1. No category

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Cloning of Murine gp91ph””cDNA and Functional Expression in a Human
X-Linked Chronic Granulomatous Disease Cell Line
By Helga Bjorgvinsdottir, Ling Zhen, and Mary C. Dinauer
The phagocyte cytochrome LI-,
a heterodimer comprised
of gp9lPn0”and p22ph””. is a flavocytochrome that mediates
the transfer of electrons from NADPH to molecular oxygen
in the respiratory burst oxidase. The human gene encoding
the glycosylated gp9lPhoxsubunit is the site of mutations
in X-linked chronic granulomatous disease (CGD). Reverse
transcriptase-polymerase chain reaction was used to obtain
a full-length clone for the murine gp9lPnox
cDNA, which was
87% identical to the human gp9lPhoxcDNA. The encoded
murine protein had 39 amino acids out of 570 that differed
from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in
binding to NADPH, flavin, and the cytosolic oxidase subunit
p47ph0x.Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycorylated
murine protein migrates with an apparent molecular mass
of 58 kD instead of 91 kD as seen for the human protein.
in a human myeloid cell line
Expression of murine gp91with a null gp9lPhoxallele using a mammalian expression
plasmid or a retroviral vector rescued stable expression of
the papho”subunit and fully reconstituted respiratory burst
activity. This suggests that the murine gp9lph0”subunit
forms a functional cytochrome & heterodimer with human
oxidase subunits, consistent with the high degree of identity
between the mouse and human proteins in domains implicated in cytochrome function.
0 1996 by The American Society of Hematology.
T
As one approach to localize important functional domains
HE PHAGOCYTE respiratory burst oxidase (NADPH
within the gp9lphoX
subunit of cytochrome b 5 5 8 , we have isooxidase) plays a central role in host defense, catalyzing
lated a cDNA for the murine gp91Ph” using reverse tranthe transfer of electrons from NADPH to molecular oxygen
scriptase-polymerase chain reaction (RT-PCR). The encoded
thereby generating the superoxide radical (0;).Superoxide
murine protein had 39 substitutions throughout the 570
is then converted into toxic oxygen derivatives that are esamino acids compared with the human gp9lPbxpolypeptide,
sential for intact microbicidal function.’ The oxidase is dormant in resting phagocytes but, upon cellular activation by
many of which were conservative. Nonconserved substituinflammatory stimuli, is rapidly assembled at the plasma
tions occurred in regions outside of the putative heme, flavin,
membrane from cytosolic and membrane components. An
and NADPH binding domains. Expression of murine
unusual b-type cytochrome, cytochrome
is located in
gp9lPh”*in a human myeloid cell line with a null gp9lPh”
the plasma membrane, where it functions as the redox center
allele rescued expression of the
protein and fully
of the oxidase. Cytochrome b 5 5 8 is a heterodimer composed
reconstituted respiratory burst activity in intact cells.
of two tightly associated polypeptides, gp91Ph”and ~22~”O’,
MATERIALS AND METHODS
and is expressed almost exclusively in phagocytic leukocytes.’-’ Two soluble oxidase subunits, p47p”Oxand ~67~“O”, Cell lines. PLB-985 cellsz4and derivatives were maintained as
translocate to the membrane upon oxidase activation, along
de~cribed.’~X-CGD PLB-985 cells were previously generated by
with a small guanosine triphosphatase, R ~ c . Mutations
~.~
in
targeted disruption of the gp9lPhUxgene, and lack endogenous
gp9lPhU*
and respiratory burst a~tivity.’~
For granulocyte differentiaany one of the four phox oxidase subunits result in chronic
tion, logarithmically growing PLB-985 and derivative cell lines at
granulomatous disease (CGD), with defects in the X-linked
a density of 1 to 3 X lo5cells/mL were exposed to 0.5% dimethylgene for gp9lPh0”accounting for the majority of cases.’ CGD
formamide (DMF) for 6 daysz5 Retrovirus packaging lines GP +
phagocytes lack respiratory burst oxidase activity and exhibit
E86” and GP+envAm12” were provided by Dr A. Bank (Columbia
impaired microbial killing, which leads to recurrent and freUniversity, New York, NY) and maintained in 50% Hams F12 and
quently life-threatening bacterial and fungal infections.
50% Dulbecco’s modified Eagles medium (GIBCO, Grand Island,
Cytochrome b 5 5 8 is a flavocytochrome that contains two
NY) supplemented with 10%fetal calf serum (Sigma, St Louis, MO),
heme groups embedded within the plasma membrane as well
as a flavin group that acts as the initial acceptor of electrons
from
The specific functional domains of the
From the H e w n B Wells Center for Pediatric Research, the
cytochrome have been only partially characterized. At least
Departments of Pediatrics (Hematology and Oncology) and of Medione of the heme groups may be shared between the gp91Ph“x cal and Molecular Genetics, James Whitcomb Riley Hospital for
and ~ 2 2 subunits,”
~ ~ ” ~whereas the flavin and NADPH bindChildren, Indiana University Medical Center, Indianapolis.
ing sites appear to be localized in the carboxyl-terminal porSubmitted August 22, 1995; accepted October 3, 1995.
Supported by ROI HL.45635 and a Ceniers for Excellence in Hetion of gp91Ph“.”~”In addition to their close functional relamatology Award (IP50 DK49218) from the National Institutes of
tionship, interactions between gp91Ph” and
appear
Health, and by a Clinical Research Awardfrom the March of Dimes.
important for normal intracellular processing and stability
Address reprint requests to Mary C. Dinauer, MO, PhD, Herman
of each cytochrome subunit.’,’’ For example, genetic defiB
Wells
Center for Pediatric Research, James Whitcomb Riley Hosciency in either gp9lPhoX
or ~
2 results
2 in ~
markedly
~ dimin~
pital for Children, Room 2600, 702 Barnhill Dr, Indianapolis, IN
ished levels of the unaffected subunit.19 Cytochrome b 5 5 8 also
46202.
acts as the focal point for assembly of the active oxidase
The publication costs of this article were defrayed in part by page
complex. Translocation of cytosolic oxidase subunits upon
charge payment. This article must therefore be hereby marked
cellular activation fails to occur in the absence of cytochrome
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
b558.5.20,21
Multiple interactions between both cytochrome
indicate this fact.
subunits and ~ 4 7 ” ~
appear
”
to be important for normal as0 1996 by The American Society of Hematology.
sembly of the oxidase.1.z2.23
0006-4971/96/8705-0022$3.00/0
Blood, Vol 87,No 5 (March 1). 1996 pp 2005-2010
2005
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2006
100 U/mL penicillin (GIBCO), 100 pg/mL streptomycin (GIBCO), 2
mmom glutamine (GIBCO), and 15 mmol/L HEPES Buffer Solution (GIBCO). NIH 3T3 fibroblasts were obtained from Dr D.W.
Clapp and mouse macrophage RAW cells were obtained from Dr
D. Skalnik (both at Indiana University School of Medicine, Indianapolis).
RT-PCR and subcloning of murine gp91Ph"'cDNA. Total RNA
was extracted from mouse macrophage RAW cells using a guanidium thiocyanate protocol.2x First-strand cDNA was generated
through reverse transcription with Moloney murine leukemia virus
(M-MuLV) reverse transcriptase and random primers using the protocol supplied by the manufacturer (Boehringer Mannheim, Indianapolis, IN). Two synthetic oligonucleotide primers, derived from
the human gp9Iph"IcDNA sequence, were prepared to attempt to
amplify the murine gp9IPh'" cDNA using PCR: S-ACAATCTCGAGACCATG GGG AAC TGG GCT GTG AAT GAA 6-3' (forward prim7and 5'-ACTTAGGATCCTTA GAA GTT TTC CTT
GTT GAA AAT GAA GTG-3' (reverserimer). Sequences from
the human gp9lPh'" cDNA are shown in bold and the initiator and
the stop codons are underlined. The synthetic oligonucleotides also
have additional nucleotides at their 5' end encoding Xho I and BumHI
restriction sites, respectively. A full-length murine gp9 lph"* cDNA
was obtained using Vent polymerase (New England Biolabs, Beverly, MA) to amplify the first-strand cDNA by PCR using the following temperature settings: 94°C x 1 minute, 65°C x I minute, 72°C
X 3 % minutes, for a total of 35 cycles. The DNA fragment was
purified using Magic PCR Preps DNA purification system (Promega,
Madison, WI), digested with Xho I and BamHI, and subcloned into
pBluescript I1 KS( +) (Stratagene, La Jolla, CA). Dideoxynucleotide
sequencing was performed in both directions. Multiple clones from
three independent amplifications were sequenced to ensure that the
sequence obtained did not include PCR-induced mutations. Translation of the nucleotide sequence and best-fit alignments with the
human gp91Ph""sequencez9were performed using the Genetics Computer Group molecular genetics software (Program Manual for the
Wisconsin Package, Version 8; Genetics Computer Group, Madison,
cDNA was subcloned into two
WI). The full-length murine gp91PhoX
different vectors after changing the 5' Xho I site to EcoRI: the
mammalian expression vector pEF-PGKneoz' to generate the plasmid, pEFneom91, and the plasmid shuttle vector for the
MSCVneoEB retrovirus3oto generate pMSCVneo-m91.
Construction of retrovirul producer cell lines. The MSCVneom91 plasmid was transfected into GP+E86 ecotropic packaging cells
using DOTAP (Boehringer Mannheim). Transfected packaging cells
were selected in G418 (GIBCO) at 0.8 mg/mL to isolate single
colonies. To estimate a quantitative titer, 3T3 cells were infected as
described3' and selected in (3418. Supernatant from a clone with the
highest titer (1.5 X IO4 infectious particles/mL) was used to crossinfect GP + envAml2 amphotropic packaging cells ("ping-pong")
to obtain amphotropic virus producer cells, m9lneoAml2. The total
G418-resistant m9lneoAml2 cell population was used for subsequent transduction of X-CGD PLB-985 cells and had an estimated
titer of -5 x lo3infectious particles/mL as quantitated on 3T3 cells.
Introduction of gp91pho'.expression vectors into X-CGD PLB cells.
Between 1 and 2 x IO7 X-CGD PLB-985 cells in logarithmic growth
were electroporated with the plasmid pEFneo-m91 and individual
clones were isolated by limiting dilution in the presence of (3418,
as previously described.z5For infection with MSCVneo-m91 retrovirus, X-CGD PLB-985 cells were transduced by coculture with
m9lneoAml2 amphotropic producer cells." After infection, cells
were grown in the presence of (3418 to select for a population of
transduced cells.
RNA and immunoblot analysis. Total cellular RNA was isolated
and analyzed by Northem blot as de~cribed.'~
RNA samples were
probed with random prime-labeled full-length murine gp9Iph""
BJORGVINSDOTTIR, ZHEN, AND DINAUER
cDNA. Triton X-100 extracts and cellular membranes of nonadherent mouse bone marrow (BM) cells and of PLB-985 and derivative
cells were prepared as previously described.25."Protein concentration was measured by BCA protein assay (Pierce, Rockford, 1L). On
a per-cell basis, membrane extracts typically yield -20% to 35%
of the protein obtained from whole-cell extracts. Immunoblot analysis was performed as describedz5using either affinity-purifiedp22ph"'
antibody or C-terminal antibody for gp9l""" as probes."
Assay ofsuperoxide formation. The nitroblue tetrazolium (NBT)
assay was performed on granulocyte-induced PLB-985 cells or derivatives.*' For quantitative measurement of superoxide formation
elicited by phorbol myristate acetate (PMA)-stimulated whole cells
after granulocyte differentiation, the continuous cytochrome c assay
was
RESULTS
To generate a full-length murine gp9lPh"' clone, synthetic
oligonucleotide primers derived from the human gp9 lphor
sequence were used to amplify the murine sequence from
first-strand cDNA prepared from mouse macrophage mRNA.
A single 1,700-bp product was generated, whose nucleotide sequence was 87% identical to the human gp91rh"'
cDNA* (not shown). The amino acid sequence of the encoded murine protein is shown in Fig 1, and is aligned with
the human gp9 lph"xsequencez9for comparison. The murine
and human sequences are identical in 531 out of 570 amino
acids, although note that the N-terminal 8 and C-terminal 9
amino acids in the murine sequence were specified by the
oligonucleotide primers. Seventeen of the nonidentical
amino acids represent conservative substitutions. Some of
the residues that are dissimilar between the two species involve consensus sequences for N-linked glycosylation (NX-S/T), with only two potential sites shared between the
murine and human sequences (Fig 1).
We investigated whether the murine gp91Ph"xprotein could
rescue ~ 2 2 " expression
~~'
and participate in superoxide generation in human X-CGD PLB-985 cells. Two different expression systems were used: a mammalian plasmid expression vector, pEF-PGKneo, in which the cDNA is under
control of the human elongation-factor 1 -apromoter,25and
a retroviral vector, MSCVneoEB, in which cDNA expression
is driven by a modified Maloney leukemia virus LTR."
pEFneo-m91 was introduced into X-CGD PLB cells by
electroporation, and stable transfectants isolated by limiting
dilution. Twelve clones were randomly chosen for analysis,
of which seven were NBT-positive, which demonstrated
both successful transfection of the entire transgene as well
as functional reconstitution of respiratory burst activity. On
Northern blot analysis, NBT-negative clones lacked mRNA
for gp91f'h0x(data not shown), which suggested that the
transgene was nonfunctional in these cells. Four of the NBTpositive clones were chosen for further detailed analysis.
Cell extracts were prepared from granulocyte-induced cells
and analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and immunoblotting to examine the expression of cytochrome b558using antibodies for both g ~ 9 1 ~ ~ ' ' ~
-
* The nucleotide sequence for the murine gp9IPh'"cDNA has been
deposited in the Genbank database (accession no. U43384).
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MURINE GP9lPHoxcDNA
2007
50
CGP-SAL
WDIPPKFFYTRJCLLGSAL 50
human 2 0 1 YF
mouse
251 W G K I K E C P V F ' K F A G N P P I V G P M ! % Y L C E R L ~ ~ K W I T K V
to that seen using the pEFneo-m91 vector. Finally, expression of recombinant murine gp91p""' resulted in markedly
increased levels of the ~22"""' subunit in human X-CGD
cells, as assessed by immunoblotting (Fig 2B).
A continuous cytochrome c reduction assay was used to
quantitate respiratory burst activity in granulocyte-induced
X-CGD PLB-985 cells that expressed recombinant murine
gp9Ip""' (Table I). As previously shown,?5.'' X-CGD PLB
cells lacked respiratory burst activity, whereas wild-type
PLB-985 cells had levels similar to those seen in normal
human neutrophils." Expression of recombinant murine
-
300
IIIIIIII:I.IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIlIII
human 2 5 1 W G K I K ~ P I W F A G N P P M I W K W I V G P E R L ~ S a ~ T K
300
V
mouse
3 0 1 WIiPFKTIELX*IKKKGFIFWCPKVSKLEWbiPFl'LTSAPEE!JF 350
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
human 3 0 1 VTHPFKT1RX)MKKKGFIFWCPWSKLEWbiPFl'LTSAPEED~
350
muse
3 5 1 F S I H I R I V G ~ E G L F E ~ ~ ~ P K I A ~400
~ F ~ A S ~ S
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
human 3 5 1 FSIHIRIVGLWESLFNACWDKQNACCCDKOEMDAWKLPKIAVDGS 400
b
mouse 4 0 1 Y E V V M L V G A G I G V T P F A S I L S W Y K Y C D ~ ? S L K L K K I Y P450
IIIIIIIIIIIIIIIIIIIIIIIIIIlI:III.IIIIIIIIIIIlIIIII
human 4 0 1
mouse
YEWMLVGAGIGVTPFASIWSWYKYCNNANATNLKLKKIY~
rl
450
4 5 1 F E W F A D L L Q L h G l Q ~ Y L ~ E S Q A N H F A V l & E E K D 500
IIIllIIIIIII.IIIIIIII.IIIIIIIIIIIIIIIIIIIIIIIIIIII
human 4 5 1 F ~ A D ~ L L E S o M Q E R N " I n n ; W D E S O A N H F A500
mouse
5 0 1 V I ~ ; L K Q K T L Y G R P I A S E ~ & ~ ? I G V F U S P ~ S K Q S 550
IIlIIIIIIIIIII1IIIIIIIIII:IIII IIIIIIIIIIIIIIIIIII
1 2 3 4 5 6 7 8
A
200
97.4
I
-
I
I
I
I
I
I
I
Lala''LADaW
68-
' +58
43-
human 5 0 1 v r ~ ~ Y ~ P " E F X T I A S Q H P ~ 1 ~ 550
~ ~ S K Q S
e
m u s e 5 5 1 ISNSESGPRGVHFIFNKENF 570
f
IIIIIIIIIIIIIIIIIIII
human 5 5 1 ISNSESGPRGVHFIF?XENF 570
Fig 1. Amino acid sequence of murine and human gp91*" subunits of phagocyte cytochrome &. The amino acid sequence of the
murine gp9lPh"" polypeptide as predicted from its cDNA sequence is
shown, and is aligned with the human gp91Ph0"forcomparison (solid
lines denote identical residues, double dots indicate high degree of
similarity, and single dots indicate lower degree of similarity). The
alignment was performed using the Genetics Computer Group software program, Best Fit. Hydrophobic regions are enclosed in shaded
rectangles. Potential N-linked glycosylation sequences (N-X-S/T) are
indicated in both the murine and human sequences using dashed
lines. Regions with homologies t o the FNR family of flavoenzymes
are indicated in the human sequence with solid lines, as follows: (a1
flavin isoalloxazine ring; lbl flavin ribityl chain; (c) NADP pyrophosphate; (d) NADP ribose; le1 NADP 2'-phosphate, NADP ribose 2'-phosphate adenine ring; (1)nicotinamide C 4 atom.
43
-
29
-
200
68
18.4
and ~22"~"'.Untransfected X-CGD PLB-985 cells lack
gp91ph""and display markedly reduced levels of ~ 2 2 " ~(Fig
"'
2A, lane 6 and Fig 2B, lane 5). NBT-positive transfectants
(Fig 2A, lanes 1 through 4) all expressed a protein of -58
kD that was immunoreactive with the gp9IPh"'antibody and
which comigrated with the gp91ph"' species detected in nonadherent murine BM cells (of which -50% are neutrophils
and -30% mononuclear phagocytes) (Fig 2A, lanes 7 and
8). There was some clone-to-clone variation in the level of
expression, which had previously been seen for expression of
recombinant human gp9 Ip"'" using this vector.'' The relative
level of recombinant murine gp9Ip""' was estimated to be
15% to 30% of that detected in murine BM cells. Murine
gp91"""' was also expressed in X-CGD PLB-985 cells using a
retroviral vector, MSCVneoEB. A (3418-resistant population
was collected for analysis. Membrane extract was prepared
from granulocyte-induced cells for immunoblot analysis of
gp9lP""' expression (Fig 2A, lane 5). which was comparable
1234 56
B
Fig 2.
I
I
I
I
I
I
- 1
lmmunoblot analysis of expression of murine cytochrome
& subunits in human PLB-985 cell lines and murine BM cells. Membrane or whole-cell extracts were prepared from granulocyte-induced
PLB-985 cells and derivatives and from wild-type mouse nonadherent
BM cells. Molecular-weight standards and the estimated size of proteins are indicated (kilodaltonsl. (A) Lanes 1through 4, pEFneo-m91transfected X-CGD PLB-985 (clones 1,4, 6, and 7, respectively); lane
5, MSCVneo-m91-transduced X-CGD PLB-985 (total G418-resistant
population); lane 6, X-CGD PLB-985; lanes 7 and 8, mouse BM cells.
Ten micrograms of membrane extract were loaded in lanes 1through
6. Whole-cell extracts were loaded in lanes 7 (2 pg) and 8 (5 pg). The
blot was probed with C-terminal antibody for gp91pho'. The band at
-110 kD in all PLB-985 membrane extracts (lanes 1 through 61 is
presumed t o be a cross-reacting protein unrelated t o gp91Pho",as it
is present in the X-CGD PLB-985 cell line and is only seen with the
C-terminal gp91Pho'antibody and not with other gp91Ph""-specificantibodies. (Bl Lanes 1through 4, pEFneo-m91-transfected X-CGD PLB985 (clones 1,4,6, and 7, respectively); lane 5, X-CGD PLB-985; lane
6, wild-type PLB-985. Twenty micrograms of whole-cell extract protein was loaded in each lane. The blot was probed with affinitypurified p22Ph0a
antibody.
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2008
ZHEN, AND DINAUER
BJORGVINSD~T~IR,
Tabla 1. Superoxide Generation by PLB-985 Cell Lines
After Granulocytic Differentiation
tion of fewer N-linked glycosylation sites is likely to explain
why the murine gp91PhoX
migrates with an apparent molecular
mass of -58 kD, compared with -91 kD for the human
Vmax
Cumulative 0;
(nmol 0;lminllo'
(nmol/30 mintlo'
protein. Upon deglycosylation, the murine and human core
Cell Line
cells)
cells)
N
proteins both migrate at -54 kD.32The C-terminal seWild-type
56 2 21
622 2 56
6
quences of gp91Ph" suggested to form the NADPH binding
0
0
4
X-CGD
site are all conserved in the murine sequence (Fig 1). Specific
X-CGD + pEFneo-m91
residues involved in flavin binding are less clear, but are
649 2 28
3
Clone 1
53 2 13
believed to reside within residues 290 through 390. This
645 2 16
3
Clone 4
66 2 28
region is identical in the mouse and human gp91Ph'" se653 4 27
3
Clone 6
48 i- 19
quences. There are only two nonconservative substitutions
68 2 21
715 2 43
3
Clone 7
within hydrophobic domains of the mouse and human proX-CGD + MSCVneo-m91
64 i- 22
654 4 57
2
teins. Some or all of these regions are likely to be transmemPLB-985 cell lines were induced to differentiate with 0.5% DMF for
brane or intramembranous domains in which the heme pros6 days. Superoxide generation in PMA-stimulated whole cells was
thetic groups are embedded. Raman and electron
measured by a continuous cytochrome c reduction assay. Data for
paramagnetic resonance spectroscopy have suggested that
superoxide formation are the mean 2 SD, and there were no statistical
the hemes are coordinated with imidazole or imidazolate
differences between wild-type PLB-985 cells and the pEFneo-m91transfected clones expressing murine gp9IPhox,using the unpaired
ligands supplied by histidine residues."' All histidines are
t-test.
conserved between the murine and human sequences (Fig
1). Finally, several regions within gp91Ph"' that may interact
with
(residues 86 through 93 and 450 through 457)''
gp9lpbXrestored respiratory burst activity to wild-type range,
are conserved between the mouse and human gp9Iph'" prowith no significant difference in either the maximal rate or
teins. The degree of similarity in a carboxy-terminal terminal
total amount of superoxide produced over 30 minutes. In
sequence of gp91Ph0"(residues 559 through 565), also impliaddition, no difference in the overall kinetics of superoxide
cated as a binding site for p47ph"x,34
cannot be
generation were noted in the hybrid mouse-human system
because most of this region of the murine sequence was
(not shown).
derived from the reverse PCR primer.
In the studies reported here, the murine gp91Ph" was capaDISCUSSION
ble of both rescuing stable expression of human ~ 2 2 ~
and~ "
of complementing other human oxidase subunits to fully
The phagocyte flavocytochrome b558 heterodimer funcreconstitute respiratory burst activity in human X-CGD cells.
tions as the redox center of the respiratory burst oxidase,
These observations strongly suggest that a functional heteromediating the transfer of electrons from NADPH to oxygen
dimeric complex is formed between murine gp9lPh'" and
upon translocation of cytosolic oxidase subunits to the
human ~ 2 2 subunits.
~ ~ " Moreover, the kinetics of superoxide
plasma membrane. Although the relative functions of each
formation
were
indistinguishable from wild-type human
subunit have not been fully defined, current data suggest that
PLB-985
cells,
suggesting
that the assembly of the multieach subunit contains a heme center and that both flavin and
subunit active oxidase complex is unaffected. These observaNADPH binding sites are localized to gp91ph"x.2~10~'5
The
tions are consistent with the conservation of sequences behuman gp91Ph" polypeptide has 570 amino acid residues,
tween human and murine gp9Iph""in domains likely to be
and contains multiple hydrophobic domains in addition to
involved
in redox function and oxidase assembly. These refive potential N-linked glycosylation sites29(Fig 1). No polysults also underscore the high degree of cross-species consermorphisms have been reported for the human sequence and
vation in the NADPH oxidase complex.
all identified amino acid substitutions have been associated
Other studies have also suggested that NADPH oxidase
with X-linked CGD, suggesting that there are stringent resubunits from different species have a high degree of similarquirements in the primary sequence to preserve structure
ity in both amino acid sequence and function. Comparisons
and/or function.'
of porcine and human ~ 2 2 showed
~ ~ "83%
~ amino acid simiHere we report cloning of a cDNA encoding the murine
larity,
and
of
murine
and
human
~
2
2
"
~
revealed
"
88% protein
gp91PhoX
subunit by RT-PCR, using oligonucleotide primers
id en tit^.",'^ Murine and human ~ 4 7 ~have
~ " "82% amino acid
derived from the human sequence. The predicted murine and
identity.38Antihuman antisera to the cytochrome bs58compohuman gp91ph" protein sequences differ by only 39 out of
nents cross-react with the guinea pig cytochrome subunits in
570 amino acids (93% identity) (Fig 1). Positions where
immunobl~ts.'~
Heterologous cell-free oxidase assays have
amino acid substitutions have been identified in X-CGD pademonstrated functional cross-species complementation betients are all conserved in the murine gp91Ph" protein.'
tween human B-cell cytosolic factors and bovine memAmino acids that are not conserved between the human and
branes, rabbit membranes, and bovine cytosolic factors, and
murine polypeptides are largely localized to hydrophilic remurine p47ph"*combined with human oxidase s~bunits.''.~".~'
gions of the polypeptide, including several substitutions that
Allogeneic BM transplantation has successfully been used
alter potential N-glycosylation sites (Fig 1). The murine
to treat some individuals with CGD and somatic gene thergp9lpbx protein contains four potential N-linked glycosylaapy is a potential treatment option in the future.42MSCV
tion sites, of which the two most carboxyl-terminal lie in
what is most likely an intracytoplasmic d ~ m a i n .Utiliza~ ~ . ~ ~ retroviral vectors have been shown to exhibit long-term ex-
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MURINE ~
~ cDNA 9
1
~
~
~
~
pression in vivo after transduction of murine hematopoietic
stem cells?' Although the level of expression of murine
gp9 lphoxconferred by the MSCVneo-m91 retroviral vector
was modest, high levels of respiratory burst oxidase reconstitution were observed in a human X-CGD cell line. We are
presently investigating the efficacy of this vector for phenotypic correction of the X-CGD mouse3' by gene transfer into
pluripotent hematpoietic stem cells.
ACKNOWLEDGMENT
We thank R. Hawley at the University of Toronto for the MSCV
neoEB plasmid, Mary Gifford for the mouse bone marrow extracts,
Ling Lin Li for help with MSCVneom91 transduced PLB-985 cells,
David Skalnik for review of the manuscript, and Donna Fischer for
assistance with manuscript preparation.
REFERENCES
1. Dinauer M: The respiratory burst oxidase and the molecular
genetics of chronic granulomatous disease. Crit Rev Clin Lab Sci
30:329, 1993
2. Parkos CA, Allen RA, Cochrane CG, Jesaitis AJ: Purified cytochrome b from human granulocyte plasma membrane is comprised
of two polypeptides with relative molecular weights of 91,000 and
22,000. J Clin Invest 80:732, 1987
3. Segal A: Absence of both cytochrome b-245 subunits from
neutrophils in X-linked chronic granulomatous disease. Nature
32638, 1987
4. Clark R,Volpp B, Leidal K, Nauseef W: Two cytosolic components of the human neutrophil respiratory burst oxidase translocate
to the plasma membrane during cell activation. J Clin Invest 85:714,
1990
5. Heyworth P, Curnutte J, Nauseef W, Volpp B, Pearson D,
Rosen H, Clark R: Neutrophil nicotinamide adenine dinucleotide
phosphate oxidase assembly. J Clin Invest 87:352, 1991
6. Quinn M, Evans T, Loetterle L, Jesaitis A, Bokoch G: Translocation of rac correlates with NADPH oxidase activation. J Biol Chem
268:20983, 1993
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1996 87: 2005-2010
Cloning of murine gp91phox cDNA and functional expression in a
human X- linked chronic granulomatous disease cell line
H Bjorgvinsdottir, L Zhen and MC Dinauer
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