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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1464-1476
REVIEW ARTICLE
NADPH Oxidase: An Update
By
Bernard M. Babior
From the Department of Molecular and Experimental Medicine, The
Scripps Research Institute, La Jolla, CA.
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INTRODUCTION |
THE NADPH OXIDASES are a group of plasma
membrane-associated enzymes found in a variety of cells of mesodermal
origin. The most thoroughly studied of these is the leukocyte NADPH
oxidase, which is found in professional phagocytes and B lymphocytes.
It catalyzes the production of superoxide
(O2 ) by the one-electron reduction of
oxygen, using NADPH as the electron donor:
The
O2 generated by this enzyme serves as the
starting material for the production of a vast assortment of reactive
oxidants, including oxidized halogens, free radicals, and singlet
oxygen. These oxidants are used by phagocytes to kill invading
microorganisms, but they also cause a lot of what the military would
call "collateral damage" to nearby tissues, so their production
has to be tightly regulated to make sure they are only generated when
and where required.
In the 40 years since Sbarra and Karnovsky first reported findings
suggesting the existence of such an enzyme in neutrophils, a great deal
has been learned about the leukocyte oxidase. Research over this period
of time has shown that the core enzyme comprises five components:
p40PHOX (PHOX for PHagocyte OXidase),
p47PHOX, p67PHOX,
p22PHOX and gp91PHOX. In the
resting cell, three of these five
components p40PHOX, p47PHOX
and p67PHOXexist in the cytosol as a complex. The
other two componentsp22PHOX and
gp91PHOXare located in the membranes of secretory
vesicles* and specific granules, where they
occur as a heterodimeric flavohemoprotein known as cytochrome
b558. Separating these two groups of components by
distributing them between distinct subcellular compartments guarantees
that the oxidase is inactive in the resting cell.
When the resting cell is exposed to any of a very wide variety of
stimuli, the cytosolic component p47PHOX becomes
heavily phosphorylated and the entire cytosolic complex migrates to the
membrane, where it associates with cytochrome b558 to
assemble the active oxidase (Fig 1). The assembled
oxidase is now able to transfer electrons from the substrate to oxygen by means of its electron-carrying prosthetic groupsits flavin and
then (according to most investigators, but not me) its heme group(s).
Activation requires the participation, not only of the core subunits,
but of two low-molecular-weight guanine nucleotide-binding proteins:
Rac2, which in the resting cell is located in the cytoplasm in a
dimeric complex with Rho-GDI (Guanine nucleotide
Dissociation Inhibitor), and Rap1A, which is located in
membranes from which it can be copurified with the cytochrome. During
activation, Rac2 binds guanosine triphosphate (GTP) and
migrates to the membrane along with the core cytosolic complex. At the
same time, cytochrome b558 and Rap1A are delivered to the
cell surface by fusion of the secretory vesicle membranes and later the
specific granule membranes with the plasma membrane of the cell. This
fusion event also releases the contents of the organelles to the
exterior. When phagocytosis takes place, the plasma membrane is
internalized as the wall of the phagocytic vesicle, with what was once
the outer membrane surface now facing the interior of the vesicle. From
this location, the enzyme pours O2 into the
vesicle, and the rapid conversion of this O2
into its successor products bathes the internalized target in a lethal
mixture of corrosive oxidants.
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| Fig 1.
Activation of the leukocyte NADPH oxidase. In the resting
cell, the subunits of the oxidase are distributed between the cytosol
(p40PHOX, p47PHOX,
p67PHOX and Rac2) and the membranes (Rap1A and
cytochrome b558, a
p22PHOX · gp91PHOX
complex). Rac2 and Rap1A are low-molecular-weight guanine
nucleotide-binding proteins that function in other processes besides
oxidase activation. The other 5 proteins are unique to the NADPH
oxidase. When the cell is activated, p47PHOX
becomes heavily phosphorylated and the cytosolic subunits migrate to
the membrane, where they bind to cytochrome b558 to
assemble the active oxidase.
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The leukocyte NADPH oxidase continues to be the object of a
considerable amount of investigation by scientists who are curious as
to how it works. Interest in the leukocyte oxidase has increased with
the growing recognition that oxidases closely related to the leukocyte
oxidase can be found in a variety of cells in which the enzymes serve a
variety of purposes. The following is a review of some of the new
information obtained through these recent studies.
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THE LEUKOCYTE NADPH OXIDASE |
Properties and Functions of the Oxidase Components
Cytochrome b558.
A question that has been under study for some time has been the exact
composition of the cytochrome b558 molecule: how many subunits of each kind does the cytochrome contain, and how many prosthetic groups? The most recent answers are that the cytochrome is a
heterodimer containing one of each kind of subunit,1 and that it contains one FAD and two hemes.2 The two heme
groups on the flavocytochrome are functionally distinct, with midpoint potentials at pH 7.0 of  225 and 265 mV.3 In the
enzyme-bound FAD, a corner of the benzene ring of the electron-carrying
isoalloxazine group is exposed to the solvent,2 suggesting
that this corner is where the electron enters or leaves.
New information has appeared concerning the redox properties of
cytochrome b558. The kinetics of electron flow through the flavocytochrome have been redetermined in a cell-free system containing purified cytochrome b558 and recombinant cytosolic
components. The turnover number for the
system was 165 heme1, s1, while the rate
of oxidation of reduced heme was 1,720 s1.4
Assuming that the concentration of cytochrome b558 in
neutrophils corresponds to 10 pmol heme/106
cells5 and that all the cytochrome b558
participates in O2 production, the observed
turnover number is equivalent to a rate of
O2 production of about 0.2 nmol/min/106 cells, only 2% of the rate seen with
maximally activated whole neutrophils. On the other hand, a rate of
heme reduction fast enough to account for O2
production by whole cells was calculated from observed levels of
reduction of the partly reduced flavin and heme in the working enzyme.
However, this calculation depended on the assumption that the observed
levels of reduction were steady-state levels; but it was later shown
that this assumption was not in accord with experimental
findings6 (see also ref 7). Moreover, earlier studies under
anerobic conditions had shown that both in whole cells and in a
cell-free system, the actual rate of reduction of the cytochrome
b558 heme by NADPH was only about 0.1% of the rate of
O2 production by the identical systems
operating in air.8,9 Because a multistep reaction can go no
faster than its slowest step, the rate of O2
production by neutrophils could be no greater than a few
pmol/min/106 cells if the heme were an obligatory
intermediate in electron transfer. Therefore, it is difficult for me to
grasp the logic of the widely held belief that a heme residue of
cytochrome b558 participates at all in electron transport
by the leukocyte NADPH oxidase, much less that it is the terminal
electron carrier. Evidence for heme participation such as the recent
demonstration that exposure of the oxidase to an activating agent
changes the spin state of the iron in cytochrome b558 from
low-spin hexacoordinate to high-spin pentacoordinate,10
though of great interest, is indirect and can be interpreted in many
ways. Kinetic competence is the gold standard, and there is no escape
from the fact that the rate of reduction of the heme is far too slow
for it to participate in any meaningful way as an electron carrier for
the oxidase.
The foregoing considerations apply only to the heme of the
flavocytochrome. There is universal agreement that its flavin is an
electron carrier. Oxidase activity is lost when FAD is removed from the
enzyme, and restored when FAD is added back.11 The oxidase
is inhibited by flavin antagonists such as deaza-FAD12 and
diphenylene iodonium13 (in contrast to the lack of effect of heme antagonists such as CN,
N3, CO, and butyl
isonitrile14). In model systems,
O2 is produced by the reaction between
oxygen and reduced flavins. Finally, recent experiments demonstrating
that under special conditions purified cytochrome b558
alone catalyzes O2 production from NADPH and
oxygen15,16 leaves little doubt that the flavin of
cytochrome b558 is an electron carrier for the leukocyte
NADPH oxidase.
The ability of the leukocyte NADPH oxidase to use artificial electron
acceptors was discovered some time ago by Green and Wu.17
Recently, iodonitrotetrazolium violet (INT) was added to the list of
oxidants that could accept electrons from the oxidase.18,19 Partial purification of the INT-reducing activity from activated neutrophil membranes showed that it contained cytochrome
b558, and that its activity was dependent on cytosol and
FAD.19 INT reduction persisted under anerobic conditions,
indicating that the dye accepted electrons directly from the enzyme.
Chronic granulomatous disease (CGD) is an inherited disorder
characterized by the failure of O2
production by phagocytes, resulting in a marked increase in the susceptibility of affected patients to bacterial and fungal infections. The disease is caused by a mutation that results in the loss or inactivation of one of the core subunits of the oxidase (CGD due to a
mutation affecting p40PHOX has not yet been
reported). Inactivating mutations (Table 1) have provided important information concerning the mechanism of the
oxidase. Several recent reports have identified new mutations leading
to inactive gp91PHOX. These include is
gp91PHOX A57E,20 E309K, C537R, P339H,
and  K31521 and gp91PHOX F215 or
216.22 Cells containing gp91PHOX
F215 or 216 have normal amounts of gp91PHOX but
show no trace of the cytochrome b558 spectrum. This finding suggests that one or both of these phenylalanine residues are essential
for the binding of the cofactors to the cytochrome.
Cytosolic Components
p47PHOX is the subunit chiefly responsible for
transporting the cytosolic complex from the cytosol to the membrane
during oxidase activation. This is evident because neutrophils that
lack p47PHOX are unable to transfer
p67PHOX from the cytosol to the membrane during
activation, although p67PHOX-deficient neutrophils
transfer p47PHOX in a normal fashion.23
Before the cytosolic oxidase components can be transferred to the
membrane, however, p47PHOX must be extensively
phosphorylated. This phosphorylation is one of the characteristic
events of oxidase activation. p47PHOX, however, is
not absolutely indispensible for oxidase activity. Even though its
deficiency results in one form of CGD, two groups have now shown that
in a detergent-activated cell-free system containing purified
cytochrome b558 and recombinant cytosolic factors,
p47PHOX can be omitted as long as the system
contains high concentrations of p67PHOX and
Rac2.24,25 The effect of p47PHOX is to
tighten by nearly 100-fold the binding of each of the other cytosolic
proteins to the assembled oxidase.
The function of p67PHOX has been a mystery. Unlike
p47PHOX, p67PHOX is absolutely
required for oxidase activity. When incubated with membranes in the
detergent-activated cell-free system, a cytosol lacking p47PHOX but containing p67PHOX
was able to support INT reduction.18 Furthermore,
p67PHOX facilitated electron transfer to the flavin
of cytochrome b558 in the absence of
p47PHOX 26; when p47PHOX was
also present, the system was said to allow electron transfer "to
proceed beyond the flavin center to the heme in cytochrome b245 and thence to
oxygen," a claim with which I am obviously not in full agreement. We
recently furnished some evidence as to the possible function of
p67PHOX in oxidase activity by showing that this
subunit contained a catalytically essential binding site for
NADPH.27 However, there is evidence that an NADPH binding
site also exists on cytochrome b558.28,29 The
relationship between these two NADPH binding sites remains to be
determined, though it should be noted that cytochrome b558
alone catalyzes active O2 production using
NADPH as the reductant,16 while catalysis of NADPH
dehydrogenation by p67PHOX has to date not been demonstrated.
A CGD patient with a functionally significant mutation of
p67PHOX has recently been described.30
This mutant, p67PHOX K58, loses its interaction
with Rac, and when phagocytes containing the
p67PHOX mutant are activated, the cytosolic complex
fails to translocate to the membrane. These results imply that Rac,
like p47PHOX, is involved in the translocation of
the cytosolic complex during oxidase activation.
The function of p40PHOX has recently been
examined.31 Oxidase activity can be established in K562
cells by transfecting them with plasmids that express recombinant
p47PHOX, p67PHOX, and
gp91PHOX.32 Cells that express
p40PHOX along with other recombinant oxidase
components produce only about half the amount of
O2 generated by cells not expressing
p40PHOX. Similarly, adding
p40PHOX to a cell-free detergent-dependent oxidase
activating system inhibits O2 production.
These results suggest that p40PHOX is an inhibitory
oxidase subunit. On the other hand, interfering with the binding of
p40PHOX to p67PHOX reduces by
50% the production of O2 in a
detergent-dependent cell-free system, a result implying that
p40PHOX is a stimulatory subunit.33
Clearly, more research on this question is needed.
Location of the Oxidase
As discussed above, the activated NADPH oxidase is associated with the
phagocyte membranes. Recent studies have indicated how the oxidase is
distributed in the membrane, and on which membranes it is located.
Immunoelectron microscopy showed that cytosolic oxidase components were
grouped together with Rac on the inner face of neutrophil plasma
membranes in 3- to 10-nm clusters34 (Fig
2). In a study of exceptional interest,
Kobayashi et al35 used cytochemical staining to show that
O2 production in phorbol-stimulated
neutrophils initially took place in small alkaline
phosphatase-containing cytoplasmic vesicles (Fig
3). These vesicles then fused together,
forming larger vesicles that then merged with the plasma membrane.
After a time, O2-producing vesicles
containing a marker of extracellular fluid appeared within the
neutrophils. These results strongly imply that in resting cells, the
membrane-associated oxidase components are located exclusively in
intracellular organelles (secretory vesicles as shown here, and
specific granules as demonstrated earlier36), and that
the active oxidase in the plasma membrane is delivered there by
membrane fusion events. They further imply that vesicles whose
membranes contain the active oxidase cycle between the interior of the
cell and the plasma membrane.
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| Fig 2.
Localization of the oxidase to the neutrophil plasma
membrane.34 Normal neutrophils were activated and unroofed,
and the cytoplasmic face of the plasma membrane was labeled by the
immunogold technique with anti-p67PHOX (large
beads) and anti-gp91PHOX (small beads). The oxidase
assemblies are gathered together in small clusters. Similar results
were obtained with
anti-p67PHOX/anti-p47PHOX and
anti-p67PHOX/anti-Rac antibody pairs. (Reprinted
with permission.34)
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| Fig 3.
Sites of O2 production in
neutrophils as a function of time after activation.35 (Top)
O2 production starts in secretory vesicles
(clumped precipitate; single arrow). The membranes of these vesicles
are distinct from the plasma membrane, because none of the
O2-forming vesicles contain the ferritin
(punctate precipitate, double arrow) that was added to the
extracellular medium during the incubation and then was internalized by
pinocytosis. (Center) The O2-forming
secretory vesicles later fuse with pinocytotic vesicles to form
secondary vesicles. The oxidase in the membranes of these secondary
vesicles continue to deliver O2 into the
vesicle lumina. (Bottom) O2-forming
secretory vesicles also fuse with the plasma membrane itself, leading
to the secretion of O2 into the
extracellular environment. (Reprinted with permission from Kobayashi T,
Robinson JM, Seguchi H: Identification of intracellular sites of
superoxide production in stimulated neutrophils. J Cell Sci 111:81,
1998, published by The Company of Biologists, Ltd, Cambridge,
UK.35)
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Inhibitors
Sulfhydryl groups are important for the function of the leukocyte NADPH
oxidase. Recently, naturally occurring sulfhydryl blockers have been
found to inhibit the oxidase. The aldehyde 4-hydroxynonenal inhibited
the oxidase with an I50 of 19 µmol/L.37 Inhibition was reversed with dithiothreitol, suggesting that the blockade of -SH groups is responsible for the action of the aldehyde. Inhibition by 4-hydroxynonenal is of physiological significance because
the aldehyde is a major product of lipid peroxidation, and therefore
will be present in tissues undergoing oxidative attack.
Nitric oxide (NO·) also inhibits the oxidase, but it
works by preventing the assembly of the oxidase during
activation.38 It has no effect on the fully active oxidase,
and does not interact with either the flavin or the hemes on cytochrome
b558. Nitrosothiols (RSNO) also inhibit oxidase activation,
preventing translocation of the cytosolic oxidase components
p47PHOX and p67PHOX through an
action on the membrane.39 The effect of nitrosothiols can
be reversed by mercaptoethanol. It is likely that both
NO· and RSNO act by combining with cysteine-SH groups to
form nitrosothiols on components of the oxidase.
It has recently been reported that PR-39, an antimicrobial peptide from
neutrophils, is able to inhibit the NADPH oxidase by binding to the SH3
domains of p47PHOX, thereby blocking the
interaction between p47PHOX and
p22PHOX that normally takes place during oxidase
activation.40 This 39-amino acid peptide is very unusual
in that it contains 10 arginines, 19 prolines, and no acidic residues.
A role for this peptide in the regulation of oxidase activity has been
proposed, but evidence supporting this proposal is scanty. It is true
that exposure of neutrophils to PR-39 inhibits
O2 production in response to phorbol
myristate acetate, but nonspecific toxicity of this peptide was not
ruled out.
The Oxidase as a Battery
The phagocyte secretes O2 as the anion,
leaving behind the proton produced in the
O2-forming reaction. In accord with this
finding are observations showing that the leukocyte NADPH oxidase is an
electrogenic enzyme, and that its activation is associated with the
opening of a channel through which the protons left behind can leave
the cell before they shut down the enzyme.41-44 In a recent
publication, the existence of an inward current associated with the
activation of the oxidase has been directly measured by patch
clamping,45 confirming earlier work by O.T.G.
Jones and associates.41 In the discussion of their report, these investigators postulate that the electric current
per se may be directly related to the function of the oxidaseie,
microbial killing. They further state that "the importance [of
O2] in microbial killing is unclear."
This statement is rather surprising in view of the very large body of
evidence showing that this O2 is the
precursor of the powerful oxidants the phagocytes use as microbicidal agents.
Activation of the oxidase.
During oxidase activation, the cytosolic oxidase subunits are
transferred to the membrane, where they bind to the membrane-associated oxidase components (cytochrome b558 and possibly Rap1A) to
assemble the active enzyme. Translocation is preceded by the
phosphorylation of certain oxidase-related proteins, some identified
and some not. Low-molecular-weight guanine nucleotide binding proteins (GNBP) are also involved in oxidase activation, but the nature of their
participation is unknown.
Protein-Protein Interactions Among the Oxidase Subunits
Examination of interactions between the various subunits of the oxidase
is a very active field of investigation, and a remarkable number of
studies on this subject have appeared in the published literature in
the last few years.2,30,31,33,46-62 These studies have
shown that in both the resting and active oxidases, the various subunits are associated with each other in the form of well-defined complexes. Resting cytosol contains an ~250-kD complex
of undetermined stoichiometry comprising the three oxidase subunits
p47PHOX, p67PHOX, and
p40PHOX. Activation of the oxidase brings this
cytosolic complex to the membrane, where it associates with cytochrome
b558, a membrane-bound dimer containing
gp91PHOX and p22PHOX that in
turn is probably interacting with Rap1A. A very large amount of effort
has been expended in mapping the protein-protein interactions
responsible for the formation of these complexes. An earlier
review63 listed those protein-protein interactions that had
been reported as of 1996. Since then, many more pairs of interactions
have been mapped in whole or in part.
The major interactions among the cytosolic subunits that have been
mapped to date are illustrated in Fig 4.
The pairwise interactions themselves are well established, but the
proposed allocation of the various interactions to the resting versus
the activated state is strictly hypothetical, although it is based on
evidence that rearrangements of the cytosolic complex take place during
oxidase activation64,65 and on the identification of
interactions that are required for translocation of the complex to the
membrane. Of particular interest are the interactions between the
C-terminal proline-rich SH3 binding domain of
p47PHOX and the SH3 domains of both
p47PHOX and p40PHOX in the
resting complex. The interaction of p47PHOX with
itself is thought to be responsible for the inability of resting
p47PHOX to bind to cytochrome b558, the
membrane-associated electron-carrying component of the oxidase. During
activation, however, the C-terminal proline-rich domain of
p47PHOX is occupied by the C-terminal SH3 domain of
p67PHOX, an interaction that is required for
translocation. This presumably means that this proline-rich domain
separates from the p47PHOX SH3 domain that it
occupies in the resting state, and perhaps from the SH3 domain of
p40PHOX as well. Another interaction required for
translocation is the one between the C-terminal SH3 domain of
p47PHOX and the N-terminal proline-rich domain of
p67PHOX; in the diagram this is shown only in the
activated complex, but it could be present in the resting complex as
well. Finally, the N-terminal SH3 domain of
p47PHOX, freed from its interaction with the distal
p47PHOX proline-rich domain, binds to the
C-terminal portion of p67PHOX. But in the active
oxidase, this p47PHOX SH3 domain has to interact
with p22PHOX. Maybe oxidase activation is a
multi-step process, one step consisting of the rearrangements shown in
the figure, and a subsequent step involving an exchange at the
N-terminal SH3 domain of p47PHOX in which the
C-terminal portion of p67PHOX is swapped for the
proline-rich domain of p22PHOX. The possibility
that oxidase activation is a multi-step process is also implied by
experiments discussed below in the section on protein phosphorylation.
These experiments suggest that oxidase activation can be dissected into
three successive events: partial phosphorylation of
p47PHOX, translocation of
p47PHOX and the other cytosolic oxidase components
to the membrane, and then final phosphoryation of
p47PHOX coupled to the acquisition of catalytic
activity by the enzyme. I must emphasize, however, that the
foregoing discussion of the changes in protein-protein interactions
undergone by the cytosolic complex during oxidase activation, though
consistent with current findings, is pure speculation.
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| Fig 4.
Proposed protein-protein interactions among the cytosolic
components of the leukocyte NADPH oxidase in the resting and activated
states. For an explanation of the figure, see text. ( ), SH3 domains;
( ), proline-rich SH3 binding domains.
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Although interactions among the cytosolic subunits, and between the
cytosolic subunits and p22PHOX, are reasonably well
defined, interacting domains between gp91PHOX and
the remainder of the oxidase subunits are uncertain. Phage display
libraries have identified certain p47PHOX peptides
that recognize gp91PHOX,66 and the
examination of hydrophobic domains67 has identified certain
peptides in gp91PHOX that could be important in
oxidase activity. At relatively high concentrations, these peptides
inhibit oxidase activity in the sodium dodecyl sulfate (SDS)-dependent
cell-free oxidase activating system and in electropermeabilized
neutrophils. However, peptide inhibition studies can be deceptive.
Illustrating this is work carried out on a peptide from the C-terminus
of gp91PHOX. For a number of years, an interaction
involving seven residues near the C-terminus of
gp91PHOX (RGVHFIF) was thought to be of
considerable functional significance because a peptide with this
sequence was found to be a potent inhibitor of oxidase activity in an
SDS-dependent cell-free oxidase activating system. Recently, however,
it was shown that mutations in this sequence had no effect on oxidase
activity in a gp91PHOX-deficient leukemia line;
O2 production by cells expressing these
mutants was no different than O2 production
in cells expressing wild-type
gp91PHOX.68 This means either that this
sequence is of no functional significance, or that its function can be
replaced by some other element in the gp91PHOX protein.
Nevertheless, this region of gp91PHOX does appear
to bind to p47PHOX, and the structure of a complex
between p47PHOX and the 17-mer
gp91PHOX 551-568 (the C-terminus of
gp91PHOX, lacking only K569) was studied by
two-dimensional nuclear magnetic resonance spectroscopy (2-D
NMR).56 This study showed that the stretch of
peptide from S555 to F564 could be clearly defined, showing an extended
bend with immobilized side chains (except for H561). This is a novel
approach to the study of interactions among the oxidase components.
Phosphorylation
Phosphorylation is an essential element in the activation of the NADPH
oxidase. Phosphorylation of p47PHOX during oxidase
activation has been recognized for many years. More recently, the
phosphorylation of p67PHOX and
p40PHOX has been shown, and the phosphorylation of
a component in the membrane has been implicated in the activation process.
The protein whose phosphorylation has been studied most thoroughly is
p47PHOX. During oxidase activation it is
extensively phosphorylated, with 8 to 9 serines in the C-terminal
quarter of the molecule acquiring phosphates. Of these target serines,
S379 is the only one whose conversion to alanine as a sole mutation
results in a major loss of oxidase activity.69
Phosphorylation of S379 is necessary for both the translocation of
p47PHOX and the activation of the oxidase. It has
recently been found, however, that the mutation of both of a pair of
target serines to alanine can inactivate p47PHOX.
Serines S303 and S304 are known to be phosphorylated during oxidase
activation. The mutation of this serine pair to alanines greatly
decreases oxidase activity, though the phosphoryation of other serines
and the translocation of the mutant p47PHOX to the
membrane is unaffected. Activity is normal, however, if these serines
are replaced by glutamates, or, surprisingly, by lysines.70
The activity of p47PHOX S303K,S304K mutant raises
the possibility that p47PHOX may in part be linked
to the rest of the active oxidase by a cation bridge. Interestingly,
the mutation to alanines of the MAP kinase target pair S345/S348 had no
effect on the activity of the oxidase.69,71
A subsequent study showed another pair of serines, S359 and S370, whose
phosphorylation was necessary for oxidase activation.72 The
mutation of both these serines to alanines results not only in the loss
of oxidase activity, but also in a complete failure of phosphorylation
of p47PHOX, both recombinant and in whole cells.
Replacement of those serines by glutamate or aspartate allows
phosphorylation and translocation to take place, but oxidase activity
is still greatly reduced. In contrast to the results obtained with
S303K,S304K, the replacement of S359 and S370 with lysines led to the
total loss of activity of the oxidase;
p47PHOX-deficient cells expressing
p47PHOX S359K,S370K showed no more
O2 production than the untransfected
p47PHOX-deficient cells. The conclusion drawn from
these studies was that during oxidase activation, S359 and/or
S370 has to be phosphorylated first; S379 then acquires a phosphate,
allowing the cytosolic complex to translocate to cytochrome
b558; and finally, S303 and/or S304 are
phosphorylated, endowing the oxidase with full catalytic activity.
The phosphorylation of p47PHOX is regulated by
protein kinase A. It had been known for some time that oxidase
activation is diminished in neutrophils containing elevated levels of
cyclic adenosine monophosphate (cAMP) or cAMP analogs. It
has now been shown that the phosphorylation of
p47PHOX in response to N-formylmethionylleucyl
phenylalanine (fMLP) is inhibited by cAMP agonists, and that this
effect is prevented by an inhibitor of protein kinase A. In contrast,
cAMP agonists had no effect on p47PHOX
phosphorylation induced by phorbol myristate acetate, an activator of
protein kinase C.73
Recently, both p40PHOX and
p67PHOX were found to take up phosphate when
neutrophils are stimulated.74,75 It was shown that
p40PHOX is phosphorylated in the resting cell,
takes up additional phosphate when the NADPH oxidase is activated, and
loses the additional phosphate when the oxidase is deactivated. Both
p40PHOX and p67PHOX acquire
phosphate when the cells are activated with either fMLP or phorbol; the
latter suggests that protein kinase C participates in the
phosphorylation of both oxidase components. On the basis of in vitro
experiments, however, it was claimed that the phosphorylation of
p40PHOX and p47PHOX involve
distinct signal transduction pathways. In regard to the phosphorylation
of p67PHOX, an earlier report76 claimed
that two 67K proteins are phosphorylated in activated neutrophils, but
that neither one is p67PHOX; their results,
however, do not rule out the possibility that a small amount of
p67PHOX is phosphorylated during oxidase
activation, a qualification that becomes important in the light of
observations showing that only ~5% of the
p67PHOX translocates to the membrane during oxidase
activation.77,78 Inhibition of MAP kinase kinase by
PD098059 prevents oxidase activation by either opsonized zymosan or
phorbol myristate acetate,79 while inhibition of p38 by
SB203580 prevents oxidase activation by fMLP.80 MAP kinase
kinase activates ERK, a member of the MAP kinase family, and p38,
another member of the MAP kinase family. To the extent that these
inhibitors are truly specific (always an open question with any
inhibitor), these results imply that both ERK and p38 participate in
the activation of the NADPH oxidase. However, the nature of their
participation is unknown.
The recent development of cell-free systems in which the NADPH oxidase
is activated by kinases81,82 offers an opportunity to study
the signal transduction pathway(s) responsible for oxidase activation
in a system that is at least partly defined. In one of these systems,
oxidase activation requires both adenosine triphosphate (ATP) and phosphatidic acid, the latter an anionic
detergent that is able to activate the oxidase by itself, although ATP
doubles O2 production in that system. A
phosphatidic acid-dependent protein kinase is postulated to account
for those findings. The other system uses no detergents, but requires
the phosphorylation of both p47PHOX and a membrane
component to activate the NADPH oxidase. The identity of the
phosphorylated membrane component is currently under investigation.
With kinase near, is phosphatase remote? Not likely, but studies of
phosphatases in relation to the leukocyte NADPH oxidase are lagging.
Three recent studies, all using whole neutrophils and inhibitors, have
implied that phosphatases are involved in the deactivation of activated
NADPH oxidase. Two studies showed an enhancement of oxidase activity
and O2 production in fMLP-activated
neutrophils treated with okadaic acid83 or calyculin
A,84 both of which are inhibitors of protein phosphatases 1 and 2A. The release of p47PHOX from the
cytoskeleton of activated neutrophils in response to protein kinase
inhibitors was prevented by both okadaic acid and calyculin
A.85 This interesting study suggests that the activity of
the oxidase is determined by the prevailing steady-state rates of
protein phosphorylation and dephosphorylation, with increases in
phosphorylation activating and increases in dephosphorylation deactivating the enzyme, lending further support to inferences that
could be drawn from a large number of earlier studies on protein
phosphorylation and oxidase activity.
Small Guanine Nucleotide Binding Proteins
Two low-molecular-weight guanine nucleotide binding proteins (G
proteins) have been implicated in the function of the leukocyte NADPH
oxidase: Rac2 (in mouse macrophages, Rac1) and Rap1A. Rac2, found
chiefly in the cytosol of resting neutrophils, is a member of the Rho
family of G proteins, a family known principally for its function in
regulating the cytoskeleton. Rap1A, located in the membranes of resting
neutrophils, is in the Ras family. Like Ras, it regulates cell
proliferation, acting as an antagonist of Ras-dependent
transformation.86
Most of the recent work on low-molecular-weight G proteins and the
NADPH oxidase has been performed on Rac2, probably because as a soluble
protein, Rac2 is much easier to deal with experimentally than Rap1A.
These studies focused on the protein-protein interactions that
developed between Rac2 and other NADPH oxidase components when the
oxidase was activated in a cell-free system using anionic detergents.
Incubation of neutrophil membranes with
p67PHOX-deficient cytosol under activating
conditions had been shown to cause p47PHOX to be
transferred to the membrane, but the reciprocal experiment with
p47PHOX-deficient cytosol did not lead to the
translocation of p67PHOX.87 In a
similar experiment but with recombinant proteins instead of whole
cytosols, the same laboratory showed that Rac2 also failed to
translocate.61 These findings suggest that during oxidase activation, the translocation of p47PHOX precedes
the translocation of both p67PHOX and Rac2. Using
chimeras between Rac1, which strongly activates the leukocyte NADPH
oxidase, and CDC42hs, a Rho-family G protein that barely activates the
oxidase, it was shown that residues 27 and 33 were particularly
important for oxidase activation.88 Both Rac1 and Rac2 were
shown to bind to p67PHOX but not to
p47PHOX. Both the effector region (residues
26-45) and the insert region (residues 125-145) of Rac1 were
important for oxidase activation by this G protein, but binding to
p67PHOX was only affected by mutations in the
effector region.2
Rap1A was first implicated in the function of the leukocyte NADPH
oxidase when it was found to copurify with cytochrome b558. Functional evidence for the participation of Rap1A in oxidase activation was not developed until much later, when it was shown using
a transfected Epstein-Barr virus (EBV)-transformed B-lymphocyte system
that both Rap1AS17N and Rap1AQ63E, which are
locked in the GDP-bound and GTP-bound conformations respectively,
inhibited phorbol-induced production of O2,
although the wild-type protein had no effect.89 The
observation that O2 production was inhibited
by both "locked" conformations of Rap1A raises the possibility
that the GTP-bound form carries the oxidase from "state 1" to
"state 2" with concomitant hydrolysis of the GTP, while the
GDP-bound form brings the oxidase back to "state 1" with the
concomitant exchange of GDP for GTP. Nothing is known about the nature
of these hypothetical states. Another ingredient in the mixture is the
finding that Rap1A can be phosphorylated by protein kinase A, and that
the phosphorylated form binds somewhat more weakly to cytochrome
b558 than the unphosphorylated form.90 Protein
kinase A is known to suppress the activity of the NADPH oxidase, and
the foregoing observations suggest that the kinase-regulated interaction between cytochrome b558 and Rap1A may be of
functional significance. On the other hand, the observations concerning
the phosphorylation of Rap1A were made in an in vitro system; there is
no evidence to date that Rap1A is phosphorylated in whole cells under
any circumstances.
A curious observation is the recent demonstration that suppressing
p120Ras-GAP biosynthesis by an antisense oligonucleotide
led to enhanced O2 production in
EBV-transformed B lymphocytes.91 Because
p120Ras-GAP binds to both Ras and Rap1A, the observed
effect could be a result of its interaction with one or both of these G
proteins. Another effect of p120Ras-GAP, however, is to
inhibit phorbol-induced cellular events.92 This seems to be
the likeliest explanation for the effect of the p120Ras-GAP
antisense oligonucleotide, because the increase in
O2 production was seen in phorbol-stimulated
cells but not fMLP-stimulated cells.
The Cytoskeleton, Integrins, and Tyrosine Kinases
The role of the cytoskeleton in the function of the leukocyte NADPH
oxidase has been recognized for some time. The two major pieces of
evidence supporting this idea are: (1) the finding that in activated
neutrophils, all the O2 producing activity
and portions of all the oxidase components are found in the cortical
cytoskeleton77,93; and (2) the demonstration that when
neutrophils adhere to a surface, a process that involves integrins and
is associated with far-reaching changes in the cytoskeleton, the time
course of O2 production becomes greatly
altered compared with the time course of O2
production in suspended cells.
Recent studies have provided more details regarding the incompletely
understood relationship between the cytoskeleton and the leukocyte
NADPH oxidase. In adherent neutrophils, stimulation with tumor necrosis
factor causes  2 integrins as well as the cytosolic
oxidase components to move slowly from the Triton-soluble to the
Triton-insoluble compartment, the latter generally regarded as
representing the neutrophil cytoskeleton.94
O2 is produced during this event, its time
course resembling that of the migration of proteins to the
cytoskeleton. A complicated series of steps involving a
2 integrin explains how antibodies and complement
cooperate in the activation of the leukocyte NADPH oxidase.95 When the activated complement protein iC3b
associates with the 2 integrin that serves as its
receptor, the Ig receptor Fc II§ becomes
attached to the cortical cytoskeleton. The occupation of the FcIII
receptor then allows the tyrosine phosphorylation of the cytoskeletally
associated FcII receptor to take place, triggering the signal
transduction pathway that leads to the activation of the leukocyte
NADPH oxidase (Fig 5).
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| Fig 5.
Mechanism of collaboration of the C3 receptor (a
2 integrin) and the FcIII receptor in the activation
of the leukocyte NADPH oxidase.95 In the resting membrane,
the C3, FcII, and FcIII receptors are empty. (1) Occupancy of the
C3 receptor causes the FcII receptor to bind to the cytoskeleton.
(2) Occupancy of the FcIII receptor activates a tyrosine kinase. (3)
The activated tyrosine kinase phosphorylates the cytoplasmic portion of
the FcII receptor, which was rendered susceptible to phosphorylation
through its interaction with the cytoskeleton. (4) The phosphorylated
FcII receptor activates the oxidase via a multistep signal
transduction pathway.
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The same laboratory that worked out antibody-complement cooperation has
also identified the "leukocyte response integrin
(LRI)."96 This protein, which recognizes the basement
membrane protein entactin, acts in association with another
membrane protein, the "integrin-associated protein." This
combination of proteins activates the leukocyte NADPH oxidase when one
of themthe LRIinteracts with entactin, or with the peptide KGAGDV
(Fig 6). O2
production activated by the LRI/integrin-associated protein (IAP) combination occurs in neutrophils deficient in CD18, the common subunit
of the 2 integrins, ruling out the participation of
these more traditional leukocyte integrins in the response of the
neutrophil to the LRI/IAP system.
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| Fig 6.
Provisional mechanism for the activation of the NADPH
oxidase by the LRI. The LRI and the IAP are thought to form a complex
in the leukocyte plasma membrane. When leukocytes are in suspension,
the signal transduction pathway between this complex and the NADPH
oxidase is blocked. The blockade is lifted by the following combination
of events: (1) the binding of LRI to entactin, a basement membrane
protein; (2) adhesion of the phagocyte to a surface (in suspended
cells, NADPH oxidase is not activated by the binding of LRI to
entactin); and (3) possibly a conformational change in IAP. These
events somehow activate protein kinase C, which then activates the
oxidase. Tyrosine phosphorylation does not participate in oxidase
activation by LRI/IAP.96
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Associations between particular cytoskeletal proteins and the NADPH
oxidase have also been described. The human counterpart of
coronin, a protein important for motility in
Dictyostelium, assocoates with p40PHOX and
accumulates around phagocytic vesicles.97 Cofilin
loses its phosphate and moves to ruffled membranes when neutrophils are
stimulated with fMLP. Oxidase components also migrate to the ruffled
membranes under those conditions, and O2
production is stimulated with kinetics that resemble the kinetics of
cofilin dephosphorylation, implying the possibility of some connection
between cofilin and the oxidase.98 Stimulation of the
oxidase in a detergent-activated cell-free system is augmented by G
actin.99
Finally, some very interesting studies relating neutrophil tyrosine
kinases to the activation of the NADPH oxidase have recently appeared
from Berton's laboratory.94,100,101 In their initial studies, this group showed that tumor necrosis factor, which activates the NADPH oxidase in adherent cells but not in suspended cells, caused
the Src family tyrosine kinase p58fgr to move from the cytosol to the
cytoskeleton. Neutrophils from knockout mice deficient in both p58fgr
and p59/61hck, another tyrosine kinase in the Src family, were unable
to produce O2 in the course of their
attachment to surfaces coated with collagen or fibronectin, though
O2 production in response to immune
complexes and phorbol myristate acetate was normal. These results
indicate that the trigger responsible for the adhesion-dependent
activation of fgr and hck, and their subsequent actions on the oxidase,
was 2 integrin. Conversely, reactive oxidizing species
were found to be required for the activation of the tyrosine kinases
p58fgr and p53/56lyn that takes place when neutrophils adhere to
surfaces, a result which indicates that at least to some extent, the
activation of these kinases is a bootstrap operation with autocatalytic features.
CGD.
CGD is an inherited immune deficiency in which phagocytes from affected
patients are unable to manufacture O2. All
cases so far have been found to result from a deficiency of one of 4 oxidase-specific proteins: p47PHOX,
p67PHOX, p22PHOX, or
gp91PHOX. Virtually all the recent work on the
oxidase in CGD has been directed toward developing a genetic cure of
the disease.
B lymphocytes and hematopoietic progenitors from patients with various
forms of CGD have been cured by transfection with vectors expressing
the oxidase component missing from the patients' cells. Various
retroviral vectors have been used to correct CGD in cells from patients
with the X-linked form of the disease, who are missing gp91PHOX. In the earliest study, the respiratory
burst was restored in 15% of transfected human myeloid cells, with the
active cells producing O2 at about 60% of
the normal rate.102 Using a different vector, the same
group transfected a CGD myeloid line as well as marrow cells from a
knockout mouse with gp91PHOX
deficiency.103 Results with the new vector were greatly
superior to results obtained previously, with oxidase activity being
largely to completely restored in both human and mouse cells.
CD34+ cells from a p67PHOX-deficient
patient were cultured after transfection with a replication-defective amphotropic retrovirus that expressed p67PHOX.
O2 production was restored in neutrophils
arising from the transfected cells.104
Two groups of investigators have cured CGD in vivo by genetic methods,
but only in mice. Knockout mice lacking gp91PHOX
were transplanted with their own marrow cells after transfection with a
murine stem cell virus vector that expressed the missing protein.105 O2 was expressed by
neutrophils from these transplanted mice, though at levels lower than
seen in wild-type mice. Despite the fact that neutrophil
O2 production was only partly corrected in
the transplanted mice, these mice withstood a challenge with
Aspergillus fumigatus that produced pneumonia in all the
untransplanted CGD mice. Similar results were obtained in
p47PHOX knockouts that were transplanted with stem
cells transfected with retroviral vectors that expressed
p47PHOX,106 except that in these mice
the challenge was with Burkholderia cepacia, a typical pathogen
of patients with CGD, though normally its activities are confined to onions.
| |
NADPH OXIDASE IN NONPHAGOCYTES |
Like all other biological macromolecules and macromolecular assemblies,
the leukocyte NADPH oxidase was created in the course of evolution. In
considering its possible origin, it was hard to believe that an enzyme
this lethal could have arisen de novo. With this in mind, it was
speculated that the oxidase might have arisen through the mutation of a
more ancient NADPH oxidase of much lower activity whose tissue
distribution was much more widespread than that of the lethal leukocyte
oxidase and whose principal function was to provide oxidants for
signaling purposes.107 Recent findings have confirmed this
speculation, showing clearly that a low-activity NADPH oxidase is
present in a variety of nonphagocytic cells, most of which are derived
from the embryonic mesoderm, and that this oxidase is a source of
second messengers.
An NADPH oxidase similar to the one found in phagocytes has been
reported to occur in all three layers of the aorta.
O2 was generated in endothelial cell
sonicates to which NADPH was added. In addition, mRNA for the four
specific oxidase subunits and protein for the two cytosolic subunits
were found, but no heme protein was detected by
spectroscopy.108 Human aortic smooth-muscle cells produced
O2 in response to platelet-derived growth
factor (PDGF). This O2 production was
inhibitable by diphenylene iodonium, suggesting that a phagocyte-like
NADPH oxidase was responsible for its production. In these cells, the
activation of NF- B by PDGF was dependent on
O2, but its activation by interleukin-1
was not.109 In fibroblasts from aortic adventitia,
O2 production occurred
constitutively.110 Its rate of production was increased by
angiotensin II110,111 but not by
norepinephrine.111 It was postulated that the
O2 generated by the aorta was functioning as
a blood pressure regulator by consuming nitric oxide, a well-known
hypotensive agent with which it reacts at a diffusion-limited
rate.110,111
In joint tissues, O2 production has been
detected in synoviocytes (both type A and type B)106 and in
chondrocytes.112 In synoviocytes,
O2 production is induced by phorbol
myristate acetate, suggesting that it is generated by an NADPH oxidase.
In chondrocytes, O2 production is elicited
by a calcium ionophore, but not by phorbol myristate acetate. In these
cells O2 production is inhibited by
diphenylene iodonium, and mRNA for p22PHOX,
p40PHOX, and p47PHOX (or their
homologs) was detected by reverse transcriptase-polymerase chain
reaction, relatively strong evidence that O2
from chondrocytes is produced by an NADPH oxidase like that in phagocytes.
There is evidence that NADPH oxidases serve as components of oxygen
sensors in various tissues. Erythropoietin production in some hepatoma
lines appears to be regulated by O2, and
p22PHOX, the  -subunit of cytochrome
b558, has been detected immunologically in renal
peritubular fibroblasts and in Ito cells of the liver, both of which
may be sources of erythropoietin.113 In the lung, pulmonary
neuroepithelial bodies have been proposed as airway oxygen
sensors.114 The cells of these organs contain mRNAs
corresponding to a subunit of a H2O2-regulated
potassium channel as well as messages for p22PHOX
and gp91PHOX, the two membrane-associated oxidase
subunits, or their homologs. H2O2 production by
the neuroepithelial body cells was constitutive, but was stimulated by
phorbol myristate acetate and inhibited by diphenylene iodonium.
(Inhibition of O2 or
H2O2 production by diphenylene iodonium is
generally regarded as presumptive evidence for an NADPH oxidase.)
Furthermore, the K+ current in these cells increased when
the cells were exposed to H2O2. These results
suggest that the cells contain an NADPH oxidase whose output is a
function of the ambient oxygen tension and whose dismuted product (ie,
H2O2) regulates the flow of current through
the K+ channel, the latter representing the signal by which
the oxygen tension is communicated to the rest of the organism.
Plants contain an NADPH oxidase that they use for host defense. Cells
from suspension cultures of Arabidopsis thaliana produced O2 when activated with phorbol myristate
acetate. Harpin, a bacterial elicitor protein,115
had the same effect, eliciting the production of
O2 from the cultured cells. The
Arabidopsis cells were found to contain proteins that were
recognized by antibodies against human p47PHOX and
p67PHOX, and membranes from the plant produced
O2 when incubated with human neutrophil
cytosol under activating conditions.
Finally, a b-type cytochrome homologous to human
gp91PHOX was discovered in yeast.116
This protein is an iron reductase that contains a low-potential heme
and sequences homologous to flavin-binding sequences in other proteins.
Binding of the heme to the apoprotein requires the presence of four
essential histidine residues, because the conversion of any of these
essential histidines to al |
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Introduction
References