-376 8 Segal,A. W. et al. (1981) Nature 290, 406-409 9 Dorseuil,O. et al. (1992) J. BioL Chem. 267, 20540-20542 10 Garcia,R. C., Whitaker, M., Heyworth,P. G. and Segal,A. W. (1992) Biochem. J. 286, 687-692 11 5egal,A. W. and Jones, O. T. G. (1978) Nature 276, 515-517 12 Segal,A. W. (1987) Nature 326, 88-91 13 Parkos,C. A., Allen, R. A., Cochrane,C. G. and Jesaitis,A. J. (1987)J. Clin. Invest. 80, 732-742 14 Segal,A. W. et aL (1983) New Engl. J. Med. 308, 245-251 15 RoyerPokora,B. et al. (1986) Nature 322, 32-38 16 Teahan,C. et al. (1987) Nature 327, 720-721 17 Segal,A. W. et al. (1992) Biochem. J. 284, 781-788
18 Abo, A. et al. (1992) J. BioL Chem. 267,
16767-16770 19 Rotrosen, D. et aL (1992) Science 256, 1459-1462 20 Hurst, J. K., Loehr,T. M., Cumutte,J. T. and Rosen,H. (1991)J. BioL Chem. 266,1627-1634 21 Dinauer, M. C. et al. (1990) J. Clin. Invest. 86, 1729-1737 22 Yamaguchi,T. et aL (1989) J. Biol. Chem. 264, 112-118 23 Nugent,J. H. A., Gratzer,W. and Segal,A. W. (1989) Biochem. J. 264, 921-924 24 Segal,A. W., Heyworth,P. G., Cockcroff,S. and Barrowman,M. M. (1985) Nature 316, 547-549 25 Volpp, B. D. et al. (1989) Proc. Natl Acad. Sci. USA 86, 7195-7199 26 Leto,T. L. et el. (1990) Science 248, 727-730 27 Pawson,T. and Gish,G. D. (1992) Cell 71, 359-362 28 Bmmberg,Y. and Pick, E. (1984) Cell Immunol.
88, 213-221 29 Knoller,S., Shpungin,S. and Pick, E. (1991) J. Biol. Chem. 266, 2795-2804 30 Abo, A. and Pick, E. (1991) J. Biol. Chem. 266, 23577-23585 31 Abo,A. et aL (1991) Nature 353, 668-670 32 Didsbufy,J. et el. (1989) J. BioL Chem. 264, 16378-16382 33 Fukumoto,Y. et el. (1990) Oncogene 5, 1321-1328 34 Knaus, U. G. et el. (1992) Science 254, 1512-1515 35 Quinn, M. T. et al. (1989) Nature 342, 198--200 36 Hart, M. J. et aL (1992) Science 258, 812-815 37 Tamura,M. et al. (1989) Arch. Biochem. Biophys. 275, 23-32 38 Karplus,P. A., Daniels,M. J. and Herriott,J. R. (1991) Science 251, 60-66 39 Hori, Y. et al. (1991) Oncogene6, 515-522 40 Boume,H. R. and Stryer,L. (1992) Nature 358, 541-543
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