Báo cáo khoa học: Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated a subunit of the heterotrimeric GTPase Gq potx

Enzymatic actions of Pasteurella multocida toxin detected
by monoclonal antibodies recognizing the deamidated
a subunit of the heterotrimeric GTPase G
q
Shigeki Kamitani
1
, Shinpei Ao
2
, Hirono Toshima
1
, Taro Tachibana
2
, Makiko Hashimoto
1
,
Kengo Kitadokoro
3
, Aya Fukui-Miyazaki
1
, Hiroyuki Abe
1
and Yasuhiko Horiguchi
1
1 Department of Molecular Bacteriology, Research Institute for Microbial Diseases, Osaka University, Japan
2 Department of Bioengineering, Graduate School of Engineering, Osaka City University, Japan
3 Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Japan
Introduction
Pasteurella multocida toxin (PMT) is a highly potent
mitogen acting on various types of cultured cells,
including fibroblasts and osteoblastic cells [1–3].
Because of this, it is referred to as cyclomodulin, which

promotes or interferes with the cell cycle of target cells
[4]. Previous studies implied that the toxin is internal-
ized by endocytosis after binding to a putative receptor
on the target cells, and escapes from endosomes to the
cytoplasm [5,6], where it activates heterotrimeric
GTPase (G
q
- and G
12 ⁄ 13
)-dependent pathways [3,7–10],
in turn, leading to upregulations in Rho, phospholipase
C (PLC)b and mitogen-activated protein kinases, such
as Jun N-terminal kinase and extracellular signal-regu-
lated kinase [5,11–14]. Recent studies indicated that,
additionally, PMT activates G
i
to inhibit adenylyl
cyclase [15,16]. PMT consists of a single polypeptide
chain of 1285 amino acids. Several lines of evidence
indicate that the N-terminal region of the toxin binds
to target cells and the C-terminal region carries the
intracellularly active moiety [17–20]. The N-terminal
region is partly homologous to Escherichia coli cyto-
toxic necrotizing factors, CNF1 and CNF2 [21,22].
Keywords
bacterial toxin, deamidation, GTPase,
heterotrimeric, in vitro assay, monoclonal
antibody, Pasteurella multocida toxin
Correspondence
S. Kamitani, Department of Molecular

Bacteriology, Research Institute for
Microbial Diseases, Osaka University 3-1
Yamada-oka, Suita-shi, Osaka 565-0871,
Japan
Fax: +81 6 6879 8283
Tel: +81 6 6879 8285
E-mail:
(Received 12 October 2010, revised 9
May 2011, accepted 25 May 2011)
doi:10.1111/j.1742-4658.2011.08197.x
Pasteurella multocida toxin (PMT) is a virulence factor responsible for the
pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through
the activation of heterotrimeric GTPase (G
q
,G
12 ⁄ 13
and G
i
)-dependent
pathways, by deamidating a glutamine residue in the a subunit of these
GTPases. However, the enzymatic characteristics of PMT are yet to be
analyzed in detail because the deamidation has only been observed in cell-
based assays. In the present study, we developed rat monoclonal antibod-
ies, specifically recognizing the deamidated Ga
q
, to detect the actions of
PMT by immunological techniques such as western blotting. Using the
monoclonal antibodies, we found that the toxin deamidated Ga
q
only

under reducing conditions. The C-terminal region of PMT, C-PMT, was
more active than the full-length PMT. The C3 domain possessing the
enzyme core catalyzed the deamidation in vitro without any other domains.
These results not only support previous observations on toxicity, but also
provide insights into the enzymatic nature of PMT. In addition, we present
several lines of evidence that Ga
11
, as well as Ga
q
, could be a substrate for
PMT.
Abbreviations
C-PMT, C-terminal region of Pasteurella multocida toxin; GST, glutathione S-transferase; IF-DMEM, inositol-free DMEM;
MEF, mouse embryonic fibroblast; PLC, phospholipase C; PMT, Pasteurella multocida toxin.
2702 FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS
Recently, we solved the crystal structure of the C-ter-
minal region (residues 575–1285) of PMT (C-PMT)
and found that C-PMT is composed of three domains
(C1, C2 and C3) [23]. In addition, we showed that the
C1 domain is involved in the plasma membrane locali-
zation of C-PMT and the C3 domain possesses a cyste-
ine protease-like catalytic triad [23,24]. Conserved
plasma membrane-targeting domains homologous to
the C1 domain were found in multiple large bacterial
toxins [24,25]. More recently, it was shown that G
i2
was activated by the deamidation of Gln
205
to Glu by
PMT from a cell-based assay and MS [15,16]. Ga

q
was
also considered to be deamidated by the toxin. The
deamidated GTPases were found to lose their GTPase
activity and, as a result, stimulate downstream signal-
ing pathways. Taken together, all these findings sug-
gest that the catalytic triad in the C3 domain conducts
the deamidation reaction. However, the enzymatic
characteristics of PMT have not been analyzed as a
result of the lack of an easily-administered assay to
detect activity of the toxin.
In the present study, we developed rat monoclonal
antibodies that specifically recognize the deamidated
a subunit of G
q
(anti-Ga
q
Q209E) and obtained results
providing new insights into the enzymatic actions of
PMT. In addition, the monoclonal antibodies enabled
us to detect PMT-induced deamidation in situ, indicat-
ing them to be powerful probes for characterizing the
actions of the toxin.
Results
Analysis of enzymatic actions of PMT with Ga
q
Q209E-specific monoclonal antibodies
According to a previous study [16], the deamidation of
Ga by PMT results in the conversion of a Gln residue
in the switch 2 region to Glu. To raise antibodies to

detect this conversion, we prepared a mutant G
q
-pep-
tide (MUT G
q
-peptide), which corresponds to the
switch 2 region of Ga
q
, with Glu substituted for the
Gln residue (Fig. 1A, Gln
209
for Ga
q
) and immunized
rats with the peptide. After screening with ELISA to
detect antibodies specific to MUT G
q
-peptide, two
hybridoma cell lines producing monoclonal antibodies,
3F6 and 3G3, were established. These antibodies recog-
nized the deamidated form of Ga
q
(Ga
q
Q209E) but
not wild-type Ga
q
, which were independently expressed
Gα
t1

FSFKDLNFRMFDVGGQRSERKKWIHCFEG
G
α
t2
FSVKDLNFRMFDVGGQRSERKKWIHCFEG
G
α
i1
FTFKDLHFKMFDVGGQRSERKKWIHCFEG
G
α
i3
FTFKELYFKMFDVGGQRSERKKWIHCFEG
G
α
i2
FTFKDLHFKMFDVGGQRSERKKWIHCFEG
G
α
o1
FTFKNLHFRLFDVGGQRSERKKWIHCFED
G
α
o2
FTFKNLHFRLFDVGGQRSERKKWIHCFED
G
α
z
FTFKELTFKMVDVGGQRSERKKWIHCFEG
G

α
s
FQVDKVNFHMFDVGGQRDERRKWIQCFND
G
α
solf1
FQVDKVNFHMFDVGGQRDERRKWIQCFND
G
α
solf2
FQVDKVNFHMFDVGGQRDERRKWIQCFND
G
α
11
FDLENIIFRMVDVGGQRSERRKWIHCFEN
G
α
q
FDLQSVIFRMVDVGGQRSERRKWIHCFEN
G
α
14
FDLENIIFRMVDVGGQRSERRKWIHCFES
G
α
15
FSVKKTKLRIVDVGGQRSERRKWIHCFEN
G
α
12

FVIKKIPFKMVDVGGQRSQRQKWFQCFDG
G
α
13
FEIKNVPFKMVDVGGQRSERKRWFECFDS
* :::.******.:*::*:.**:.
WT Gq peptide IFRMVDVGGQRSERRKWIHC
MUT Gq peptide IFRMVDVGGERSERRKWIHC
A
194 200 210 220
BC
+ mGαq WT
None
+ mGα
q
Q209E
Anti-Gαq
Anti-Gα11
+ mGαq/11 105–113
+ mG
α
11
Anti-β-actin
3G3
3F6
Anti-Gα
q Q209E
MEF
(–)
complemented by

WB:
+ rG
α
s
None
+ rGα
s Q227E
+ hG
α
13
293T
+ rGα
i2
+ rGαi2 Q205E
+ hGα
13 Q226E
+ mGα11
+ mGα11 Q209E
MEF
(–)
+ mGα
q
Q209E
anti-Gαq Q209E
anti-β-actin
anti-Gα
13
anti-Gα11
anti-Gαs
anti-Gαi2

WB:
Fig. 1. Isolation of Ga
q
Q209E-specific antibodies. (A) Alignment of amino acid sequences of the switch 2 region in the a subunits of mouse
heterotrimeric GTPases by
CLUSTALW. Sequences of synthetic oligopeptides for the generation of antibodies are shown at the bottom of the
panel. The sequences corresponding to the oligopeptides are highlighted. The nucleotide sequences are obtained from NCBI; Ga
t1
(accession
number NP_032166), Ga
t2
(NP_032167), Ga
i1
(NP_034435), Ga
i2
(AAH65159), Ga
i3
(NP_034436), Ga
o1
(P18872), Ga
o2
(P18873), Ga
z
(NP_034441), Ga
s
(P63094), Ga
solf1
(NP_034437), Ga
solf2
(NP_796111), Ga

11
(NP_034431), Ga
q
(NP_032165), Ga
14
(NP_032163), Ga
15
(NP_034434), Ga
12
(NP_034432) and Ga
13
(NP_034433). The numbers above the alignment indicate the amino acid positions of Ga
q
.Ga
q
and
Ga
11
are highlighted by a yellow background. Identical amino acid residues are denoted by asterisks, highly conserved residues by double dots,
and modestly conserved residues by dots. (B) Western blot analysis using anti-Ga
q
Q209E, 3F6 and 3G3, to detect Ga
q
Q209E. Lysate of
Ga
q ⁄ 11
-deficient MEF cells (MEF
())
) complemented with the plasmids expressing wild-type Ga
q

, mutant Ga
q
Q209E, Ga
11
or Ga
q ⁄ 11 105–113
was subjected to 15% SDS ⁄ PAGE and western blotting with monoclonal rat anti-Ga
q
Q209E (3F6 or 3G3), polyclonal rabbit anti-Ga
q
, polyclonal
rabbit anti-Ga
q11
or polyclonal rabbit anti-b-actin. (C) The substrate specificity of rat anti-G a
q
Q209E monoclonal for the key members of Ga su-
bunits. The deamidated forms of each mutant Ga subunits were detected by anti-Ga
q
Q209E (3G3). 293T cells were transfected pEF6-based
plasmids expressing the indicated Ga subunits. After 24 h of incubation, the cells were lysed and subjected to 15% SDS ⁄ PAGE followed by
western blotting with monoclonal rat anti-Ga
q
Q209E (3G3), polyclonal rabbit anti-Ga
s
, polyclonal rabbit anti-Ga
i-2
, polyclonal rabbit anti-Ga
13
,
serum polyclonal rabbit anti-Ga

11
and polyclonal rabbit anti-b-actin as described in the Experimental procedures. Similar results were obtained
with 3F6, the monoclonal anti-Ga
q
Q209E (3F6). The extracts of MEF
())
cells expressing Ga
q
Q209E were loaded as the positive control.
S. Kamitani et al. Enzymatic actions of P. multocida toxin
FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS 2703
in Ga
q ⁄ 11
-deficient mouse embryonic fibroblast (MEF)
cells [designated as MEF
())
cells] (Fig. 1B). Next, we
investigated the substrate specificity of these Ga
q
Q209E-specific antibodies for other key members of Ga
subunits, including Ga
s
,Ga
i
and G a
13
. The antibodies
recognized all the deamidated forms of Ga subunits we
tested (Fig. 1C). By using the Ga
q

Q209E-specific anti-
bodies, we attempted to detect the deamidation of the
recombinant Ga
q
caused by in vitro treatment with
PMT or PMT variants under various conditions. In
these experiments, Ga
i ⁄ q
was used in place of Ga
q
because the former chimera was more stable and solu-
ble and more readily prepared than the latter wild-type
[26]. The deamidation of Ga
i ⁄ q
was detected by the
antibody when Ga
i ⁄ q
b
1
c
s
was treated with wild-type
C-PMT and the full-length PMT (Fig. 2A). C-PMT,
which consists of only the intracellularly active domains
[23], appeared to deamidate Ga
i ⁄ q
b
1
c
s

approximately
ten-fold more efficiently than PMT. PMT C1165S, in
which the active core Cys
1165
is replaced with Ser, did
not cause the deamidation, indicating that the antibody
recognizes the deamidation resulting from the enzy-
matic actions of PMT. Thereafter, we aimed to charac-
terize the deamidation in vitro by C-PMT. The
deamidation was observed only under reducing condi-
tions (Fig. 2B). Similar to PMT C1165S, C-PMT
C1165S did not cause the deamidation under reducing
or nonreducing conditions. By contrast, C-PMT
C1159S, in which Cys
1159
is replaced with Ser, deami-
dated Ga
i ⁄ q
even under nonreducing conditions
(Fig. 2B). C-PMT is composed of three distinct
domains (C1, C2 and C3) [23]. The in vitro assay
revealed that C-PMT DC1(4H), in which the first four
helices are deleted from the C1 domain, deamidated
Ga
i ⁄ q
, although the catalytic efficiency was approxi-
mately 100-fold lower than that of C-PMT (Fig. 2C). A
glutathione S-transferase (GST)-fused form of the C3
domain, GST-C3 WT, deamidated Ga
i ⁄ q

in vitro,
whereas GST-C3 C1165S and GST alone showed no
deamidation activity (Fig. 2D). GST-C3 WT was
approximately 100-fold less efficient than C-PMT.
C-PMT deamidated Ga
i ⁄ q
in both the monomeric
and heterotrimeric state in vitro, although the mono-
meric Ga
i ⁄ q
was approximately 100-fold less sensitive
than the heterotrimeric form (Figs 3A and S1A). Ga
i ⁄ q
was also deamidated when the concentration of mono-
meric Ga
i ⁄ q
increased (Fig. 3B).
Ga
11
as another target for PMT
The sequence of the WT G
q
-peptide is completely con-
sistent with the corresponding region of Ga
11
(Fig. 1A).
Indeed, the deamidated form of Ga
11
(Ga
11

Q209E)
was also recognized by the Ga
q
Q209E-specific anti-
body (Fig. 1C). Therefore, the Ga
q
Q209E-specific
antibody should detect the deamidation of Ga
11
,if
Ga
11
serves as a substrate of PMT. A previous study
reported Ga
q
, but not Ga
11
, to be a substrate for PMT,
and attributed the sensitivity to PMT to the helix aBof
the helical domain comprising amino acid residues 105–
113 of Ga
q
[8,10]. It was also shown that Ga
q ⁄ 11 105–113
,
which has the Ga
q
backbone with the helix aB of the
helical domain of Ga
11

, was insensitive to PMT. We
constructed Ga
q ⁄ 11
-deficient MEF cells expressing
either Ga
11
or the chimeric Ga
q ⁄ 11 105–113
(Fig. 1B) and
examined their sensitivity to PMT. As shown in
Fig. 4A, both Ga
11
and Ga
q ⁄ 11 105–113
were deamidated
by PMT. Furthermore, we examined whether each of
the cells responds to the PMT treatment by determining
intracellular PLC activity (Fig. 4B). In addition to Ga
q
,
Ga
11
and the chimeric Ga
q ⁄ 11 105–113
conferred sensitivity
to PMT on Ga
q ⁄ 11
-deficient MEF cells, although the
magnitude of the response to PMT was small in the cells
expressing Ga

11
or Ga
q ⁄ 11 105–113
compared to those
expressing Ga
q
. We also found that a weak band
appeared on the western blot of Ga
q ⁄ 11
-deficient MEF
cells treated with PMT, suggesting an additional sub-
strate besides Ga
q
and Ga
11
(Fig. 4A). According to the
previous study [16], PMT-induced deamidation causes pI
shift of native Ga proteins. We analyzed the Ga
11
from
MEF Ga
q ⁄ 11
-deficient cells expressing Ga
11
with or with-
out treatment of PMT by native gel electrophoresis. The
results obtained confirmed that PMT increased the
migration of Ga
11
, as well as Ga

q
, in native gel electro-
phoresis, as detected by Ga
q ⁄ 11
-specific immunoblot
analysis, and the Ga
q
Q209E-specific antibody only
recognized the migration-increased Ga
11
(Fig. S2A).
Furthermore, using immunoprecipitation of Ga
11
and
western blotting, we confirmed that PMT deamidated
Ga
11
expressed in MEF Ga
q ⁄ 11
-deficient cells (Fig. S2B).
Application of Ga
q
Q209E-specific monoclonal
antibodies to detect PMT activity
The Ga
q
Q209E antibodies also detected the deamida-
tion of the endogenous Ga caused by PMT in
Swiss3T3 cells (Fig. 4C), although the subtype of Ga
could not be identified. On the basis of the immuno-

precipitation of Ga
q
and western blotting, we con-
firmed that the intracellular Ga
q
was deamidated
(Fig. S3A). Furthermore, we examined whether the
Ga
q
Q209E antibodies detect the deamidated Ga in
PMT-treated cells. The combination of Ga
q
Q209E-
antibody, 3G3, and fluorescent-labeled anti-rat IgG in
immunofluorescent microscopy recognized the cells
affected by PMT (Fig. 4D).
Enzymatic actions of P. multocida toxin S. Kamitani et al.
2704 FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS
Discussion
Orth et al. [15,16] recently reported that PMT activates
heterotrimeric GTPase-dependent signaling pathways
by deamidating Ga
i2
,Ga
i1
and Ga
q
. Although the
deamidation of Ga
i2

by PMT was identified by MS
[16], that of Ga
i1
and Ga
q
was only supported by indi-
rect evidence, such as the alteration of isoelectric
points demonstrated by 2D or native gel electrophore-
sis [16].
In the present study, we aimed to analyze the enzy-
matic characteristics of PMT by using monoclonal rat
antibodies that specifically recognize the deamidated
Ga
q
. Previously, we succeeded in detecting the small
GTPase Rho deamidated by dermonecrotic toxin from
Bordetella bronchiseptica by using rabbit antibodies
specifically recognizing the deamidated residues [27].
The deamidation catalyzed by PMT and by dermone-
crotic toxin occurs on a Gln residue that is conserved
among GTPases and essential for GTPase activity. We
therefore expected a similar strategy for detecting
PMT-catalyzed deamidation to be successful. Indeed,
we could detect PMT activity both in vitro and in situ
by using the monoclonal antibodies.
75
37
50
MW (kDa)
Gαq Q209E

(μM)
10 0.01 0.1
10.10.01
C-PMT
WT
10.10.01
PMT
WT
PMT
C1165S
B
C
50
PMT: Gαi/qβ1γs
= 10 nM : 1 μM
MW (kDa)
37
75
25
DTT
50
37
75
25
Gα
q Q209E
Gα
q Q209E
D
GST

C-PMT WT
Mock
0.01 0.01
GST-C3
C1165S
GST-C3
WT
0 0.1 1 0.1 10.01 0.01 10.1
50
37
75
25
Protein (μM)
Gα
q Q209E
MW (kDa)
MW (kDa)
C-PMT
WT
Mock
C-PMT
C1165S
C-PMT
C1159S
–+ –+ –+–+
PMT: Gαi/qβ1γs
= 100 nM : 1 μM
Gαi/qβ1γs = 1 μM
Gαi/qβ1γs = 1 μM
A

C-PMT
ΔC1
C-PMT
WT
Mock
0.01 0.1
C-PMT
C1165S
C-PMT
C1159S
0 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1
50
37
75
25
C-PMT (nM)
Gα
q Q209E
MW (kDa)
MEF
(–)
+ mGα
q
Q209E
50
WB:
CBB:
Gαq
WB:
CBB:

WB:
CBB:
50
Gαq
Gαq
50
WB:
CBB:
50
WB:
CBB:
50
Gαq
Gαq
Fig. 2. In vitro deamidation of Ga
i ⁄ q
b
1
c
s
by
PMT. Ga
i ⁄ q
b
1
c
s
and PMT or PMT variants
were incubated at 37 °C overnight under
various conditions and subjected to 15%

SDS ⁄ PAGE and subsequently western blot-
ting with rat anti-Ga
q
Q209E (3F6) (upper
panel). Recombinant Ga
i ⁄ q
b
1
c
s
proteins after
incubation with PMT or PMT variants were
applied at 4.5 lg per each lane. The loaded
recombinant Ga
i ⁄ q
was visualized by
Coomasie Brilliant Blue staining (lower
panel). (A) C-PMT is more efficient as a
deamidase than PMT. C-PMT and PMT
variants at the indicated concentrations and
1 lmGa
i ⁄ q
b
1
c
s
were incubated in the pres-
ence of 5 m
M dithiotreitol. One hundred
micrograms of the lysate of MEF Ga

q ⁄ 11
-
deficient cells expressing Ga
q
Q209E was
used as the positive control. (B) In vitro
deamidation of Ga
i ⁄ q
by PMT under reduc-
ing conditions. Ga
i ⁄ q
b
1
c
s
at 1 lM was incu-
bated with C-PMT at 10 n
M (upper panel) or
100 n
M (lower panel) in the presence or
absence of 5 m
M dithiotreitol. (C) C-PMT
DC1(4H) deamidates Ga
i ⁄ q
in vitro. C-PMT,
C-PMT C1165S, C-PMT C1159S or C-PMT
DC1(4H) at the indicated concentrations and
1 l
M Ga
i ⁄ q

b
1
c
s
were incubated in the pres-
ence of 5 m
M dithiotreitol. (D) Deamidation
of Ga
q
by the C3 domain. The indicated con-
centrations of GST-C3 WT, GST-C3 C1165S
or GST and 1 l
M Ga
i ⁄ q
b
1
c
s
were incubated
in the presence of 5 m
M dithiotreitol.
S. Kamitani et al. Enzymatic actions of P. multocida toxin
FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS 2705
The in vitro assay with the monoclonal antibodies
provided insights into the enzymatic action of PMT.
(a) C-PMT deamidates Ga
q
at least ten-fold more
efficiently than the full-length PMT (Fig. 2A). Almost
all bacterial toxins exerting toxic effects through their

enzymatic actions are known to undergo intracellular
cleavage after binding to specific receptors on target
cells. Similarly, intramolecular cleavage may occur on
PMT and C-PMT encompassing the catalytic domain
may be liberated into the cytoplasm, where the sub-
strates, Ga proteins, reside. Thus, the N-terminal
region of PMT may hamper the action of the cata-
lytic C-PMT. (b) The C3 domain of C-PMT alone
showed the deamidation activity (Fig. 2D). It was pre-
viously reported [24] that C-PMT is the minimum
unit required for intracellular toxicity after transloca-
tion into the cytoplasm. Indeed, when expressed in
cells, C-PMT lacking the C1 domain, which functions
as the membrane-targeting domain [24], no longer
affected the cells. These results indicate that the C1,
C2 and C3 domains must coordinate in the cytoplasm
for the cytotoxicity to occur, although the enzymatic
action is attributable to the C3 domain per se. (c)
C-PMT deamidated the Ga proteins only under
reducing conditions, whereas C-PMT C1159S did so
under both reducing and nonreducing conditions
(Fig. 2B). These results confirm that cleavage of the
disulfide bond between Cys
1159
and Cys
1165
in the C3
domain is essential for formation of the catalytic triad
comprising Cys
1165

, His
1205
and Asp
1220
(Fig. S4) [21].
(d) Ga in the heterotrimeric state was a more prefera-
ble substrate for PMT than monomeric Ga
q
. Hetero-
trimeric GTPases are known to be in a resting state
and to dissociate into an a subunit and a bc dimer in
response to extracellular signals transduced by ligand-
bound seven-transmembrane receptors. These results
imply that PMT mainly targets the a subunit of
heterotrimeric GTPases.
The Ga
q
Q209E-specific antibodies also detected
deamidated Ga proteins in PMT-treated cells (Fig. 4)
and, by using them, we found Ga
11
to be a substrate
for PMT. Ga
q ⁄ 11 105–113
, which has the Ga
q
backbone,
along with the helix aB of the helical domain of Ga
11
,

was also deamidated by PMT. Moreover, MEF
())
cells
complemented with Ga
q
or Ga
q ⁄ 11 105–113
responded
to PMT with an increase in intracellular inositol phos-
phates, indicating the activation of PLCb downstream
of Ga
q
or Ga
11
. Furthermore, PMT increased the
migration of Ga
11
protein in native gel electrophoresis,
probably as a result of PMT-catalyzed deamidation.
The combination of immunoprecipitation and western
blotting by using the Ga
q
Q209E-specific antibody also
revealed that Ga
11
expressed in MEF
())
cells was
deamidated by PMT (Fig. S2). These results were
inconsistent with the previous observation that Ga

11
did not serve as a substrate for PMT [8,10]. This dis-
crepancy may occur as a result of clonal variations of
MEF
())
cells because Ga
11
-orGa
11
derivative-comple-
mented MEF
())
cell strains were independently estab-
lished in each study. Furthermore, whether the PLC
assay is proper for the detection of PMT action must
also be examined because activation of PLC followed
by inositolphosphate accumulation is an indirect event
occurring downstream of Ga subunit and may be
influenced by other factors. This issue remains to be
addressed, although it is conceivable that both Ga
q
and Ga
11
are sensitive to PMT because they share
approximately 90% homology [28]. Furthermore, a
weak band appeared on the western blot of Ga
q ⁄ 11
-
deficient MEF cells treated with PMT, suggesting an
additional substrate besides Ga

q
and Ga
11
(Fig. 3A).
These cells did not show an increase in inositol phos-
phate levels in response to the toxin and, thus, the
additional substrate could not be upstream of PLCb.
A
Gαq Q209E
10 0.01 0.1 10.01 0.10
75
37
50
MW (kDa)
Gαi/qβ1γs
Gαi/q
C-PMT (μM)
B
75
37
50
MW (kDa)
Gαq Q209E
515 1 5511G protein (μM)
C-PMT
C-PMT
Buffer
Buffer
Gαi/qβ1γs
Gαi/q

Gαq
50
50
Gαq
WB:
CBB:
WB:
CBB:
Fig. 3. Ga
q
monomer serves as a substrate for PMT. (A) Ga
i ⁄ q
b
1
c
s
or Ga
i ⁄ q
at 1 lM was incubated with C-PMT at various concentra-
tions. Recombinant Ga
i ⁄ q
or Ga
i ⁄ q
b
1
c
s
proteins after incubation
with C-PMT were respectively applied at 1.1 or 4.5 lg per each
lane. (B) Ga

i ⁄ q
b
1
c
s
,orGa
i ⁄ q
at 1 and 5 lM was incubated with
10 n
M C-PMT. Recombinant Ga
i ⁄ q
proteins after incubation with
C-PMT were respectively applied at 1.1 or 5.5 lg per each lane,
and recombinant Ga
i ⁄ q
b
1
c
s
were at 4.5 and 22.5 lg per each lane.
In all experiments, the reaction mixture after incubation at 37 °C
overnight was subjected to 15% SDS ⁄ PAGE and western blotting
with rat anti-Ga
q
Q209E (3F6) (upper panel). The loaded recombi-
nant Ga
i ⁄ q
was visualized by Coomasie Brilliant Blue staining (lower
panel).
Enzymatic actions of P. multocida toxin S. Kamitani et al.

2706 FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS
Ga
i1
,Ga
i2
,Ga
12
and Ga
13
, known as substrates for
PMT, are not linked to PLCb. Therefore, the weak
band in PMT-treated MEF
())
cells may represent them
because the G a
q
Q209E antibodies recognized the
deamidated Ga
i2
and Ga
12 ⁄ 13
, although the switch 2
regions comprise distinct amino acid sequences. In
addition, Ga
14
or Ga
15
could comprise candidate
because the sequence of the WT G
q

-peptide is com-
pletely consistent with or highly homologous to the
corresponding region of Ga
14
or Ga
15
, and the anti-
bodies recognized the Ga
14
Q205E mutant protein in
the lysate of cells expressing mouse Ga
14
Q205E on
A
C
WB:
+PMT
No stimulation
Deamidated Gα
q
Nucleus
None
+ mG
α
q
WT
PMT
– + –+ –+–+ – +–+
+ mGα
11

WT
+ mGα
q/11 105–113
Swiss3T3
MEF
(–)
+ mGα
q
Q209E
MEF
(–)
DB
0
10
20
30
40
[
3
H]-Total inositol phosphates
(×10
3
cpm/well)
PMT (ng/ml)
0
100
None
+ mG
α
q WT

+ mGα
11 WT
+ mG
α
q/11 105–113
+ mGαq Q209E
Swiss3T3
MEF
(–)
n.s.
n.s.
P = 0.0040
P = 0.0056
P = 0.0174
P = 0.0019
Anti-Gαq Q209E
Anti-Gα
q
10000101001
WB:
100010 1001
MEF
(–)
+ mGαq Q209E
WT C1165S
PMT
(ng/ml)
Anti-β-actin
Anti-β-actin
Anti-Gα

q Q209E
Anti-Gα
q
Fig. 4. Ga
11
as another target for PMT. (A) Swiss3T3 cells and Ga
q ⁄ 11
-deficient MEF cells [MEF
())
] complemented with Ga
q
or Ga
11
were
treated with 100 ngÆmL
)1
PMT for 4 h. After incubation, the cells were lysed and subjected to 15% SDS ⁄ PAGE followed by western blotting
with monoclonal rat anti-Ga
q
Q209E (3F6) as described in the Experimental procedures. (B) PLC activity in Ga
q ⁄ 11
-deficient MEF cells com-
plemented with Ga
q
and Ga
11
. The cells that had been labeled with [
3
H]myo-inositol for 48 h were treated with 100 ngÆmL
)1

PMT, and intra-
cellular [
3
H]inositol phosphates, which are products of the enzymatic action of PLC, were measured as described in the Experimental
procedures. Each bar represents the mean of triplicate measurements, with the error bar indicating the SD. Representative results from
three independent experiments are shown. The statistical significance of differences between PMT-treated and untreated cells was evalu-
ated by a paired t-test. P < 0.05 was considered statistically significant. (C) PMT deamidated Ga
q
in Swiss3T3 cells. Swiss3T3 cells were
treated with PMT WT or PMT C1165S at the indicated concentrations. After 4 h of treatment, cells were lysed and subjected to 15%
SDS ⁄ PAGE followed by western blotting with monoclonal rat anti-Ga
q
Q209E (3F6) as described in the Experimental procedures. The lysate
of MEF Ga
q ⁄ 11
-deficient cells expressing Ga
q
Q209E was used as the positive control. (D) Immunofluorescent microscopy of Swiss3T3 cells
treated with 100 ngÆmL
)1
PMT for 4 h. After fixing and permeabilization of the cells, the deamidated Ga and the nucleus were visualized
with anti-Ga
q
Q209E 3G3 (green) and 4¢,6-diamidino-2-phenyl-indole (blue), respectively. Images are presented at the same magnification.
S. Kamitani et al. Enzymatic actions of P. multocida toxin
FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS 2707
western blotting (Fig. S3B). Taken together, the anti-
bodies could be useful for detecting the PMT-catalyzed
deamidation of Ga proteins. It is noteworthy that they
detected localization of the tissues or cells influenced

by PMT during Pasteurella infections, although their
use might be limited to Ga proteins encompassing the
switch 2 region that is highly homologous to the MUT
G
q
-peptide.
Experimental procedures
Construction of plasmids
Plasmids for retroviral transduction
The retroviral vector plasmids pCXbsr-mGa
q
and pCXbsr-
mGa
11
for a subunit cDNAs of mouse heterotrimeric GTP-
ases, G
q
and G
11
, were constructed by PCR cloning. PCR
was performed with a sense primer with a HindIII site and
an antisense primer with a KpnI site (Table S1). pMEpyori-
Ga
q
and pMEpyori-Ga
11
, previously constructed in our
laboratory from pCISGa
q
and pCISGa

11
[29], were used as
the template DNA. Consequently, each amplified DNA
fragment was once cloned into pEF6-V5 ⁄ His-TOPO TA
(Invitrogen, Carlsbad, CA, USA), and then sequenced.
Plasmid clones containing the correct sequence of Ga
q
or
of Ga
11
were respectively designated pEF6-mGa
q
and
pEF6-mGa
11
. pEF6-mGa
q
or pEF6-mGa
11
was excised
with BamHI and NotI and cloned into the BamHI-NotI
site of pCXbsr [30]. The resultant plasmids were desig-
nated pCXbsr-mGa
q
and pCXbsr-mGa
11
. pCXbsr-Ga
q
Q209E and pCXbsr-G a
q ⁄ 11 105–113

were constructed by a
QuikChange II site-directed mutagenesis kit (Stratagene, La
Jolla, CA, USA) with the mutagenized primers listed in
Table S1 and pEF6-mGa
q
as the template DNA in accor-
dance with the manufacturer’s instructions.
Plasmids for transfection into 293T cells
A series of pEF6-V5 ⁄ His-Ga plasmids for a subunit
cDNAs of heterotrimeric GTPases, G
s
,G
i-2
,G
13
and G
11
,
was constructed by PCR cloning. PCR was performed with
a sense primer and an antisense primer as shown in
Table S1. pCMV6-SPORT-rGa
s
(Origene, Rockville, MD,
USA) for pEF6-V5 ⁄ His-rGa
s
, pCIS-Ga
i-2
[29] for pEF6-
V5 ⁄ His-rGa
i-2

and pCMV5-hGa
13
[31] for pEF6-V5 ⁄ His-
hGa
13
were used as the template DNA. Consequently, each
amplified DNA fragment was cloned into pEF6-V5 ⁄ His-
TOPO TA (Invitrogen) and then sequenced. Plasmid clones
containing the correct sequence of each Ga subunit were
designated pEF6-V5 ⁄ His-Ga
s
, pEF6-V5 ⁄ His-Ga
i-2
and
pEF6-V5 ⁄ His-Ga
13
, respectively. All deamidated mutant
plasmids of pEF6-V5 ⁄ His-Ga plasmids were constructed by
a QuikChange II site-directed mutagenesis kit (Stratagene)
with the mutagenized primers listed in Table S1 and pEF6-
V5 ⁄ His-Ga
s
, pEF6-V5 ⁄ His-Ga
i-2
and pEF6-V5 ⁄ His-Gas
13
as the template DNA in accordance with the manufac-
turer’s instructions.
Plasmids for expression in E. coli
pPROEX-1-C-PMT [32], pPROEX-1-C-PMT C1165S [23],

pPROEX-1-C-PMT C1159S [23], pPROEX-1-PMT [23],
pPROEX-1-PMT C1165S [23] and pPROEX-1-C-PMT
DC1(4H) [24] were constructed previously. pGEX-FLAG-C3
and pGEX-FLAG-C3 C1165S were constructed by PCR
using primers shown in Table S1. PCR was performed with
a sense primer with a BamHI site and an antisense primer
for pPROEX-1-PMT as the template DNA. The conse-
quently amplified DNA fragment was once cloned into
pCR2.1-TOPO TA and sequenced. The FLAG-C3 fragment
with the correct sequence of the PMT gene was excised with
BamHI and NotI and inserted into the BamHI-Not I sites of
pGEX-4T3 (GE Healthcare, Amersham, UK).
Cell culture, transfection, retrovirus production
and transduction
Swiss3T3 cells were cultured in DMEM supplemented with
10% fetal bovine serum, and maintained at 37 °C under
an atmosphere of 95% air ⁄ 5% CO
2
. 293T cells were trans-
fected with the plasmids by using Lipofectamin 2000 (Invi-
trogen) in accordance with the manufacturer’s instructions.
In brief, 2 · 10
5
cells were seeded in each well of a 24-well
plate. The next day, 1.0 lg of the plasmid was transfected.
After 24 h of incubation, the cells were lysed and subjected
to 15% SDS ⁄ PAGE followed by western blotting with
antibodies as described in the Experimental procedures.
MEFs derived from Ga
q

⁄ Ga
11
or Ga
12
⁄ Ga
13
gene-defi-
cient or wild-type mice were cultured as described previ-
ously [10,33,34]. For production of the retroviral vector,
Plat-E cells were transfected with the retroviral transfer vec-
tor used in the plasmid construction by Lipofectamin 2000
(Invitrogen) in accordance with the manufacturer’s instruc-
tions. In brief, 2 · 10
5
cells were seeded in each well of a
six-well plate. The next day, 1.0 lg of the retroviral transfer
vector was transfected. The supernatant was collected after
2 days and centrifuged to spin down cellular debris.
Ga
q
⁄ Ga
11
gene-deficient MEF cells (2 · 10
5
cells) were
infected in the presence of 5 lgÆmL
)1
polybrene (Nacalai
tesq, Kyoto, Japan) after filtration of the virus-containing
medium with a 0.22 lm membrane (Millipore, Billerica,

MA, USA). The expression of the a subunit of each hetero-
trimeric GTPase was monitored by western blotting.
Production of monoclonal rat antibody
The anti-Ga
q
Q209E monoclonal rat antibodies were gener-
ated based on the method established by Kishiro et al. [35].
Enzymatic actions of P. multocida toxin S. Kamitani et al.
2708 FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS
A 10-week-old female WKY ⁄ Izm rat (SLC, Shizuoka,
Japan) was immunized with an emulsion containing a syn-
thesized mutant G
q
-oligopeptide (MUT G
q
-peptide) conju-
gated with KLH (Fig. 1A) and Freund’s complete adjuvant
(Invitrogen). After 3 weeks, cells from the lymph nodes of
a rat immunized with the antigen were fused with mouse
myeloma Sp2 ⁄ 0-Ag14 cells. At 5 days postfusion, the
hybridoma supernatants were screened by an ELISA
against the MUT G
q
-peptide-conjugated BSA. Finally, two
independent hybridoma clones producing monoclonal anti-
bodies, 3F6 and 3G3, were selected. Large-scale in vitro
production and purification of these antibodies was carried
out by culturing clones in Hybridoma-SFM medium (Invi-
trogen) containing interleukin-6 and Hitrap SP ion
exchange chromatography (GE Healthcare).

Fluorescence microscopy
Swiss3T3 cells were seeded into 24-well plates containing
glass coverslips (Matsunami, Osaka, Japan). After incuba-
tion overnight, the cells were treated with 100 nm PMT for
4 h, and fixed with 3.7% formaldehyde in NaCl ⁄ Pi for
15 min. After treatment with 0.1% Triton X-100 in
NaCl ⁄ Pi for 5 min at room temperature and subsequently
with 3% skimmed milk in NaCl ⁄ Pi for 30 min, the cells
were stained with monoclonal rat the Ga
q
Q209E-specific
antibody, 3G3, for 1 h at room temperature. They were
washed with NaCl ⁄ Pi three times, stained with Alexa Flour
488-conjugated anti-rat IgG serum (Invitrogen) for 30 min
at room temperature, washed again with NaCl ⁄ Pi, and
treated with Slow Fade GOLD antifade reagent with
4¢,6-diamidino-2-phenyl-indole (Invitrogen). The cells were
examined under microscopy with an epifluorescence micro-
scope (BX50; Olympus, Tokyo, Japan). Images were cap-
tured and analyzed by SlideBook 4.0 (Roper Industries,
Inc., Sarasota, FL, USA) to control the fluorescent decon-
volution microscopy.
Purification of heterotrimeric Ga
i ⁄ q
b
1
c
s
and
monomeric Ga

i ⁄ q
Baculovirus amplification for Ga
i ⁄ q
b
1
c
s
and monomeric
Ga
i ⁄ q
For preparation of the heterotrimeric Ga
i ⁄ q
b
1
c
s
and mono-
meric Ga
i ⁄ q
, we used three baculoviruses constructed by
Kozasa et al. [26]. The Ga
i ⁄ q
gene was a chimeric gene, in
which the native N-terminus of Ga
q
was replaced with that
of Ga
i1
. The expressed Ga
i ⁄ q

has an N-terminal His
6
tag,
followed by a TEV cleavage site, amino acids 1–28 of rat
Ga
i1
, a linker of Arg and Ser, and the 37–359 amino acid
region of mouse Ga
q
. The bovine Gb
1
gene and bovine
His
6
-tagged soluble Gc
2
gene (C68S mutant, henceforth
referred to as Gc
s
) were used for the expression of Gb
1
and
Gc
s
. Baculoviruses were amplified by infection of Sf9 insect
cells [36] in Sf9-SFM select medium in accordance with the
manufacturer’s instructions.
Expression and purification of Ga
i ⁄ q
b

1
c
s
and Ga
i ⁄ q
For preparation of the Ga
i ⁄ q
b
1
c
s
, baculoviruses for His
6
-
Ga
i ⁄ q
, bovine Gb
1
and bovine His
6
-Gc
s
were co-infected
into High 5 cells (Invitrogen) and the cells harvested after
36–48 h. All purification steps were performed at 4 °C. The
cell pellet was resuspended in lysis buffer (20 mm Hepes, pH
8.0, 100 mm NaCl, 3 mm MgCl
2
, 100 lm EDTA, 10 mm
b-mercaptoethanol and 50 lm GDP) and lysed with a doun-

ce homogenizer followed by sonication. The sample was
centrifuged for 40 min at 186 000 g, and the supernatant
was filtered and diluted to a final protein concentration of
5mgÆmL
)1
with buffer A (20 mm Hepes, pH 8.0, 100 mm
NaCl, 1 mm MgCl
2
,50lm GDP and 10 mm b -mercapto-
ethanol) and loaded onto a 10 mL Nickel-NTA column
(Sigma, St Louis, MO, USA) pre-equilibrated with the same
buffer. The column was washed with 200 mL of buffer A
followed by 100 mL of buffer B (buffer A with 300 mm
NaCl and 10 mm imidazole, pH 8.0). Ga
i ⁄ q
b
1
c
s
was eluted
with buffer A supplemented with 150 mm imidazole (pH
8.0). The eluate was dialyzed against buffer A in which
2mm dithiotreitol was substituted for 10 mm b-mercapto-
ethanol. The protein was concentrated using a VIVASPIN2
30 (GE Healthcare) to approximately 1.0 mgÆmL
)1
, and
analyzed by 15% SDS ⁄ PAGE, followed by Coomasie bril-
liant blue staining (Fig. S1A). For preparation of the mono-
meric Ga

i ⁄ q
, baculovirus for His
6
-Ga
i ⁄ q
was introduced into
High 5 cells and the purification was performed as for
Ga
i ⁄ q
b
1
c
s
after harvesting of the cultured cells (Fig. S1A).
Purification of recombinant PMT and mutants
All the recombinant proteins were produced by E. coli
BL21-CodonPlus (DE3)-RIL (Stratagene). pPROEX-1-
PMT, pPROEX-1-PMT C1165S, pPROEX-1-C-PMT,
pPROEX-1-C-PMT C1165S, pPROEX-1-C-PMT C1159S
and pPROEX-1-C-PMT DC1(4H) were used for the expres-
sion of C-PMTs, PMTs and C-PMT DC1(4H). Recombi-
nant PMTs, C-PMTs and C-PMT DC1(4H) were purified
by affinity chromatography with Nickel-NTA agarose
(Sigma) in accordance with the manufacturer’s instructions.
GST-C3 from pGEX-C3, GST-C3 C1165S from pGEX-C3
C1165S and GST from pGEX-4T3 were purified by affinity
chromatography with glutathione sepharose 4B FF (GE
Healthcare) in accordance with the manufacturer’s instruc-
tions (Fig. S1B).
In vitro PMT deamidation assay

The recombinant Ga
i ⁄ q
b
1
c
s
or Ga
i ⁄ q
alone was incubated
with purified recombinant PMT and its mutants at a molar
S. Kamitani et al. Enzymatic actions of P. multocida toxin
FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS 2709
ratio of 100 : 1, 10 : 1 or 1 : 1 in 20 mm Tris-HCl (pH 7.5),
10 mm MgCl
2
and 1 mm EDTA with or without 5 mm
dithiotreitol at 37 °C overnight. After incubation, the reac-
tion mixture was subjected to 15% SDS ⁄ PAGE, followed
by western blotting. The deamidated Ga
i ⁄ q
was detected by
the monoclonal rat antibody 3F6, isolated as above.
PLC assay
Swiss3T3 cells were seeded at 5 · 10
4
cellsÆwell
)1
into a
24-well plate and incubated at 37 °C for 2 days. The cells
were washed with inositol-free DMEM (IF-DMEM) twice

and labeled with [
3
H]myo-inositol (Moravek Biochemicals
Inc., Brea, CA, USA) at 37 kBqÆmL
)1
in IF-DMEM con-
taining 0.3% BSA for 48 h. The cells were then washed
twice with IF-DMEM containing 0.3% BSA and 5 mm
LiCl and treated with the HVJ envelope vector (Ishihara
Co. Ltd, Osaka, Japan) loaded with C-PMT or C-PMT
DC1(4H) in accordance with the manufacturer’s instruc-
tions. The cells were incubated at 37 °C for 4 h after treat-
ment with the toxins, and lysed by incubation in
200 lLÆwell
)1
of 0.1 m formic acid for 20 min at room tem-
perature. The amount of total [
3
H]inositol phosphates was
determined by the yttrium silicate scintillation proximity
assay [37]. Twenty microliters of cell extract was mixed with
80 lL of yttrium silicate scintillation proximity assay beads
(GE Healthcare) in water to give a final concentration of
1.0 mg of the beadsÆmL
)1
Æwell
)1
of white 96-well plates
(Picoplate-96; Packard, Palo Alto, CA, USA), and the
plates were sealed with adhesive and clear plastic cover

sheets (Topseal-A, Packard). The contents were mixed by
shaking for 1 h. The beads were allowed to settle for 2 h,
and the radioactivity of each well was determined using a
TopCount microplate scintillation counter (Packard).
Other materials and methods
The protein concentration in each sample was measured by
Protein Assay CBB Solution (Nacalai Tesque, Kyoto,
Japan) and the Micro BCA Protein Assay Kit (Pierce,
Rockfold, IL, USA). SDS ⁄ PAGE was carried out by the
method of Laemmli [38] in a 15% and a 5–20% gradient
polyacrylamide gel. The 5–20% gradient polyacrylamide gel
was obtained from ATTO (Tokyo, Japan). For western
blotting, the samples in the gel after SDS ⁄ PAGE were elec-
trophoretically transferred onto poly(vinylidene difluoride)
membranes (Bio-Rad Laboratories, Hercules, CA, USA).
The membranes were then treated with 5% skim milk and
the transferred proteins were probed with proper antibodies
and visualized on Fuji Medical film (Fujifilm, Minato-ku,
Japan) with an enhanced chemiluminescence system in
accordance with the manufacturer’s instructions (ECL plus;
GE Healthcare). Antibodies for western blotting were
purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA, USA) for anti-Ga
q
(E-17), anti-Ga
q ⁄ 11
(C-19),
anti-Ga
11
(D-17), anti-Ga

i-2
(T-19) and anti-Ga
13
(A-20);
from Merck KGaA (Darmstadt, Germany) for anti-Ga
s
,
(371732); and from IMGENEX (San Diego, CA, USA) for
anti-b-actin (IMG-5142A).
Statistical analysis
Values are expressed as the mean ± SD. The statistical sig-
nificance of differences between PMT treated and untreated
cells was evaluated by a paired t-test. P < 0.05 was consid-
ered statistically significant. All experiments were performed
independently in triplicate.
Acknowledgements
We greatly appreciate the gift of baculovirus for the
expression of Ga
i ⁄ q
b
1
c
s
from Dr T. Kozasa (University
of Illinois, Chicago, IL, USA), of plasmids for Ga
q
,
Ga
11
, and Ga

i-2
from Dr M. I. Simon (California
Institute of Technology, CA, USA), of Ga
13
from Dr
H. Itoh (Nara Institute of Science and Technology)
and of Ga
q ⁄ 11
-deficient MEF cells from Drs S. Offer-
manns and B. Zimmermann (University of Heidelberg,
Heidelberg, Germany). We would like to thank Ms
Tomoko Suzuki for secretarial assistance. This work
was supported in part by Grants-in-aid for Scientific
Research from the Ministry of Education, Culture, Sci-
ence and Technology of Japan.
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Supporting information
The following supplementary material is available:
Doc. S1. Supplemental methods.
Fig. S1. SDS ⁄ PAGE of purified recombinant Ga
q
and
GST-C3.
Fig. S2. Ga
11
was deamidated by PMT.
Fig. S3. (A) Ga
q
was deamidated in Swiss3T3 cells
treated by PMT. (B) Ga
14
Q205E was detected by the
Ga
q
Q209E-specific antibody.
Fig. S4. Model of the activation of PMT by the
breaking of the disulfide-bond between Cys1159 and
Cys1165.
Table S1. Primers for construction of plasmids in the

present study.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
Enzymatic actions of P. multocida toxin S. Kamitani et al.
2712 FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS