In response to a wide range of stimuli, neutrophils and other
microbicidal phagocytes undergo a respiratory burst in which molecular
oxygen is converted to superoxide (O
) (
)and other reactive species(1) . The enzyme
responsible for this activity, NADPH oxidase, is localized to the
plasma membrane of the cells, which also forms the phagolysosomal
membrane. The function of this oxidase system in phagocytes is to
participate in the destruction of bacteria, fungi, parasites,
virus-infected cells, and tumors by directing the reactive oxygen
species against the target cells. In order to respond to a wide range
of dissimilar stimuli and to minimize damage to normal tissues, the
neutrophil has evolved a complex signal transduction mechanism to
regulate NADPH oxidase. This signaling pathway is not yet fully
understood. In part, the oxidase is regulated by separation of
component parts in different cellular localizations. Thus, assembly of
the oxidase at the plasma membrane, promoting electron transfer between
the redox components of the oxidase, is one of the important steps in
the activation process. An essential component of this oxidase system
is a unique low potential b-type cytochrome, cytochrome b
(also known as cytochrome b
or cytochrome b
)(2) . The cytochrome is a
heterodimeric protein consisting of a 22-kDa
- subunit, termed
p22
(phagocyte oxidase), and a heavily
glycosylated 91-kDa
- subunit termed gp91
.
Both a flavin and a heme redox center are probably contained within the
cytochrome heterodimer (3, 4) . Electrons supplied by
the donor, NADPH, are believed to be transferred to oxygen as shown
below.

We have recently provided evidence for the direct transfer of
electrons from the reduced flavin center to the dye acceptor INT and
have suggested that autoxidation of the reduced flavin results in low
levels of intracellular hydrogen peroxide production in some forms of
CGD(5) . In this inherited disorder, there is a failure of the
phagocytic cells to generate O
. Affected
individuals suffer from severe recurrent infections due to the
inability of the phagocytes to effectively kill infecting
micro-organisms. Since the realization that defects in NADPH oxidase
are the cause of CGD, it has proved possible to identify four essential
polypeptide components of this system, defects in any one of which can
lead to the clinical phenotype of CGD. Individuals with genetic lesions
in either gp91
(type X91° CGD) or
p22
(type A22° CGD) usually have phagocytes
that do not express detectable levels of either cytochrome polypeptide,
suggesting that the individual subunits are unstable in the absence of
their partners(6, 7) . In a very few cases, missense
mutations have resulted in normal levels of a spectrally normal, but
nonfunctional cytochrome b
(type
X91
CGD) or a decreased abundance of a fully or partly
functional cytochrome b
(X91
CGD)(8) .
In addition to the membrane-bound redox
centers of NADPH oxidase, there are at least three other components
required for the fully active enzyme that are present in the cytosolic
fraction of disrupted neutrophils. Defects in the genes coding for two
of these proteins, p47
and
p67
, result in two of the recognized forms of
CGD(8) . The third cytosolic factor is the low molecular weight
GTP-binding protein, Rac. Initially, Rac1 was isolated from guinea pig
macrophages (9) and Rac2 from human neutrophils(10) ,
based on their ability to substantially augment cell-free
O
production. Subsequent studies using
wholly or partially recombinant systems have shown that Rac (either
form) is absolutely required for NADPH oxidase activity.
Here we
describe a new patient (patient A) with neutrophils that express normal
levels of a cytochrome that is non functional in terms of
O
production, but retains normal
dye-reductase activity. By comparison with another patient (patient B)
previously described by Dinauer et al. (patient RC in (11) ) whose neutrophils express a cytochrome b
in which there is a Pro
His substitution in gp91
, we can
infer that patient A has a mutation affecting electron transfer
processes subsequent to flavin reduction, whereas patient B has a
mutation affecting the initial electron transfer processes preventing
enzyme flavin reduction.
EXPERIMENTAL PROCEDURES
Preparation of Neutrophils and Their Subcellular
Fractions
For experiments with intact neutrophils (including
translocation studies) blood was collected by venipuncture and the
neutrophils purified by dextran sedimentation, hypotonic lysis of
erythrocytes, and centrifugation through Ficoll-Hypaque(12) .
For the preparation of subcellular fractions neutrophils were obtained
from normal subjects and CGD patients by leukapheresis (13) and
purified as described above with the omission of the dextran
sedimentation step. Neutrophils were treated with 2.5 mM diisopropyl fluorophosphate for 10 min at 4 °C, disrupted in
relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl
, 1 mM ATP, 1.25 mM EGTA, 10
mM PIPES, pH 7.3) (14) by N
cavitation,
and fractionated on discontinuous Percoll gradients. These methods,
described in detail elsewhere(15, 16) , produce
cytosol and plasma membrane fractions whose final concentrations were
adjusted to 9
10
and 1.25
10
cell equivalents (cell eq)/ml, respectively. Fractions were
stored at -80 °C for up to 1 year without loss of activity.
Translocation of Cytosolic Oxidase
Components
Experiments to study the translocation of
p47
and p67
from the soluble to the
particulate neutrophil fractions were performed exactly as described by
Heyworth et al.(17) , using PMA (100 ng/ml) as the
stimulus.
Gel Electrophoresis and Protein
Immunoblotting
Neutrophil fractions were subjected to
SDS-polyacrylamide gel electrophoresis on homogeneous 10 or 13%
acrylamide gels (see figure legends) with prestained Rainbow protein
markers (Amersham Corp.) as molecular mass standards. Separated
proteins were electrophoretically transferred to PVDF membrane
(Bio-Rad) in a Transblot cell (Bio-Rad). Nonspecific binding sites were
blocked with 3% bovine serum albumin, 10% goat serum in HEPES-buffered
saline and the blots probed with antibodies to the appropriate subunits
of NADPH oxidase. Antisera to the Phox proteins were raised in rabbits
using synthetic peptides (coupled to keyhole limpet hemocyanin)
corresponding to amino acid residues 340-355 of p47
(RPGPQSPGSPLEEERQ), 437-450 of p67
(DEPKESEKADANNQ), 153-170 of p22
(SNPPPRPPAEARKKPSEE), and 537-556 of gp91
(CGPEALAETLSKQSISNSES). The antisera to p47
and
p22
were used without further purification at dilutions
of 1:2000 and 1:1000 respectively. Antibodies to p67
and
gp91
were affinity-purified on Affi-Gel columns
(Bio-Rad) to which the cognate peptides had been coupled. A goat
anti-rabbit IgG antibody-alkaline phosphatase conjugate (1:1000
dilution) (Bio-Rad), together with 5-bromo-4-chloro-3-indolyl phosphate
and nitro blue tetrazolium substrates (Bio-Rad) were used to detect
bound primary antibody. The protein content of neutrophil membrane
fractions was estimated using the BCA microassay (Pierce) with bovine
serum albumin as a standard.
Functional Assays
Whole cell
O
production was determined
spectrophotometrically by the superoxide dismutase-inhibitable
reduction of cytochrome c(15) . NBT slide tests were
performed as described previously(18) . Intracellular
production of oxidants was assayed using a flow cytometric assay of
dichlorofluorescein fluorescence, using the method described by Bass (19) with minor modifications. Briefly, neutrophils were
preincubated at a cell density of 10
/ml for 15 min at 37
°C in phosphate-buffered saline (138 mM NaCl, 2.7 mM KCl, 8.1 mM Na
HPO
, 1.47 mM KH
PO
) supplemented with glucose (5
mM) and gelatin (0.1%) containing 5 µM DCFH-DA.
Tubes were mixed every 5 min. Stock solutions (5 mM) of
DHFH-DA (Eastman Kodak Co.) in EtOH were prepared fresh daily and kept
in the dark at room temperature. After preincubation, catalase was
added (275 units/ml), and the samples were divided into two 1-ml
aliquots. PMA (100 ng/ml in Me
SO) was added to one tube and
Me
SO to the other. After 10- and 20-min incubation at room
temperature, samples were read on a FACScan (Becton Dickinson, San
Jose, CA) equipped with a 15-milliwatt argon laser and FACScan Research
Software. 10,000 events were collected; cellular aggregates and debris
were excluded by gating on the neutrophil population. Mean channel
fluorescence was calculated for each sample using a linear scale, the
unstimulated (+Me
SO) control values were subtracted
from the stimulated (+PMA) fluorescence.
Biochemical Assays
INT reductase activities of
NADPH oxidase activated in the cell-free system were assayed
spectrophotometrically in 96-well microtiter plates using a kinetic
plate reader as described previously(5) .
O
assays were performed on membranes
prepared from PMA-activated neutrophils and on membranes and cytosols
prepared from unstimulated neutrophils using the cell-free activation
system in microtiter plates by determination of the superoxide
dismutase-inhibitable rate of cytochrome c reduction(20) . These assays were also used to determine
the Michaelis parameters for NADPH oxidase.
Genetic Analysis
Molecular genetic analyses were
undertaken as described previously using genomic DNA extracted from 3
ml of whole blood(21) . Details of these methods will be
published fully elsewhere. (
)
Spectroscopy
Reduced minus oxidized
difference spectra were recorded as described previously using 2% (w/v)
Triton X-100 extracts of whole neutrophils (15) or 2% (w/v) OG
extracts of neutrophil plasma membranes that had been washed with 1 M NaCl to remove myeloperoxidase(22) . CO difference
spectra were recorded by reduction of a detergent extract of membranes
with a few grains of sodium dithionite, storing the spectrum in the
memory of the spectrophotometer, then gently bubbling the contents of
the cuvette with CO for 60 s (one to two bubbles/s through a fine
pipette attached to the gas line) and re-recording the spectrum. The
subtraction of the original trace results in the (reduced + CO)
minus (reduced) spectrum. Reoxidation of reduced cytochrome b
by oxygen was assessed by reducing an
aliquot of detergent-solubilized membranes with a slight excess of
crystalline sodium dithionite (0.5 mg), recording the absorbance
spectrum, and reoxidizing the reduced flavin and cytochrome b
by gentle end over end mixing of the
contents of the cuvette to introduce atmospheric oxygen; after 30-s
agitation the spectrum was re-recorded to determine changes in the
degree of cytochrome b
reduction.
NADPH-dependent reduction of FAD and cytochrome b
in 2% OG extracts of neutrophil plasma
membranes after cell-free activation of NADPH oxidase activity was
determined under conditions of reduced oxygen tension using the
galactose and galactose oxidase system described
previously(23) .
RESULTS
Patient A had been diagnosed with CGD at 7 years of age
following a history of recurrent infections and an abnormal NBT slide
test. The family history was highly suggestive of X-linked inheritance.
On evaluation at Scripps Clinic, no O
production could be detected from the patient's neutrophils
using either PMA or formylmethionylleucylphenylalanine as the stimulus,
confirming the clinical diagnosis of CGD.
NBT Slide Test
NBT slide tests performed on normal
subjects show 98-100% of PMN strongly staining with purple NBT
formazan after stimulation of the cells with PMA(8) . Typical
CGD patients (both X-linked (X91° and X91
,
including patient B) and autosomal recessive forms (A22°, A47°,
and A67°)) show no detectable NBT staining. In contrast,
neutrophils and monocytes from patient A displayed a uniform population
(98-100%) of weakly staining cells (not shown). This staining
pattern is similar to that observed in a very few other variant
X-linked CGD patients (X91
CGD), with diminished
levels (3-25%) of a functional cytochrome b
. (
)Unlike patient A, these
X91
CGD patients have neutrophils that are capable of
diminished levels of O
production that
broadly correlate with their degree of cytochrome expression.
DCFH-DA Fluorescence
Measurements of intracellular
oxidant production using the fluorescent probes DCFH-DA and rhodamine
123 have shown that in marked contrast to patients with mutations in
gp91
or p22
in which cytochrome b
is missing (X91° or A22° CGD)
and who have essentially no production of intracellular oxidants,
neutrophils from patients with p47
deficiency have
levels of intracellular oxidant production intermediate between
cytochrome b
-deficient patients and
normal controls. (
)The reactive species responsible for the
oxidation of the DCFH is probably H
O
. It should
be emphasized that with neither cytochrome-deficient nor
p47
-deficient CGD neutrophils can any extracellular O
or H
O
be
detected. Analysis of intracellular oxidant production in the
neutrophils of patient A gave a high intermediate fluorescence value of
85 after 10 min, and 231 after 20 min, PMA stimulation, compared with
the normal control of 246 and 495 at 10 and 20 min, respectively (Table 1). These values are higher than that observed in
p47
deficient CGD patients (35 and 129 at 10 and 20 min,
respectively). In contrast, the PMN of patient B gave a low mean
fluorescence values of 10 at 10 min and 55 at 20 min, similar to the
values of 7 at 10 min and 16 at 20 min observed in cytochrome-deficient
X91° CGD patients (Table 1).
Localization of the Defect in Patient A to the PMN
Membrane
In order to define more closely the type of CGD,
cell-free O
assays were performed in
microtiter plates using combinations of membranes and cytosol from
patient A and normal controls. By these means the defect in patient A
was localized to the membranes, since the neutrophil cytosol from the
patient A could support O
production in
normal membranes, whereas the neutrophil membranes from patient A were
incapable of O
generation under any
conditions (see below).
Effects of Increasing NADPH Concentration
The
mutation in patient B forms part of the region postulated to be the
NADPH-pyrophosphate binding loop(3) . We hypothesized that this
mutation might increase the K
of the oxidase for
NADPH to such an extent that no activity would be seen in whole PMN
(where the NADPH concentration is 34 µM(24) ) or
under the conditions normally employed in the cell-free assay (160
µM NADPH). However, no superoxide dismutase-inhibitable
cytochrome c reductase activity could be obtained at
concentrations of NADPH up to 3 mM using membranes from either
patient A or B. Above this concentration the rate of superoxide
dismutase-insensitive activity made estimations of
O
-dependent activity unreliable.
Cytochrome b
Spectroscopy
Localization of the defect in patient A to
the neutrophil membrane suggested a genetic lesion in one of the genes
encoding cytochrome b
(either
p22
or gp91
). Most commonly such
mutations result in the absence of cytochrome, but reduced minus
oxidized difference spectra of Triton X-100 extracts of neutrophils
isolated on three different occasions from patient A, disclosed the
presence of normal quantities of cytochrome b
(51.8, 68.6, and 62.7 pmol/10
cells, compared with
59.8, 67.3, and 63.7 pmol/10
cells for normal controls
processed in parallel). On first inspection, the absorbance maxima of
the patient's cytochrome appeared grossly normal, but it was
noted that the Soret band of the reduced minus oxidized spectrum was
shifted slightly toward the red. In order to examine the cytochrome
spectrum in more detail, 2% OG extracts were made of neutrophil plasma
membrane fractions, and the absolute oxidized, absolute reduced (Fig. 1B) and reduced minus oxidized (Fig. 1A) difference spectra determined. The Soret band
of the oxidized cytochrome b
of patient A
is shifted 1 nm hypsochromically from 414.6 to 413.6 nm. The reduced
spectrum is shifted bathochromatically, from 426 to 426.5 nm. As a
result, the Soret peak and trough are shifted in the reduced minus
oxidized difference spectrum from 427 to 427.5 nm (peak) and from 412
to 411.6 nm (trough). No differences in absorption maxima were observed
for the
(558 nm) or
(529 nm) bands. The ratio of cytochrome b
Soret/
absorbance was not
significantly altered in the patient. In contrast, patient B (with a
Pro
His mutation in gp91
) had no
apparent abnormalities in any of the cytochrome b
visible absorption spectra (Refs. 11,
25, and 26 and this work). Cytochrome b
has been shown to bind CO with low affinity(27) . As a
further probe of the heme environment of the two mutant cytochromes b
, the ability of each cytochrome to form
a complex with CO was compared with that of normal cytochrome b
. In all three cases, CO was bound
weakly with 30-40% of the cytochrome in each sample forming a CO
complex (not shown). As a further test of cytochrome function, the
ability of the reduced cytochrome to become reoxidized by atmospheric
oxygen was approximated as described under ``Experimental
Procedures.'' In all cases, cytochrome b
was fully reoxidized within 30 s of mixing in air.
Figure 1:
A, reduced minus oxidized difference
spectra of normal and X91
CGD membranes. OG extracts
were made of neutrophil plasma membranes, and reduced minus oxidized
difference spectra were recorded as described under ``Experimental
Procedures.'' Solid line, solubilized membranes from a
normal donor; dashed line, solubilized membranes from patient
A. Each spectrum contains approximately 290 nM cytochrome b
and 180 nM FAD. The dashed
spectrum has been offset for clarity. B, absolute
absorption spectra of normal and patient A X91
CGD membranes. Absolute oxidized (solid and short dashed traces) and absolute dithionite reduced spectra (dotted and long dashed traces) were recorded of
normal (solid and dotted traces) and X91
CGD neutrophil membrane extracts from patient A (dashed
traces) as described under ``Experimental Procedures.''
The cytochrome b
concentration was 290
nM.
Immunocytochemistry
Immunochemical methods
confirmed the presence of cytochrome b
in
the neutrophils of CGD patient A and patient B (with the Pro
His mutation in gp91
). As shown in Fig. 2, neutrophil plasma membrane fractions from both patients
contained both the p22
and gp91
subunits
of the cytochrome. Although the amount of cytochrome present in the
patients neutrophils and plasma membranes was normal as determined by
visible spectroscopy of the heme (see above and Table 1), and the
same preparations of membranes were used for both spectroscopic
determination of cytochrome b
and
immunostaining, the apparent intensity of the immunostaining of the
gp91
bands in both patients A and B was unexpectedly
decreased. The peptide used to raise the gp91
antibody
used in these experiments did not correspond with the regions of either
mutation (see below and ``Experimental Procedures'').
Figure 2:
Protein immunoblots showing presence of
flavocytochrome b
subunits in neutrophil membranes
from CGD patients A and B. Neutrophil plasma membranes isolated from:
normal control (lane 1), flavocytochrome b
-deficient CGD patients (lane
2) (p22
-deficient in A and
gp91
-deficient in B), CGD patient B (lane 3), CGD patient A (lane 4) (all 10 µg of
protein/well) were subjected to SDS-polyacrylamide gel electrophoresis
on 13% (A) and 10% (B) acrylamide gels. After
electrophoretic transfer of the proteins to PVDF, the blots were probed
with antipeptide antibodies to either p22
(1:1000 dilution of antiserum) (A) or
gp91
(affinity-purified antibody) (B).
The location and molecular mass (kDa
10
) of
the marker proteins are indicated to the left of each blot.
The glycoprotein gp91
runs as a broad band as
indicated.
Genetic Analysis
Based on the localization of the
defect in patient A to the membrane, the shifts in the Soret absorbance
band of the cytochrome b
spectrum, and
the X-linked inheritance of the disease, we considered the underlying
mutation in patient A was most likely to reside in the gene for
gp91
. Consequently, genomic DNA from the patient was
sequenced revealing a single base substitution of a cytosine for
guanine 173 in exon 2 of gp91
(AGG
ACG). The
predicted effect of this mutation is the nonconservative replacement of
arginine 54 with a serine residue. This is unlikely to be a
polymorphism as it has not been observed in more than 200 normal and
CGD patients alleles evaluated at Scripps. No other abnormalities in
the sequence were detected. The patients mother and two sisters were
found to be heterozygous for the mutation, in keeping with their NBT
test results as reported to us, other family members; including another
sister, had normal gp91
sequences and NBT tests.
Translocation of p47
and p67
during Activation
We have previously shown that the
absence of cytochrome b
, in both the
X91° and A22° forms of CGD, prevents the association of both
p47
and p67
with the plasma membrane
fraction of neutrophils(17) . In the case of patient A we were
interested in determining if the Arg
Ser mutation,
which leads to normal levels of nonfunctional cytochrome b
, disrupted the assembly of cytosolic
components at the membrane. As shown in Fig. 3, translocation of
p47
and p67
was normal in patient A, with
both proteins appearing in the particulate (pellet) fraction upon
stimulation of the normal and CGD neutrophils with PMA. Thus the
mutation that renders the cytochrome inactive does not appear to
disrupt the interaction with other oxidase components.
Figure 3:
Difference absorption spectra showing the
reduction of cytochrome b
and flavin in
detergent-solubilized membranes after the cell-free activation of NADPH
oxidase. Membrane extracts, cell-free activation of NADPH oxidase, and
spectrophotometry were performed as described under ``Experimental
Procedures.'' in the presence of galactose and galactose oxidase. a, absorption spectrum obtained from mixing normal cytosol and
normal membrane extract. b, absorption spectrum obtained from
mixing X91
CGD membrane extract from patient A with
normal cytosol.c, absorption spectrum obtained from
mixing X91
CGD membrane extract from patient A with
p47
-deficient cytosol. d,
dithionite-reduced minus oxidized difference absorption spectrum of 14
nM FAD.
Dye Reductase (Diaphorase) Activity
We have
recently described a dye (INT) reductase (diaphorase) activity
associated with NADPH oxidase (5) and have provided evidence
that electrons are transferred to the dye at the level of the FAD redox
center(5, 23) . INT diaphorase activity was absent
from the membranes of patients with X91° or A22° forms of CGD
(in which there is an absence of cytochrome b
heme and a marked decrease or absence of flavin). We have
examined the ability of the two X91
CGD patients
neutrophil membranes to support INT reductase activity in the cell-free
activation system (Table 1). The X91
patients A
and B (who have normal levels of both cytochrome b
and flavin) differ with regard to
diaphorase activity, patient A has completely normal activity, whereas
patient B has close to zero activity, typical of that of
cytochrome-deficient membranes (Table 2). The cytosol from both
patients was capable of supporting normal levels of dye-reductase
activity in normal membranes, demonstrating the defect was
cytochrome-associated. This suggests that the mutation in each of these
patients affects different points in the pathway of electron flow,
despite the fact that they affect the same (gp91
)
subunit of cytochrome b
. We have recently
provided evidence that electrons are transferred to INT from the FAD
center of NADPH oxidase(5) . Thus, it appears that in patient
B, electrons from NADPH cannot be transferred to the flavin, whereas in
patient A, the flavin center can become reduced and electrons can be
transferred to INT, but electrons cannot flow to oxygen. This could
arise either because cytochrome b
does
not become reduced or because the reduced cytochrome does not react
with oxygen. In order to resolve this question we examined the ability
of the heme and flavin redox centers of the patients' cytochrome b
to undergo reduction in the cell-free
activation system in the presence of NADPH, using the methods we have
developed previously(23) . In accord with the results of the
INT diaphorase experiment, in patient A the NADPH oxidase-associated
flavin underwent reduction in the cell-free activation system in the
presence of NADPH (Fig. 4). In contrast, there was no observable
reduction of flavin in the case of patient B (not shown). In neither
patient was there any evidence for the reduction of cytochrome b
, unlike that observed in extracts of
normal membranes (Fig. 4, trace a). We have recently
provided evidence that p67
and p47
have
individual roles in controlling electron flow in NADPH oxidase. We
showed that the presence of p67
is sufficient, in the
absence of p47
, to activate NADPH-oxidase to a state
were electrons reduce the flavin center of the oxidase, but
p47
was required for electron transfer to continue to
the heme center. Consistent with that proposal, we found that
p47
-deficient cytosol was capable of inducing electron
flow into the flavin redox center of NADPH oxidase in extracts of the
neutrophil membranes of patient A (Fig. 4, trace c).
Figure 4:
Translocation of p47
and p67
upon stimulation of
neutrophils from a normal control and an X91
CGD
(patient A). Neutrophils from a normal control and CGD patient A were
incubated for 5 min with either PMA (+) or an equal volume of
Me
SO carrier(-), disrupted by sonication, and
fractionated as described under ``Experimental Procedures.''
Cytosol (1.5
10
cell eq) and particulate (pellet)
(4.5
10
cell eq) fractions were subjected to
SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels, the
proteins transferred to PVDF membrane, and the blots probed with
antibodies to p47
and
p67
. The location and molecular mass (kDa
10
) of the marker proteins are indicated on
the left of the figure, and p47
and
p67
are identified on the right.
DISCUSSION
The results reported above identify mutations in
gp91
that give insight into the functions of particular
regions of the protein. The Arg
Ser mutation in
patient A is in a region of gp91
that is predicted to be
on the cytoplasmic side of the membrane, close to a transmembranous
domain. The result of this mutation appears to mainly effect the
function of the heme moiety of cytochrome b
rather than the NADPH-binding or flavin-binding regions since: (a) there is a subtle shift in the optical absorbance
properties of the heme; (b) the heme fails to become reduced
on activation but the flavin is reduced normally; (c) the
flavin is functional with regard to INT diaphorase activity; and (d) there is a high level of DCFH-DA fluorescence (possibly by
autoxidation of reduced flavin generating intracellular
H
O
as we have recently proposed).
The ability of the FAD to donate electrons to external acceptors
is probably the cause of the slight positive staining observed in the
NBT slide test of patient A. We (and others) have previously speculated
that at least part of the NBT reduction seen in the NBT slide test,
although NADPH-oxidase dependent, is
O
-independent, since NBT reduction is
only partly inhibited by saturating concentrations of superoxide
dismutase or under anaerobic
conditions(28, 29, 30) . The finding that the
PMNs of patient A display a degree of NBT reduction in the absence of
O
production supports this proposal. In
contrast to patient A, patient B has a mutation that appears to
principally effect the flavin and/or NADPH-binding domains, since (a) the physicochemical properties of the heme appear normal
with regard to spectrum, redox potential, and CO binding ( (26) and this work); (b) the flavin does not become
reduced on activation of NADPH oxidase; (c) no diaphorase
activity is seen with INT (or NBT staining in the slide test); and (d) the intracellular oxidant production measured by DCFH-DA
fluorescence is low. These findings are consistent with the proposal
that the mutation in this patient is within a NADPH-pyrophosphate
binding loop that was identified by comparison of the sequence of
gp91
with the sequences of enzymes of the ferredoxin
NADP
reductase
class(3, 4, 31) .
The experiments
described above support the proposal that NADPH-derived electrons do
not reduce the flavin center of patient B, and electrons cannot pass
from the flavin to cytochrome b
in
patient A. The correlation of INT reductase activity with the ability
of NADPH oxidase-associated FAD to become reduced in neutrophil
membrane extracts from patient A is strong confirmatory evidence that
the diaphorase activity we have previously identified is due to
electron transfer from the reduced FAD to INT. Furthermore, the ability
of p47
-deficient cytosol to permit electrons to reduce
the flavin in this patient provides further support for our proposal
that p47
and p67
have separate roles in
the control of electron flow in NADPH oxidase.
Recently, another
X91
CGD patient was reported with a mutation resulting
in the substitution of a glycine residue for Asp
toward
the C terminus of gp91
(32) . The functional
defect in this patient apparently arises from the failure of the
cytosolic factors to translocate to the membrane during the activation
process. The C terminus of gp91
has previously been
implicated as being essential for translocation of the cytosolic
factors p67
and p47
and activation of
NADPH oxidase(17, 33, 34) . Translocation of
these factors appeared to be completely normal in patients A and B.
Thus the study of three naturally occurring mutations in gp91
has led to the identification of three separate functional
domains within cytochrome b
.