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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2098-2104
Missense Mutations in the gp91-phox Gene Encoding Cytochrome
b558 in Patients With Cytochrome b Positive
and Negative X-Linked Chronic Granulomatous Disease
By
Mizuho Kaneda,
Hitoshi Sakuraba,
Akira Ohtake,
Akira Nishida,
Chika Kiryu, and
Katsuko Kakinuma
From the Departments of Inflammation Research and Clinical Genetics,
The Tokyo Metropolitan Institute of Medical Science, Tokyo; the
Department of Clinical Pathology, Kitasato University School of
Medicine, Kanagawa; the Department of Veterinary Clinical Sciences,
Graduate School of Veterinary Medicine, Hokkaido University, Sapporo;
and the Biophotonics Information Laboratories, Yamagata Advanced
Technology Research Center, Yamagata, Japan.
 |
ABSTRACT |
Chronic granulomatous disease (CGD) is a disorder of host defense
due to genetic defects of the superoxide (O2-)
generating NADPH oxidase in phagocytes. A membrane-bound
cytochrome b558, a heterodimer consisting of
gp91-phox and p22-phox, is a critical component of the
oxidase. The X-linked form of the disease is due to defects in the
gp91-phox gene. We report here biochemical and genetic analyses
of patients with typical and atypical X-linked CGD. Immunoblots showed
that neutrophils from one patient had small amounts of p22-phox
and gp91-phox and a low level of O2-
forming oxidase activity, in contrast to the complete absence of both
subunits in two patients with typical CGD. Using polymerase chain
reactions (PCR) on cDNA and genomic DNA, we found novel missense
mutations of gp91-phox in the two typical patients and a point
mutation in the variant CGD, a characteristic common to two other
patients with similar variant CGD reported previously. Spectrophotometric analysis of the neutrophils from the variant patient
provided evidence for the presence of heme of cytochrome b558. Recently, we reported another variant CGD
with similar amounts of both subunits, but without oxidase activity or
the heme spectrum. A predicted mutation at amino acid 101 in
gp91-phox was also confirmed in this variant CGD by PCR of the
genomic DNA. These results on four patients, including those with two
variant CGD, are discussed with respect to the missense mutated sites
and the heme binding ligands in gp91-phox.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
CHRONIC GRANULOMATOUS disease (CGD) is a
severe clinical syndrome characterized by recurrent bacterial and
fungal infections with granuloma formation.1 The disease is
caused by failure of the NADPH oxidase in phagocytes (neutrophils,
eosinophils, monocytes, and macrophages), a superoxide
(O2-) forming enzyme required for microbicidal
activity. This enzyme system consists of multiple factors including
membrane-associated cytochrome b558 and cytosolic
components for oxidase activation.2-6 Cytochrome
b558 is a heterodimer formed by a
91-kD glycosylated heavy chain, gp91-phox, and a
22-kD light chain, p22-phox. CGD is now known to be caused by
deficiency of one of four proteins; gp91-phox,
p22-phox, p47-phox, and p67-phox,
respectively.2,7,8 Several mutations of gp91-phox
with X-linked recessive CGD have been reported: deletional, splicing,
missense, nonsense, and duplicational mutations.6,9
Here we report molecular and biochemical studies on three patients with
X-linked CGD: one variant and two typical. The variant CGD has small
amounts of both p22-phox and gp91-phox with
O2- forming NADPH oxidase activity. Sequence
analyses of the gp91-phox gene showed novel point mutations in
the two typical cases of CGD and a conservative point mutation in the
patient with variant CGD. Spectrophotometric studies on neutrophils
from these patients were performed using an accurate, sensitive
technique for analysis of the heme spectrum of cytochrome
b558.10 Our results on the variant CGD
are compared with those on another variant X-linked CGD that showed
similar amounts of both subunits, but no oxidase activity or the heme
spectrum.11 In the latter CGD, the predicted mutation at
amino acid 101 replacing His by Tyr was further confirmed by polymerase
chain reaction (PCR) of genomic DNA. Taken together with the
spectrophotometric data, the results on all four CGDs are discussed
with respect to the heme binding histidines and the mutated sites in
the gp91-phox protein.
| |
MATERIALS AND METHODS |
Case reports.
Three male patients, a 10-year old (T.M.), a 19-year old (T.H.), and a
25-year old (J.O.), all had recurrent episodes of bacterial infection
and were diagnosed as having X-linked recessive CGD by functional
analysis of their and their mothers' neutrophils at the age of 6 months, 5 years, and 6 years, respectively. Patients T.H. and J.O. had
received operations for liver abscesses before the diagnosis of CGD.
Although they often suffered from perianal abscesses, inguinal
lymphadenitis, stomatitis, and skin abscesses, they were relieved of
severe infection by administration of sulfamethoxazole-trimethoprim.
Materials.
Ficoll-Paque and Dextran T-500 were purchased from Pharmacia Biotech,
Uppsala, Sweden; diisopropyl fluorophosphate (DFP) was from Wako Pure
Chemicals, Osaka, Japan; Sodium-p-tosyl-L-lysine chloromethyl
ketone (TLCK), zymosan, and phorbol 12-myristate 13-acetate (PMA) were
from Sigma, St Louis, MO; phenylmethylsulfonyl fluoride (PMSF) was from
Nakarai Co, Kyoto, Japan. All other reagents were of analytical grade.
Reagents for the analysis of RNA and DNA are described below. Opsonized
zymosan (op-zy) (20 mg/mL) was prepared as described
previously.12 PMA was dissolved in dimethyl sulfoxide (20 mg/mL) and diluted with Ca2+-free Krebs Ringer phosphate
buffer [KRP: 122 mmol/L NaCl, 4.9 mmol/L KCl, 1.2 mmol/L
MgCl2, 17 mmol/L sodium phosphate buffer (pH 7.4)] to 20 µg/mL before use.
Isolation of neutrophils.
Neutrophils from the three CGD patients, their parents, and healthy
controls were collected from 10 to 20 mL samples of heparinized venous
blood by Dextran sedimentation, followed by hypotonic treatment for
lysis of red blood cells, and then stepwise separation with Ficoll-Paque according to Böyum's method13 with some
modifications.10 The cells, consisting of 98% to 99%
neutrophils and less than 1% eosinophils, were suspended in ice-cold
Ca2+-free KRP before use. All donors gave their informed consent.
Assay of cellular O2- formation.
Neutrophils were suspended in a narrow-cuvette at 1.0 to 2.0 × 106 cells/700 µL in KRP containing 5 mmol/L glucose and
0.6 mmol/L CaCl2 and were continuously stirred with a
wind-mill cell mixer14 for the assay of
O2- release from stimulated cells. The rates of
O2- formation by neutrophils from donors in the
presence of PMA (0.14 µg/106 cells) or op-zy (0.4 mg/106 cells) were measured at 37°C by recording
superoxide dismutase inhibitable-reduction of ferricytochrome
c12 at the absorbance difference of 550 to 540 nm
in a Hitachi dual wavelength spectrophotometer, model 557 (Hitachi, Tokyo, Japan).
Preparation of membrane and cytosol fractions.
Neutrophils from each donor were treated with 2 mmol/L DFP on ice and
were suspended in sonication buffer consisting of 1 mmol/L PMSF, 1 mmol/L TLCK, and 20 mmol/L sodium phosphate buffer (pH 7.4) at a final
concentration of 2 × 107 cells/mL. Aliquots of the
cell suspensions were placed in microtubes and sonicated in a
Bioruptor, model UCD-200TM (Cosmo-Bio, Tokyo, Japan) at
0°C, setting its controller at an optimal sonication time (40 to 50 × 1.5 seconds with intervals of 1 second). After sedimentation of
cell debris and nuclei at 400xg for 10 minutes, the
supernatants were centrifuged at 2.3 × 105g
for 15 minutes at 2°C in a table top ultracentrifuge (Beckman model
TL-100; Beckman, Fullerton, CA) to separate the membrane and cytosol fractions as precipitates and supernatants.
Immunoblot analysis.
Rabbit polyclonal antibodies were raised against synthetic polypeptides
corresponding to the COOH-terminal peptides (17 residues) of
p22-phox and gp91-phox of cytochrome
b558 of human neutrophils. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as
described by Laemmli with 4% stacking gel and 10% or 11% separating
gel.15 Membrane fractions (2 × 106 cells
equivalent) were mixed with an equal volume of Laemmli buffer and
incubated for 3 minutes at 90°C. After electrophoresis, proteins
were transferred to a nitrocellulose membrane, which was treated with
Ponceau and rabbit antiserum against each peptide.16 The
specific bindings of the antibodies to protein bands were detected
using goat antirabbit IgG conjugated with horseradish peroxidase. Color
was developed with 0.01% H2O2 and 0.03%
4-chloro-1-naphthol, and the color density was measured with an image
analyzer (FAS II, TOYOBO, Osaka, Japan), as reported
recently.11
Spectrophotometric measurement.
The sonicated neutrophils were exposed to carbon monoxide (CO) as
follows.10 A soft rubber ball (diameter, 7 cm), which had
been deflated with a needle, was inflated through the needle with CO
gas from a small bomb containing 99.95% CO gas. An aliquot of the
sonicated sample (200 to 300 µL) was loaded into a sterile syringe,
into which CO gas in the ball was then withdrawn through a needle to be
a volume ratio of 1:3 (sample, CO). After blocking the needle with a
piece of silicon, the syringe was turned slowly upside down several
times for 10 minutes to allow binding of contaminating hemoglobin with
CO. All of the above procedures were performed in a draft chamber. The
CO-treated sample was delivered by syringe into a microcuvette plugged
with a rubber septum. Absorption spectra were measured in a Unisoku
spectrophotometer model USP-530 (Unisoku Co, Hirakata).
Heme proteins in the CO-treated sample were reduced with a
few grains of sodium dithionite and the reduced minus oxidized difference spectra were measured in the range of 400 to 700 nm. The
spectra obtained were further processed to obtain differential spectra
in the range of 540 to 580 nm.10
Preparations of RNA and DNA.
RNA was isolated from Epstein-Barr virus (EBV)-transformed cell lines
by first dissolving the cells in 4.0 mol/L guanidine thiocyanate;
subsequently, RNA was precipitated through 5.7 mol/L cesium chloride by
centrifugation. The mRNA was then isolated using an oligo d(T) column
as described by Aviv and Leder.17 First strand cDNA was
synthesized using a cDNA synthesis Kit (SuperscriptTMII;
GIBCO, Rockville, MD). The protein coding region of the
gp91-phox cDNA was amplified by PCR in three overlapping
fragments using three pairs of synthetic oligo nucleotide primers as
described previously.18 Cycle parameters were: 30 cycles of
1 minute at 94°C, 1 minute at 60°C, and 4 minutes at 72°C.
The PCR products were purified with a Geneclean II kit (BIO 101, Inc, Vista, CA) to remove the primers and then subcloned
into a plasmid pGEMT (Promega, Madison, WI). Sequence
analyses were performed by the dideoxy chain terminating method using
an automated DNA sequencer (Model 373A; Applied Biosystems, Foster,
CA) according to the manufacturer's instructions.
Multiple clones of each fragment were sequenced in both directions.
Genomic DNA was isolated from circulating blood leukocytes from donors
to confirm each mutation. Exons plus intron boundary sequences were
amplified by PCR using primers corresponding to sequences on the
flanking introns7 as indicated in Table 1. The conditions
for PCR were as described above. The PCR product was digested with Taq
I (Takara, Kyoto, Japan) for exon 9 of patient T.H. and with MspI for
exon 12 of patient T.M. The sizes of the fragments were analyzed in 3%
agarose gel. The PCR products for exon 10 of patient J.O. and exon 4 of
patient T.K. were sequenced as described above.
| |
RESULTS |
Neutrophils from the two patients (T.M. and J.O.) had no
O2- generating activity, whereas those from the
other patient (T.H.) exhibited slight O2-
generation (6% to 7% and 8% to 9% of the control activities on stimulation with PMA and op-zy, respectively).
Figure 1 shows traces of
O2- generation by phagocytosing cells from the
patient (T.H.) and his mother and father. The rates were 3.9 ± 0.7, 25 ± 1 and 44 ± 3 nmol
O2-/minute/107 cells (n = 3), which were 8.8%, 55%, and 100% of the control value,
respectively.
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| Fig 1.
O2- formation by neutrophils from
patient T.H. (P) and his father (F) and mother (M). The rate of
O2--dependent reduction of ferricytochrome
c was recorded by spectrophotometric measurement of the 550 to
540 nm absorption difference. Neutrophils (1.0 to 2.0 × 106 cells/700 µL KRP) were stimulated by addition of
op-zy. The numbers on traces are cell numbers added to cuvettes.
|
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Figure 2 shows immunoblots of the membrane
fractions from neutrophils of the CGD patients. Two patients (T.M. and
J.O.) had no p22-phox or gp91-phox (Fig 2A and C),
characteristic of typical X-linked recessive CGD, whereas the membranes
from the variant CGD patient (T.H.) had small amounts of both
p22-phox and gp91-phox (Fig 2B). The glycosylated heavy
chain, gp91-phox, showed broad electrophoretic migration, and
so the stained band appeared faint, but detectable in the gel, in
contrast to its complete absence in the two typical cases of CGD.
Densitometric analysis of the stained bands of the variant CGD showed
the presence of p22-phox and gp91-phox at 14% and 13%
of the control, respectively (Table 1).
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| Fig 2.
Immunoblot analyses of neutrophils from CGD
patients, T.M. (A), T.H. (B), and J.O. (C). The membrane fractions from
neutrophils (1 to 2 × 106 cell equivalents) were
electrophoresed and transferred to nitrocellulose membranes, which were
treated with antiserum against polypeptides of p22-phox and
gp91-phox and stained with Ponceau. (A) Patient T.M. (3),
mother (2), and controls (1 and 4). (B) Patient T.H. (2) and controls
(1 and 3). (C) Patient J.O. (2) and controls (1 and 3). The protein
concentration of the variant CGD (B) loaded was higher than those in
other proteins (A and C).
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Figure 3 shows the reduced minus oxidized
absorption difference spectra of neutrophils from a typical CGD
patient; J.O. (Fig 3A), the variant CGD patient; T.H. (Fig 3B), and a
healthy donor (Fig 3C). The spectrum of the variant CGD (Fig 3B) showed
a small peak at 558 nm of cytochrome b, in contrast to the complete
absence of the peak in the typical CGD (Fig 3A). The large absorption band of myeloperoxidase with a peak at 475 nm gave no evidence for the
presence of eosinophils, because eosinophil peroxidase gives a distinct
peak at 453 nm, causing a shoulder even if the preparation is
contaminated with only a few percent of this cell fraction. Indeed, the
cell fraction from the variant CGD patient contained 99% neutrophils,
but undetectable eosinophils. Figure 4
shows the reduced minus oxidized difference spectra of neutrophils from
the variant CGD family: father (F), mother (M), and the patient (P).
The spectra in the  -band region were five times enlarged (inset of
Fig 4). The spectra obtained (Fig 4) were differentiated as
d[ A]/d , where A is the absorption difference and is the wavelength, as shown in Fig 5. The
differential spectrum of the variant CGD (T.H.) shows a small peak in
the range of 550 to 560 nm in contrast to that of typical cases of
X-CGD (J.O. or T.M.), which show only a trough, but no
peak,10 as shown in the inset. The differential spectra
gave further evidence for the indication of the presence of heme in the
spectrum of the variant CGD. The heme contents of samples of the
variant P, and theMand F were about 12%, 48%, and 100% of the
control value, respectively.
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| Fig 3.
Reduced minus oxidized difference spectra of neutrophils
from patient J.O. (a), patient T.H. (b) and a healthy control (c).
Neutrophil sonicates (6 × 106 cells/250 µL) were
treated with CO and then the reduced minus oxidized
difference spectra were measured. The inset shows the  -band region
enlarged 5.4-fold.
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| Fig 4.
Reduced minus oxidized difference spectra of neutrophils
from variant CGD patient T.H. (P) and his mother (M) and father (F).
The inset shows the -band region enlarged fivefold. The difference
spectra were measured as in Fig 3.
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| Fig 5.
Differential spectra. The spectra in Fig 4 were
differentiated as d[ A]/d (A = absorption difference of the
spectra, = wavelength) in the region of the -band: patient
T.H. (P), mother (M) and father (F). The inset shows differential
spectra of the patient with cytochrome b558
deficiency; P (-) and of a control (C).
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The protein coding regions of overlapping fragments of
gp91-phox cDNA from CGD patients, carriers, and healthy
controls were amplified by PCR and the products were subjected to
sequence analysis. All three patients showed normal sized fragments on
an agarose gel (data not shown). The sequence of patient T.M. showed a
point mutation at bp 1558 of thymine to cytosine, predicting an amino acid substitution at residue 516 of Trp to Arg. In the sequence of
patient T.H. with variant CGD, a point mutation at bp 937 of guanine to
adenine was found, predicting an amino acid substitution at residue 309 of Glu to Lys. The sequence of patient J.O. showed a point mutation at
bp 1271 of thymine to cytosine, predicting a Leu to Pro conversion at
amino acid position 420.
To confirm the mutations found, genomic DNA fragments containing these
mutated sites were amplified by PCR and then analyzed by means of
restriction digestion or direct sequencing. As expected, the PCR
products from the patients and their carriers had the predicted sizes
on the gel. Incubation of exon 12 of patient T.M. with MspI resulted in
fragmentation into two pieces: distinct 84 bp and weak 40 bp bands
(Fig 6, lane 2) in contrast to the two
controls, which gave a single band of 124 bp (lanes 1 and 4 in Fig 6).
Exon 12 of the mother of T.M. (Fig 6, lane 3) showed partial
fragmentation into weak 84 and 40 bp bands, providing evidence for the
carrier state. The restriction enzyme TaqI did not digest the PCR
product of exon 9 of patient T.H. (Fig 7,
lane 2), but completely digested those of exon 9 of his father and a
healthy control to yield a 228 bp product (with slight 26 bp) (Fig 7,
lanes 1 and 4). The PCR product of his mother showed partly digested
products as evidence of the carrier state (Fig 7, lane 3). The PCR
products of exon 10 of patient J.O. and his mother were directly
sequenced because no available restriction enzyme was found for the
mutated site. The T1271 to C substitution of patient J.O. was
identified in the read sequence of exon 10, and the mother was
heterozygous for the mutation (data not shown).
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| Fig 6.
Restriction digestion analysis of DNA fragments from
patient T.M. and his mother. DNA samples (exon 12) from the patient
(lane 2) and his mother (lane 3) and healthy controls (lanes 1 and 4)
were digested with MspI. DNA from the CGD patient showed abnormal
restriction digestion patterns of the gp91-phox gene and that
from the mother showed evidence of the carrier state.
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| Fig 7.
Restriction digestion analysis of DNA fragments from CGD
patient T.H. and his parents. DNA samples (exon 9) from patient T.H.
(lane 2), the father (lane 1), mother (lane 3), and a control (lane 4).
When digested with Taq I, DNA from patient T.H. showed abnormal
restriction fragment patterns and that from the mother showed evidence
of a carrier state.
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We identified missense mutations in the gene encoding gp91-phox
in the three patients with X-linked CGD, as summarized in Table 1. In
addition to the three families, we recently reported another variant
CGD (patient T.K.) in which neutrophils have similar amounts of both
p22-phox and gp91-phox, but show no
O2- forming activity or the heme spectrum of
cytochrome b558,11 in contrast to the
present variant CGD (T.H.). The mutated site in patient T.K. was found
at residue 101 causing replacement of His by Tyr, probably one of the
heme binding residues for bis-histidine coordination of cytochrome
b558. In the present study, the predicted mutated
site, H101Y, was confirmed at a position in exon 4 with the PCR product
of genomic DNA, as shown in Table 1.
| |
DISCUSSION |
All four missense mutations are illustrated in
Fig 8 with the predicted domains. Sequence
analyses of the two typical cases of X-linked CGD (T.M. and J.O.)
showed novel missense mutations; W516R and L420P, respectively. The
absence of the heme spectrum (X910) in the two patients
suggests that a replacement at residue 420 or 516 causes a very
unstable gp91-phox protein, leading to the complete absence of
cytochrome b558 in the membranes. Residue 420, where Leu is replaced by Pro in patient J.O. is involved in the
predicted NADPH-binding domain.19-21 The substitution of a
positively charged Arg for the nonpolar hydrophobic residue Trp at
residue 516 might alter the folding properties of the peptide backbone.
In the other variant CGD patient (T.H.), the missense mutation at
residue 309 is interesting, having common characteristics with two
other patients with variant CGD reported previously, showing the same
replacement of Glu by Lys.22 This conservative substitution
of a positively charged residue for a negatively charged one might
alter the ionic properties of the peptide, whereas all E309K cases have
a small amount of cytochrome b558 and a low level
of O2- generating oxidase activity. This fact
suggests that residue Glu 309 and its adjacent residues in
gp91-phox are neither involved in the domain forming a stable
complex with p22-phox nor in the domain holding flavin adenine
dinucleotide (FAD) because the conversion of charged
residues is critical for electron and proton transfer reactions.
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| Fig 8.
A topological scheme of the heme binding histidines and
the mutated residues in gp91-phox. Mutations in the gene encoding
gp91-phox were detected in the four patients; H101Y (patient
T.K.), E309K (patient T.H.), L420P (patient J.O.), and W516R (patient
T.M.). A cytochrome P450-like alignment: residues 78-85 FLRGSSAC85.27 A cytosolic loop: residues 87-94:
STRVRRQL.28 Taken together with previous studies on ESR and
MCD spectra,29 the spectrophotometric studies on the CGD
neutrophils provided a topological scheme for the locations of the
hemes in gp91-phox. For details, see text.
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|
Finegold et al23 suggested from the similarity in residues
between the yeast iron reductase (FRE1) and gp91-phox that the corresponding residues coordinating heme may be two pairs of histidines (101 and 115; 209 and 222) buried in the separate hydrophobic domains.
However, there is no direct evidence for the heme binding-ligands in
gp91-phox except our results on the heme spectra of phagocyte cytochrome b558 from the two variant CGD patients.
From the differential spectra, it is possible to distinguish between
the presence and absence of heme, even in the presence of a minute
amount of cytochrome b558 from CGD patients.
Neutrophils from the variant CGD with a novel mutated site at
His101 showed a similar differential spectrum11
to those of typical X-linked CGD patients whose phagocytes have no
subunits and show no heme spectrum (Fig 5, inset). In contrast, the
present variant CGD showed a distinct differential spectrum with a peak
in the left of the -band (550 to 560 nm) (Fig 5), indicating the
presence of the heme of cytochrome b558, which may
account for the O2- forming oxidase activity.
Comparative studies on these heme spectra confirmed that one of the
heme binding histidines is His101. Bolscher et
al24 reported a typical X-linked CGD patient with a point
mutation at the same His101 to arginine in
gp91-phox, suggesting that this part is important in binding of
the heme or for formation of a stable complex with p22-phox.
Although patients with many other mutations of histidine residues at
the N-terminal have been reported,6,9 most of these
patients had typical X-linked CGD and their phagocytes had neither
gp91-phox nor p22-phox, so the heme binding ligands
could not be identified by spectrophotometry.
In addition to immunoblot analyses, we detected the gp91-phox
protein in neutrophils from the two variant CGD patients (T.H. and
T.K.) by flow cytometry of the cells using monoclonal antiboby 7D525 (data not shown), which has recently been identified
as an anti-gp91-phox antibody (M. Nakamura, personal
communication, March 1998). Yu et
al26 have recently been investigating the biosynthesis of
cytochrome b558. Their results suggest that heme incorporation is important in the assembly of cytochrome
b558 to form a stable complex of gp91-phox
and p22-phox. Consistent with their report, in the present
variant CGD (T.H.), the proteins of both subunits and the heme in the
cytochrome b558 were closely associated, as shown
by immunoblots and spectra. In contrast, the other variant CGD (T.K.)
showed similar amounts of the two subunits, but no heme.11
The point mutation at residue 101 with replacement of His by Tyr is
probably similar to FRE1 mutants that express FRE1 protein, but do not
show a heme spectrum.23
Previous electron spin resonance (ESR) studies on
neutrophil cytochrome b558 provided indirect
evidence that His101, located in the vicinity of
Cys85 in a cytochrome P450-like alignment
(residues 78-85: FLRGSSAC85), is the fifth (proximal) heme
binding ligand, and the sixth (distal) ligand may be
His209, because pyridine treatment of cytochrome
b558 changed the ESR signal of the bis-histidine
coordination to a cytochrome P450-like signal, possibly
through the replacement of His101 by the nearby residue
Cys85.27 As shown in Fig 8, Cys85
may be adjacent to His101 in the two transmembrane regions
because a cytosolic loop of gp91-phox (residues 87-94:
STRVRRQL) is located between Cys85 and
His101.28 The results of comparative studies on
the heme spectra of the two variant CGDs support the prediction from
previous ESR studies. Because near-infrared magnetic circular dichroism
(NIR-MCD) spectra demonstrated the presence of two distinct forms of
the heme with bis-histidine coordination,29 another pair of
heme-binding histidines may be present, probably within a short
distance (4 to 5 Å) from the outer surface,30 close to the
glycosylation sites31 (Fig 8).
| |
ACKNOWLEDGMENT |
We thank Dr M. Nakamura (Institute of Tropical Medicine, Nagasaki
University, Nagasaki) for supplying monoclonal antibody 7D5.
| |
FOOTNOTES |
Submitted February 19, 1998; accepted November 11, 1998.
Supported in part by grants from the Ministry of Education, Science,
and Culture of Japan and the Tokyo Metropolitan Institute of Medical Science.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Katsuko Kakinuma PhD, Department of
Inflammation Research, The Tokyo Metropolitan Institute of Medical
Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo, 113-8613 Japan.
| |
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