Biochem. J.

(2014) 462, 337345 (Printed in Great Britain)

337

doi:10.1042/BJ20131620

Recombinant form of mammalian gp91phox is active in the absence of p22phox

Aymen EZZINE*, Hager SOUABNI*, Tania BIZOUARN* and Laura BACIOU*1

p22phox . Collected information on the maturation and the activity

of the recombinant gp91phox and the participation of individual
cytosolic subunits in the active complex allowed us to propose,
in the absence of p22phox , an unconventional stabilized complex
compared with the heterodimer.

INTRODUCTION

However, until now, no heterologous expression of an active form

of the monomer gp91phox has been described.
Using a yeast heterologous expression system, we aimed at
producing gp91phox and p22phox in distinct cells in order to better
understand their relationship. To our surprise, we engineered
an active recombinant monomeric gp91phox . Moreover when
reconstituted in liposomes, it had NADPH oxidase activity with
no prerequisite for other protein partners. Our findings also shed
a new light on the proteinprotein interactions essential for their
membrane recruitment.

The NADPH oxidase complex generates ROS (reactive oxygen

species) by transferring electrons from NADPH across biological
membranes to dioxygen. These reactions occur through the
membrane catalytic subunit which presents conserved structures
in all members of the NADPH oxidase family [1,2]. It harbours
an NADPH- and a FAD-binding site in the cytosolic C-terminus
region and two non-identical b-type haem groups [3,4] localized
in the six-transmembrane domain that mediate the final steps
of electron transfer to molecular oxygen [5]. In phagocytes,
NOX2 (NADPH oxidase 2), also known as gp91phox , is a highly
glycosylated protein [6] and is localized in plasma membranes
in close association with its membrane partner p22phox [79],
together forming the so-called cytb558 (flavocytochrome b558 ).
NOX2 appears to be the most widely distributed among the NOX
isoforms and has been localized in a large number of cells and
tissues (phagocytes, neurons, cardiomyocytes, endothelial cells
etc.) [10]. The stimulus-dependent activation of gp91phox occurs
through multiple proteinprotein interactions with cytosolic
factors (p67phox , p47phox p40phox and a small GTP-binding protein,
Rac1/Rac2). Previous studies demonstrate that phagocytes from
p22phox -deficient patients have no detectable gp91phox [1113].
In transfected CHO (Chinese-hamster ovary) cells, Dinauer and
colleagues demonstrated that p22phox -dependent maturation of
gp91phox carbohydrates, cell surface expression of gp91phox and the
enzymatic function of cytb558 are relatively correlated. In these
cells, low levels of gp91phox are expressed at the cell membrane
in the absence of p22phox co-expression [14] and it was concluded
that when p22phox is absent, no oxidase activity was possible [15].
Until now, all studies and interpretations of the phagocyte NADPH
oxidase function have agreed that the presence of the p22phox
membrane subunit is crucial for the activity of gp91phox [14,16].
Nevertheless, many studies demonstrated that the C-terminally
truncated forms of gp91phox obtained in heterologous expression
systems can perform diaphorase activity by electron and protons
transfer from NADPH to FAD in the absence of p22phox [17,18].

Key words: heterologous expression, NADPH oxidase,

Pichia pastoris, post-translational modification, proteinprotein
interaction, reactive oxygen species (ROS).

MATERIALS AND METHODS

Expression of bovine gp91phox in Pichia pastoris cells

We created the transgenic yeast strains X33/ gp91, X33/ p22

and X33/ gp91-p22 by the integration of recombinant vectors
in the genomic DNA of Pichia pastoris cells strain X33 (Life
Technologies). The coding sequences of the bovine gp91phox
and p22phox were subcloned separately into pPICZA vectors
at the XhoI and XbaI restriction sites, in-frame with the factor signal peptide (to enhance the membrane addressing of the
recombinant proteins) under the control of the AOX1 (alcohol
oxidase 1) promoter. Then the expression cassette from the
recombinant pPICZA/p22phox was cut out using BamHI/BglII
restriction enzymes and then inserted at the BamHI site of
the recombinant pPICZA/His-gp91phox to obtain the chimaeric
vector containing the two expression cassettes (containing their
own AOX1 promoter) in the same vector. Transgenic strains
were confirmed by PCR amplification using specific primer sets
described previously [19]. Expression of recombinant proteins
was carried out by growing the transgenic yeast clones under
methanol induction for 72 h as described previously [19]. A
negative control X33/ strain was generated by transforming X33
cells with the empty vector pPICZA. After culture, harvested
cells were stored at 80 C.

Abbreviations: AA, arachidonic acid; AOX1, alcohol oxidase 1; CHO, Chinese-hamster ovary; cytb 558 , flavocytochrome b 558 ; DDM, n-dodecyl-DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPI, diphenyliodonium; FRE1, ferric reductase 1; Ni-NTA, Ni2 + -nitrilotriacetate; NOX2,
NADPH oxidase 2; PL, proteoliposome; PNGase F, peptide N-glycosidase F; SH3, Src homology 3; SOD, superoxide dismutase.
1
To whom correspondence should be addressed (email laura.baciou@u-psud.fr).
D-maltoside;


c The Authors Journal compilation 
c 2014 Biochemical Society

Biochemical Journal

The flavocytochrome b558 of the phagocyte NADPH oxidase

complex comprises two membrane proteins, a glycosylated
gp91phox and a non-glycosylated p22phox . Gp91phox contains all
of the redox carriers necessary to reduce molecular oxygen to
superoxide using NADPH. The capacity of gp91phox to produce
superoxide in the absence of its membrane partner p22phox has been
little studied. In the present study, we have generated in Pichia
pastoris for the first time an active form of bovine gp91phox able
to carry out the entire NADPH oxidase activity in the absence of

www.biochemj.org

*Laboratoire de Chimie Physique, Universite Paris Sud, UMR8000 CNRS, Bat. 350, 91405 Orsay Cedex, France

338

A. Ezzine and others

Membrane preparation and protein analysis

Cell pellets were thawed, broken using glass beads and membrane
fractions were collected by centrifugation at 100 000 g for 120 min
at 4 C. The pelleted membrane fractions were resuspended in
50 mM Tris/HCl (pH 8), 120 mM NaCl, 10 % glycerol and 1 mM
PMSF and loaded on to a discontinuous sucrose gradient (60 %,
40 % and 20 % sucrose) as described previously [20]. After
overnight centrifugation at 110 000 g, the essential pure plasma
membranes were removed from the 60 % and 40 % interface,
diluted 4-fold and pelleted at 130 000 g for 90 min. The cytb558 enriched plasma membrane fraction were resuspended in 1 mM
EDTA, 30 % sucrose and 20 mM Tris/HCl (pH 8.0) and stored
at 20 C. Reduced-minus-oxidized spectra were performed
using a double beam Uvikon 943 spectrophotometer (Kontron
Instruments). Excess dithionite was added to the cuvette to
reduce the sample before obtaining the spectra. The absorbance
difference between the peak at 428 nm and the trough at
411 nm was used to determine the cytb558 concentration using
the molar absorption coefficient 428411 = 200 mM 1 cm 1 [21].
Deglycosylation was performed with the PNGase F (peptide Nglycosidase F; New England Biolabs) treatment as described by
the manufacturer.
For the Western blotting assay, anti-His antibody conjugated to
HRP (horseradish peroxidase; Clontech), rabbit anti-gp91 (54.1)
and anti-p22 (FL-195) antibodies (Santa Cruz Biotechnology)
were used for specific identification at a dilution of 1:1000. Antirabbit (NA934V; GE Healthcare) and anti-mouse (NA931; GE
Healthcare) IgG monoclonal antibodies were used at a 1:15 000
dilution as secondary antibodies to detect the anti-gp91 and antip22 primary antibodies respectively. Signal development was
performed by the ECL Plus Advance Western Blotting Detection
Kit (GE Healthcare).
NADPH oxidase activity in cell-free assay

Plasma membranes isolated on sucrose gradient or PLs

(proteoliposomes) were used for the oxidase activity assays.
Unless indicated, 2.24 nM gp91phox was mixed with recombinant
cytosolic factors [p47phox (0.89 M), p67phox (0.42 M) and
Rac1Q61L (0.34 M)] and AA (arachidonic acid; Sigma
Aldrich) at the required concentrations in a total reaction volume
of 500 l. Cytosolic proteins were prepared as described in [22].
After 5 min of incubation at 25 C, the superoxide production
was initiated by the addition of 400 M NADPH and followed
by the SOD (superoxide dismutase)-inhibitable reduction of
ferricytochrome c (100 M). The rate of generated superoxide
was calculated using a value of  550nm (cytc) of 21.1 mM 1 cm 1
[23]. The plasma membrane fraction isolated from the Pichia
strain transformed by the empty vector was used as control for the
cell-free oxidase activity under the same experimental conditions
as described above.
Detection of cytosolic proteins interacting with gp91phox membrane

The cytosolic proteins were incubated combined or separately

for 5 min at 25 C with 3 g of membrane fraction containing
gp91phox (2.24 nM, 0.16 g) with 650 M AA. Cytosolic proteins
were used at the following concentrations: p47phox (0.89 M),
p67phox (0.42 M) and Rac1Q61L (0.34 M). The protein mixture
was loaded on to a sucrose gradient (20 60 %). After overnight
centrifugation at 110 000 g, the membrane-assembled and nonassembled fractions were separated. Co-localized cytosolic
subunits with membrane were found in the pellet and soluble
(unassembled) proteins remained mostly in the supernatant. The

c The Authors Journal compilation 
c 2014 Biochemical Society

pellet and supernatant were analysed by Western blotting using

mouse anti-human p47phox , mouse anti-human p67phox (kindly
provided by Dr Florence Lederer) or mouse anti-Rac1 (ARC03,
Cytoskeleton) antibodies.

Purification of the gp91phox and PL reconstitution

Gp91phox -containing membranes were solubilized with 39 mM

DDM (n-dodecyl--D-maltoside; 2 %). The extract was
purified using a column of Ni-NTA (Ni2 + -nitrilotriacetate)
superflow Sepharose (GE Healthcare) followed by gel-filtration
chromatography using S200 Sephadex resin (GE Healthcare)
in TBS buffer [20 mM Tris/HCl (pH 7.5) and 50 mM NaCl]
supplemented with 0.49 mM (0.025 %) DDM. The purity of the
protein was checked by SDS/PAGE (10 % gel).
To reconstitute the purified protein into PLs, a stock solution
of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine; P6354;
SigmaAldrich; purity 99 %) was diluted in PBS (pH 7.4) and
sonicated to induce the formation of lipid vesicles. Purified His
gp91phox (200 g) was mixed with vesicles to obtain a 5:1 lipid
to protein ratio (w/w). Detergent was removed by incubating the
proteoliposome mixture with 1 % (w/v) Bio-Beads SM2 (BioRad Laboratories) under rotating condition over 2 h at room
temperature.

RESULTS
Gp91phox maturation is partially determined by the presence of
p22phox

On the basis of the successful expression of bovine cytb558

in P. pastoris [19], we intended to improve the expression
level of recombinant proteins by using another Pichia strain
(X33). X33/ gp91, X33/ p22 and X33/ gp91-p22 transgenic clones
were generated by integration of the coding sequences of the
bovine gp91phox and p22phox , separately or simultaneously, into
the genomic DNA of X33 cells using the pPICZA vector
(Figure 1A). For X33/ gp91 and X33/ gp91-p22 cell membranes,
we have clearly identified the characteristic peaks of the neutrophil
cytb558 in the reduced-minus-oxidized difference spectra; the
Soret band at 428 nm and band at 558 nm indicating haem
incorporation in gp91phox proteins (Supplementary Figure S1
at http://www.biochemj.org/bj/462/bj4620337add.htm). Unfortunately these peaks were not easily distinguishable from similar,
but smaller, peaks measured in the membrane spectrum of the
negative control strain containing the empty vector (X33/ ) and
which have been attributed to the endogenous FRE1 (ferric
reductase 1) protein described in yeast cells [24]. Interestingly,
we observed that the contribution of FRE1 to the spectra was
largely decreased in X33/ p22 cell membranes suggesting that
heterologous expression of membrane proteins that are targeted
to the yeast plasma membranes may reduce the presence of FRE1.
Analysis of transgenic cell membrane contents showed that
the expression of gp91phox protein occurred under different
forms depending on the presence or absence of p22phox protein
(Figure 1B). After 72 h of methanol induction, when co-expressed
with p22phox , gp91phox was predominantly in two N-glycosylation
states (80 and heavily glycosylated >100 kDa) as shown
previously [19]. However, without p22phox gp91phox was found with
molecular masses of approximately 65 and 80 kDa (Figure 1B).
The 80 kDa form is likely to correspond to the above mentioned
glycosylated form, whereas the 65 kDa form might correspond
to the theoretically expected size (68 kDa) of the gp91phox
protein without post-translational modifications. In support of

Functional gp91phox in the absence of p22phox

Figure 1

339

Constructs in X33 Pichia strains and analysis of the expression and glycosylation of recombinant gp91phox

(A) Transgenic Pichia strains X33/ Hisgp91, X33/ Hisp22 and X33/ Hisgp91/p22His were obtained by the integration of the respective recombinant vectors pPICZA/His-gp91phox ,
pPICZA/His-p22phox and a chimaeric pPICZA/His-gp91phox /p22phox in the genomic DNA of P. pastoris cells strain X33 respectively. (B) Western blots analyses (anti-His antibodies) of
membrane fractions. Lane 1, X33/ (control strain containing the empty vector); lane 2, X33/ gp91; and lane 3, X33/ gp91p22. All fractions were recovered from cells cultured for 72 h. (C)
Deglycosylation of the recombinant gp91phox with the PNGase F. The membrane expressing gp91phox (lane 1) was treated by 10 units of enzyme PNGase F under denaturing conditions (lane 2).
Proteins were detected by Western blot analysis using the monoclonal anti-gp91 antibody (54.1).

this hypothesis, PNGase F treatment followed by Western blot

analysis using anti-gp91 monoclonal antibodies (Figure 1C) gave
rise to the major 65 kDa deglycosylated form.
These findings demonstrate that an incomplete glycosylation of
gp91phox subunit correlated with the absence of the p22phox protein.

activity assay mixture with the X33/gp91 membrane (Figure 2C),

although at a higher concentration than for neutrophil cytb558 . It is
likely that midpoint redox potentials of FAD (Flox /Flred ) might be
different in gp91phox without p22phox and thus might have modified
the kinetics of inhibition.

Functional activity of gp91phox in the absence of p22phox

How does gp91phox interact as a monomer with the cytosolic

partners?

In the literature it has been proposed that gp91phox is not functional

without p22phox [15]. To find out whether this is also the case
for Nox2 expressed in P. pastoris, the capacity to produce
superoxide by the recombinant gp91phox monomer was tested
and compared with the X33/ gp91p22 heterodimer under the
same cell-free assay conditions, i.e. incubation with an anionic
amphiphile AA (commonly used as an NADPH oxidase activator
in cell-free systems) and the cytosolic partners (p67phox , p47phox
and Rac). Surprisingly, a significant rate of superoxide was
measured with X33/ gp91 membranes, whereas the control X33/
membranes, also tested for superoxide production under the same
experimental conditions, showed no SOD-inhibitable NADPH
oxidase activity. We therefore investigated more thoroughly the
behaviour of X33/ gp91 membranes and their activation by AA.
The X33/gp91phox membranes displayed the AA-dependent bellshaped activation curve (Figure 2A) commonly described for
neutrophil NADPH oxidase, except that a lesser amount of
AA, compared with X33/gp91p22 membranes, was needed for
optimal oxidase activity.
To further characterize the recombinant form of gp91phox , we
determined its kinetic parameters deduced from the Michaelis
Menten fitting of the NADPH concentration dependence of
oxidase activity (Figure 2B). We obtained a K m value of 20 M
and an average turnover of 181 mol of superoxide/s per mol
of gp91phox . These values are in the range of those obtained
for cytb558 neutrophils [25,26] or recombinant heterodimer (H.
Souabni and L. Baciou, unpublished work) indicating that the
absence of p22phox did not affect the apparent substrate affinity
and the superoxide-generation capacity. Known as a good in vitro
inhibitor of NADPH oxidase, DPI (diphenyliodonium) showed
an inhibition effect on the oxidase activity when added in the

The existence of an active monomeric gp91phox in Pichia

membranes brings, for the first time, the opportunity to investigate
its interactions with the cytosolic proteins individually omitting
the interactions related to p22phox . For that purpose, the activation
of gp91phox by each cytosolic partner singly or by pairs was
measured (Figure 3A). In the absence of the in vitro stimulant
AA, but in combined presence of all cytosolic partners, almost
no activity was detected confirming the prerequisite of the
presence of AA for optimal oxidase activity. AA, itself, leads to a
basal activity of approximately 20 % compared with the entire
complex although statistical analysis indicates no significant
difference with the activity in the presence of only cytosolic
partners (2 %).
In the presence of AA, when p47phox or Rac1 were added as the
sole cytosolic partner, the oxidase activity reached approximately
40 % and 60 % compared with the entire complex respectively.
In contrast, the presence of p67phox alone had no significant effect
suggesting an AA-inactive form of p67phox . The total activity
was recovered only when p67phox and Rac1 partners were present
together, although the couples of p47phox /p67phox or Rac1/p47phox
both restored high oxidase activities.
To correlate gp91phox activation by individual interactions with
cytosolic proteins, we performed co-localization experiments
(Figure 3B). In these experiments, Western blots were performed
to identify if the cytosolic subunits individually or together
translocate to X33/gp91phox or X33/ membranes. The p47phox ,
p67phox and Rac proteins were incubated either individually
or together with the membrane fractions in the presence of
AA. Then, the membrane-translocated cytosolic proteins were
separated from unassembled proteins on a sucrose gradient. The

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340

Figure 2

A. Ezzine and others

Functional properties of the recombinant gp91phox

Activity assays were performed in a cell-free system using membrane fractions expressing gp91phox (2.24 nM) incubated for 5 min in the presence of recombinant cytosolic proteins [p47phox
(0.89M), p67phox (0.42 M) and Rac1Q61L (0.34 M)]. All results in this Figure are means +
S.D. of three independent experiments (n = 3). (A) Activation of NADPH oxidase complex by AA of
gp91phox and of gp91phox /p22phox . Activities with gp91phox (black line) or heterodimer gp91/p22 (dashed line) membrane fractions (3 g and 8.25 g of total membrane proteins respectively) were
determined using standard cytochrome c assays, as described above, after the addition of 400 M NADPH at different cis -AA concentrations. (B) Kinetic parameters of the gp91phox . Activity assays
were performed in a cell-free system in the presence of cis -AA (650 M). The reaction was initiated by the addition of increasing concentrations of NADPH in the presence of 100 M cytochrome c .
The fitting of the determined values and K m and V max calculation were performed by Origin software. (C) NADPH oxidase inhibition by DPI. Two different concentrations of DPI (200 and 400 M) were
used to determine its inhibitory potential on the NADPH oxidase activity. Assays were carried out under the same conditions as described above except that an optimal concentration of AA (650 M)
was used. Results are means +
S.D. of three independent experiments (n = 3). Statistical analysis for multiple comparisons was performed with GraphPad software. ***P < 0.001, compared with
the control using one-way ANOVA followed by Dunnetts test.

membrane co-migrating proteins (assembled fraction) were found

in the pellet, whereas the unbound proteins remained in the
supernatant. Rac, partially p47phox and to a less extent p67phox
co-localized with the membranes of X33/gp91phox , whereas in the
same experiments using X33/ membranes p47phox and p67phox
do not translocate with the P. pastoris membranes. In contrast
Rac interacts with X33/ membranes probably through unspecific
polybasic domain interaction. When all cytosolic proteins (p47phox ,
p67phox and Rac) are added together, p47phox and p67phox colocalized more efficiently with the X33/gp91phox membranes. The
absence of co-localization with the X33/ membrane indicates that
the interactions of the cytosolic subunits with the X33/gp91phox
membrane were specific to the presence of gp91phox .
These findings corroborate the activation process of individual
cytosolic protein described above, since the low activity detected
in the presence of AA-activated p67phox with gp91phox derived
from its low capacity to develop proteinprotein interactions with
gp91phox . In the absence of p22phox , the active complex interactions
might have been modified in which Rac appeared to be a good
stabilizer of the complex.

c The Authors Journal compilation 
c 2014 Biochemical Society

Is the recombinant gp91phox still active after detergent extraction

and relipidation?

To assess the stability of gp91phox in the presence of commonly

employed detergent, the protein was solubilized in DDM, purified
and the absorption spectrum of the purified protein was recorded.
The purified protein has been identified by Western blot analysis
using anti-gp91phox monoclonal antibodies (Figure 4).
To measure the oxidase activity of gp91phox following
purification, different experimental conditions were performed.
The purified gp91phox in detergent showed almost no activity
(Table 1). However, when complemented with the X33/ and
X33/ p22 membranes (which are inactive and have no capacity
to produce any superoxide anions) by incubation for 30 min
with DDMgp91phox , 60 % and 75 % of the NADPH oxidase
activity was restored respectively (Table 1). This finding indicates
a successful rescue of the oxidase activity of the non-active
DDMgp91phox by its membrane re-insertion. Moreover, the
complementation with p22phox -containing membranes leading to
higher oxidase activity suggests possible interactions of both

Functional gp91phox in the absence of p22phox

Figure 3

341

Activation of gp91phox by the cytosolic subunits and co-sedimentation analysis with membranes expressing gp91phox

In a 500 l total reaction volume, 3 g of membrane protein fractions (containing 2.24 nM of gp91phox ) were incubated for 5 min in the presence of recombinant cytosolic proteins [p47phox (0.89 M)
and/or p67phox (0.42 M) and/or Rac1Q61L (0.34 M)]. (A) Superoxide production determined in a cell-free system with alternative presence of cytosolic subunits. Results are means +
S.D. of three
independent experiments (n = 3). Statistical analyses for multiple comparisons were performed with GraphPad software using one-way ANOVA followed by Tukeys test in order to pinpoint where the
real differences lie. ***P < 0.001 and *P < 0.05 compared with the control (entire complex); + + P < 0.01 for comparison within each group (i.e. only one cytosolic protein added or two by two);
and $P < 0.05 and $$$P < 0.001. n.s., not significant difference. (B) The cytosolic proteins were incubated independently or together in the presence of AA (650 M) with either X33 /gp91phox
or X33 / membranes. After sucrose gradient separation, the cytosolic proteins translocated to the pelleted membrane (membrane-assembled fractions; AF) or remained in the supernatant of the
sucrose gradient (S) were revealed by Western blotting with anti-p47phox , anti-p67phox or anti-Rac1 antibody. Experiments were repeated twice.

membrane proteins and, furthermore, underlines the putative

effect of p22phox on gp91phox activity.
The purified DDMgp91phox protein was also incorporated into
DOPC lipid bilayers previously shown to allow NADPH oxidase
activity [19]. The liposome reconstitution restored the SODsensitive NADPH oxidase activity and the similar AA-dependent
bell-shaped activity as for X33/gp91 membranes, except that no

cytosolic proteins were necessary (Figure 5). Incubation with or

without cytosolic proteins did not improve the NADPH oxidase
activity. Our results show that the superoxide production detected
in yeast X33/gp91 membranes does originate from the monomeric
gp91phox , which displays kinetic properties very similar to that
of neutrophils or recombinant heterodimer p22phox gp91phox and
can be activated in a reconstituted lipid environment by AA

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342

A. Ezzine and others

Table 1

Functional analysis of membrane-re-inserted purified gp91phox

Purified fractions of gp91phox were incubated with membranes prepared from X33 / cells or
X33 cells expressing only p22phox (X33/ p22) for 45 min and then used for cell-free assays.
The same quantity of protein was used in all measurements. In addition, we used the maximum
concentration of activating AA (650 M) and NADPH (400 M) in these measurements. Activity
measurements with the dimer gp91p22 were considered as the reference for 100 % activity.
Type of membrane tested DDMgp91phox Mol of O2 /s per mol of gp91phox Activity (%)

Figure 4

Purification and identification of the recombinant gp91phox subunit

(A) SDS/PAGE analysis. The total membrane fraction (25 g; lane 1) was solubilized in 2 %
DDM and the recombinant gp91phox protein was purified on a Ni-NTA column (lane 2) followed
by gel filtration (15 g; lane 3). Gels were stained with Coomassie Blue. (B) The characteristic
peaks of the purified protein were identified in the reduced-minus-oxidized spectral analysis
using sodium dithionite. (C) Following SDS/PAGE separation of 15 g of purified protein and
transfer on to membranes, specific identification by the monoclonal anti-gp91 antibody (54.1;
Santa Cruz Biotechnology). M, Magic Mark standard protein (Invitrogen) used as a molecular
mass reference.

alone. These results stressed the importance of the membrane

environment for the stability and activity of gp91phox .

X33 /
X33 /p22
X33 /
X33 /p22
X33 /gp91p22
X33 /gp91

+
+

7.5 +
5.4
8+
6.3
116.1 +
3.2
149.2 +
4.5
198.5 +
7
181 +
5
20 +
3.6

3.7
4
58.4
75
100
91.4
10

The heterologous expression of gp91phox reported to date produced

only truncated forms containing the C-terminal domain of gp91phox
that exhibits diaphorase activity [17,18]. On the basis of a new
expression of cytb558 in the methylotrophic yeast P. pastoris [19],
we produced separately gp91phox and p22phox and provided a model
for the study of the activation process involving only the catalytic
subunit gp91phox .
p22phox is not required but contributes to gp91phox glycosylation

DISCUSSION

In phagocytes, cytb558 is the key component of the NADPH

oxidase complex since it contains all redox intermediates for
electron transfer and can produce superoxide in the absence
of the other cytosolic partners [2729]. However, there was no
evidence to date that gp91phox can produce superoxide alone
independently of p22phox association. Rather it was postulated
that the heterodimerization was essential for functional cytb558 .

Figure 5

The impact of the presence of p22phox on the expression and

maturation of gp91phox was studied by immunoblot analysis. As
shown in Figure 1, the co-expressed p22phox and gp91phox in yeast
results in a mature glycosylated (mannosylation) form of gp91phox
that migrates as a 100 kDa protein as previously described in [19].
The absence of p22phox failed to promote the maturation of gp91phox
carbohydrate at the same level. Instead the mature glycosylated
form of gp91phox migrates as an 80 kDa protein that is shifted to
a focused 65 kDa band after deglycosylation. The heterodimer

NADPH oxidase activity of the reconstituted gp91phox in DOPC liposomes

Measurements of the superoxide production by PLs (2.24 nM of gp91phox ) were performed in a cell-free system using a standard cytochrome c reduction assay in the absence of any cytosolic
proteins and increasing concentrations of AA. For comparison the activation curve obtained with the gp91phox membrane fraction (Figure 2A) is also shown. Results are means +
S.D. (n = 3).

c The Authors Journal compilation 
c 2014 Biochemical Society

Functional gp91phox in the absence of p22phox

formation is not required to initiate the maturation and to target

the protein to the plasma membrane, but increases significantly
the abundance of the mature gp91phox glycoprotein [3033]. In
contrast with what was observed in CHO gp91 cells [31,34], in
Pichia the absence of p22phox does not prevent the glycosylation,
but affects its pattern (80 kDa instead of 100 kDa) and does not
reduce the expression of the mature gp91phox and its targeting
to plasma membranes. A possible explanation could be that, in
P. pastoris, a p22phox -like protein could replace p22phox in the
maturation process of gp91phox . We have performed bioinformatics
analyses (BLAST) to identify a putative p22phox -like protein in the
P. pastoris genome; however, no significant homologies have been
found. In addition Western blots using anti-p22phox monoclonal
antibody have been performed with the P. pastoris membrane that
does not express p22phox (X33/ control membranes). Under those
experimental conditions, no signals were detected indicating, at
the least, no recognition of p22phox epitopes. We also observed for
gp91phox , but not for the heterodimer, a decrease in thermal stability
and duration of the oxidase activity, but also enhancement of
sensitivity to proteolysis. These observations are consistent with
the idea that, in the absence of heterodimerization in COS cells,
the low expression of the mature form of gp91phox (91 kDa) is due
to protein instability [33].
Although the glycosylation pattern associated with mammalian
proteins from P. pastoris is different from that of higher
mammalian proteins, the question remains whether the different
glycosylation pattern are dependent of the presence of p22phox .
Three glycosylation sites (Asn131 , Asn148 and Asn239 ) have been
identified in two different extracellular loops of gp91phox (loops
C and E) [35]. An additional glycosylation site in loop C was
discovered for patients with variant CGD (chronic granulomatous
disease) in which p22phox was absent [36]. We can speculate
that the locations of glycosylation (i.e. external loops) point to
structural elements that are functionally linked to the gp91phox
p22phox association processes. This hypothesis is supported by
modelling approaches that identified a large extracellular loop
in p22phox as an extensive interacting region with gp91phox
[37]. We propose that the proteins expressed in yeast benefit
heterodimerization processes which adapt to the evolving glycan
shield.

343

residues in the vicinity of the glycosylation site. The modulation

of local structure might serve to enhance the overall stability of
gp91phox or might enable gp91phox to perform some required
function at given sites, such as NADPH or cytosolic protein
binding. Hence, this can explain the ability of this new form
of gp91phox to produce superoxide in the absence of p22phox .
Alternatively, it has been reported in the literature that the
presence of anionic phospholipids promotes NADPH oxidase
activity [27,40,41]. Comparison of anionic phospholipid content
between plasma membranes indicates a higher content of
negatively charged phospholipids in yeast [4244] than in
neutrophils [45]. This would suggest that P. pastoris membranes
provide, naturally, a remarkable environment for gp91phox
activation and may reflect the phagocyte-activated state of gp91phox
observed within mammal cells, which have been shown to change
its lipid composition during neutrophil activation.
Following detergent extraction, the relipidation with
zwitterionic DOPC rescued NADPH oxidase activity. However,
this enzyme activity depends only on the presence of the AA
as activator. This result suggests that, in the liposomes, anionic
phospholipid contaminants might be present arising either from
impure DOPC preparations, as was previously shown by Koshkin
and Pick [27] for relipidated cytb558 in PC (phosphatidylcholine)
vesicles, or from P. pastoris lipids co-purified with gp91phox .
The mixing assay of DDMgp91phox purified with X33/ and
X33/p22 membranes is suitable for proper assembly of a
functional heterodimer and leads to superoxide production. The
restored NADPH oxidase activity was higher with membrane
containing p22phox , but still did not reach X33/gp91p22 or
X33/gp91membrane activity levels. This result raises the question
of a structural role for p22phox and of the membrane allowing a
restructuration of gp91phox that was inactive in detergent. It has
been shown that structural changes in cytb558 are observed by cisAA activation [4648], but not by the isomer trans-AA [20],
underlining important structural determinant in the activation
process. The present study provides strong evidence that AA can
directly target and activate gp91phox .

Interaction pattern of gp91phox with oxidase cytosolic factors

p22phox is not required for superoxide production
phox

Previous studies have shown that p22 is functionally important

for enzyme activity and that particular regions, such as the Nterminal 11 amino acids and the C-terminal proline-rich-region
of p22phox , are required [15]. We have studied the impact of
the absence of p22phox on the capacity of gp91phox to produce
superoxide. In contrast with all expectations, gp91phox exhibited
a classical NADPH oxidase activity. When expressed in Pichia
in the absence of p22phox , gp91phox showed kinetic properties (K m
value of 20 M and turnover of 180 s 1 ) similar to what is
measured in neutrophils [38] or with recombinant heterodimer
(H. Souabni and L. Baciou, unpublished work), showed also
sensitivity to DPI and was activated by the cytosolic proteins
and AA. Similarly, the AA activation follows a characteristic
bell-shape.
The active form of gp91phox in the absence of p22phox might
be correlated with structural changes mediated by different
glycosylation levels. As reviewed by Imperiali and OConnor
[39], glycosylation can affect the local secondary structure of
proteins by facilitating the formation of segments of secondary
structure and can play a crucial role in directing the protein folding
pathway. In turn, glycosylation can increase the stability of the

Because p22phox possesses a C-terminal proline-rich region known

to provide a high affinity for the SH3 (Src homology 3)
domains of p47phox during NADPH oxidase assembly [49,50],
we studied to what extent the interaction of monomeric gp91phox
with the cytosolic partners has been modified. This behaviour
was important to clarify since we provide for the first time the
opportunity to highlight the possible interactions of the cytosolic
partners with gp91phox in the absence of p22phox . Our data show
that in the Pichia membrane, p67phox alone cannot activate gp91phox
(absence of a membrane attachment signal), but are required to be
tethered by Rac. This is consistent with in vitro experiments and
the proposed model in which Rac must associate simultaneously
both with p67phox and the membrane to activate NADPH oxidase
[51,52]. However, our data also showed that the presence of
p47phox , and in particular Rac, alone enhances substantially the
production of superoxide anion by gp91phox expressed in P.
pastoris membranes. We also observed that gp91phox in the P.
pastoris membrane can be activated with quasi-equal potency by
AA combined with Rac and p47phox or p47phox and p67phox . We
can conclude that, in the absence of p22phox , in the P. pastoris
membranes Rac and p47phox are the essential elements for the
stabilization of an active complex.

c The Authors Journal compilation 
c 2014 Biochemical Society

344

A. Ezzine and others

Figure 6 Schematic representation of the active gp91phox subunit and potential interactions with cytosolic proteins in the absence of p22phox in P. pastoris
membranes
The p67phox subunit bind to the dehydrogenase domain allowing interaction of its activation domain (AD) required for NADPH oxidase activation [53], but, in the absence of p22phox , it must be
tethered (Rac or p47phox ) to be stabilized at the membrane. This can be accomplished via a tail-to-tail interaction involving the SH3 domain of p67phox and the proline-rich region (PRR) in the
C-terminal region of p47phox . p67phox contains four tetratricopeptide (TPR) motifs which create a binding surface for activated Rac. p47phox contains the regulatory PX (phox homology) domain that
binds phosphoinositides as well as tandem SH3 domains for binding its target PxxP motif in p22phox . In the absence of p22phox , the p47phox subunit might interact directly with gp91phox through two
putative binding sites of in the C-terminal region of gp91phox as predicted in [54].

In P. pastoris membranes, the interactions are likely to be

different compared with the scheme currently proposed when
p22phox is associated to gp91phox . As shown in Figure 6, we propose
a model in which gp91phox acts as the central docking component
interacting directly with the cytosolic proteins p47phox , p67phox
and Rac, independently of the presence of p22phox . Despite being
unconventional, this assembly leads to active NADPH oxidase.
This indicates that the presence of p22phox offers interacting
domains to stabilize p67phox on gp91phox . In turn, the Rac protein
serves as a membrane tether for the juxtaposition of p67phox
to gp91phox protein. Thus, in addition to its active form, this
recombinant gp91phox conserves and consolidates the potential
interactions with their partners in the oxidase complex. The
proposed proteinprotein interaction might help to extend our
understanding of the molecular mechanisms that govern oxidase
regulation in particular for Nox proteins such as Nox5, where no
p22phox , was identified or Nox4, where the presence of p22phox is
not necessary.

AUTHOR CONTRIBUTION
Aymen Ezzine and Laura Baciou designed the research; Aymen Ezzine and Hager Souabni
performed the research; Aymen Ezzine, Hager Souabni, Tania Bizouarn and Laura Baciou
analysed the data; and Aymen Ezzine and Laura Baciou wrote the paper.

ACKNOWLEDGEMENT
We thank Florence Lederer for stimulating discussions and careful reading of the paper
before submission.

FUNDING
This work was supported by the Agence Nationale de la Recherche [project number
ANR-2010-BLAN-1536-01].

c The Authors Journal compilation 
c 2014 Biochemical Society

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Received 10 December 2013/27 May 2014; accepted 3 June 2014

Published as BJ Immediate Publication 3 June 2014, doi:10.1042/BJ20131620


c The Authors Journal compilation 
c 2014 Biochemical Society

Biochem. J. (2014) 462, 337345 (Printed in Great Britain)

doi:10.1042/BJ20131620

SUPPLEMENTARY ONLINE DATA

Recombinant form of mammalian gp91phox is active in the absence of p22phox

Aymen EZZINE*, Hager SOUABNI*, Tania BIZOUARN* and Laura BACIOU*1
*Laboratoire de Chimie Physique, Universite Paris Sud, UMR8000 CNRS, Bat. 350, 91405 Orsay Cedex, France

Figure S1

Spectroscopic analysis of the X33 /gp91phox membranes during induced culture

Cell fractions were recovered every 24 h of methanol induction and reduced-minus-oxidized spectra of the total membrane fractions were determined. Green, control strain after the 72 h culture
period; blue, 24 h of culture; red, 48 h of culture; and violet, 72 h of culture.

Received 10 December 2013/27 May 2014; accepted 3 June 2014

Published as BJ Immediate Publication 3 June 2014, doi:10.1042/BJ20131620

To whom correspondence should be addressed (email laura.baciou@u-psud.fr).


c The Authors Journal compilation 
c 2014 Biochemical Society