by monoclonal antibodies recognizing the deamidatedShigeki Kamitani1, Shinpei Ao2, Hirono Toshima1, Taro Tachibana2, Makiko Hashimoto1, Kengo Kitadokoro3, Aya Fukui-Miyazaki1, Hiroyuki A
Trang 1by monoclonal antibodies recognizing the deamidated
Shigeki Kamitani1, Shinpei Ao2, Hirono Toshima1, Taro Tachibana2, Makiko Hashimoto1,
Kengo Kitadokoro3, Aya Fukui-Miyazaki1, Hiroyuki Abe1and Yasuhiko Horiguchi1
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 (Gq- and G12⁄ 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 Gi 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: skami@biken.osaka-u.ac.jp
(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 (Gq, G12⁄ 13 and Gi)-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 Gaq, to detect the actions of PMT by immunological techniques such as western blotting Using the monoclonal antibodies, we found that the toxin deamidated Gaq 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 Ga11, as well as Gaq, 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.
Trang 2Recently, 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 Gi2
was activated by the deamidation of Gln205to Glu by
PMT from a cell-based assay and MS [15,16] Gaqwas
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 findsignal-ings
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 Gq(anti-GaqQ209E) 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 characterizindicat-ing the actions of the toxin
Results
Analysis of enzymatic actions of PMT with Gaq 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 Gq -pep-tide (MUT Gq-peptide), which corresponds to the switch 2 region of Gaq, with Glu substituted for the Gln residue (Fig 1A, Gln209 for Gaq) and immunized rats with the peptide After screening with ELISA to detect antibodies specific to MUT Gq-peptide, two hybridoma cell lines producing monoclonal antibodies, 3F6 and 3G3, were established These antibodies recog-nized the deamidated form of Gaq (Gaq Q209E) but not wild-type Gaq, which were independently expressed
Gαt1 FSFKDL NFRMFDVGGQRSERKKWIHC FEG
Gαt2 FSVKDL NFRMFDVGGQRSERKKWIHC FEG
Gαi1 FTFKDL HFKMFDVGGQRSERKKWIHC FEG
Gαi3 FTFKEL YFKMFDVGGQRSERKKWIHC FEG
Gαi2 FTFKDL HFKMFDVGGQRSERKKWIHC FEG
Gαo1 FTFKNL HFRLFDVGGQRSERKKWIHC FED
Gαo2 FTFKNL HFRLFDVGGQRSERKKWIHC FED
Gαz FTFKEL TFKMVDVGGQRSERKKWIHC FEG
Gαs FQVDKV NFHMFDVGGQRDERRKWIQC FND
Gαsolf1 FQVDKV NFHMFDVGGQRDERRKWIQC FND
Gαsolf2 FQVDKV NFHMFDVGGQRDERRKWIQC FND
Gα11 FDLENI IFRMVDVGGQRSERRKWIHC FEN
Gαq FDLQSV IFRMVDVGGQRSERRKWIHC FEN
Gα14 FDLENI IFRMVDVGGQRSERRKWIHC FES
Gα15 FSVKKT KLRIVDVGGQRSERRKWIHC FEN
Gα12 FVIKKI PFKMVDVGGQRSQRQKWFQC FDG
Gα13 FEIKNV PFKMVDVGGQRSERKRWFEC FDS
* :::.******.:*::*:.**:.
WT Gq peptide IFRMVDVGGQRSERRKWIHC
MUT Gq peptide IFRMVDVGG E RSERRKWIHC
Anti-Gα q Anti-Gα 11
Anti- β-actin
3G3 3F6 Anti-Gα q Q209E
MEF (–) complemented by
293T
α q
anti-Gα q Q209E
anti-β-actin
anti-Gα 13 anti-Gα 11
anti-Gα s anti-Gα i2 WB:
Fig 1 Isolation of GaqQ209E-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; Gat1(accession number NP_032166), Gat2 (NP_032167), Gai1 (NP_034435), Gai2 (AAH65159), Gai3 (NP_034436), Gao1 (P18872), Gao2 (P18873), Gaz (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), Ga12(NP_034432) and Ga13(NP_034433) The numbers above the alignment indicate the amino acid positions of Gaq Gaqand
Ga11are 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
Gaq⁄ 11-deficient MEF cells (MEF())) complemented with the plasmids expressing wild-type Gaq, mutant GaqQ209E, Ga11or Gaq⁄ 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-Gaq, polyclonal rabbit anti-Ga q11 or polyclonal rabbit anti-b-actin (C) The substrate specificity of rat anti-Ga q Q209E monoclonal for the key members of Ga su-bunits The deamidated forms of each mutant Ga subunits were detected by anti-GaqQ209E (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-GaqQ209E (3F6) The extracts of MEF())cells expressing GaqQ209E were loaded as the positive control.
Trang 3in Gaq⁄ 11-deficient mouse embryonic fibroblast (MEF)
cells [designated as MEF()) cells] (Fig 1B) Next, we
investigated the substrate specificity of these Gaq
Q209E-specific antibodies for other key members of Ga
subunits, including Gas, Gaiand Ga13 The antibodies
recognized all the deamidated forms of Ga subunits we
tested (Fig 1C) By using the GaqQ209E-specific
anti-bodies, we attempted to detect the deamidation of the
recombinant Gaq caused by in vitro treatment with
PMT or PMT variants under various conditions In
these experiments, Gai⁄ q was used in place of Gaq
because the former chimera was more stable and
solu-ble and more readily prepared than the latter wild-type
[26] The deamidation of Gai⁄ q was detected by the
antibody when Gai⁄ qb1cs 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 Gai ⁄ qb1cs approximately
ten-fold more efficiently than PMT PMT C1165S, in
which the active core Cys1165 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 Cys1159 is replaced with Ser,
deami-dated Gai⁄ 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
Gai ⁄ 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 Gai⁄ 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 Gai⁄ q in both the monomeric
and heterotrimeric state in vitro, although the
mono-meric Gai⁄ q was approximately 100-fold less sensitive
than the heterotrimeric form (Figs 3A and S1A) Gai⁄ q
was also deamidated when the concentration of
mono-meric Gai⁄ qincreased (Fig 3B)
Ga11as another target for PMT
The sequence of the WT Gq-peptide is completely
con-sistent with the corresponding region of Ga11(Fig 1A)
Indeed, the deamidated form of Ga11 (Ga11 Q209E)
was also recognized by the Gaq Q209E-specific anti-body (Fig 1C) Therefore, the Gaq Q209E-specific antibody should detect the deamidation of Ga11, if
Ga11 serves as a substrate of PMT A previous study reported Gaq, but not Ga11, to be a substrate for PMT, and attributed the sensitivity to PMT to the helix aB of the helical domain comprising amino acid residues 105–
113 of Gaq[8,10] It was also shown that Gaq⁄ 11 105–113, which has the Gaqbackbone with the helix aB of the helical domain of Ga11, was insensitive to PMT We constructed Gaq⁄ 11-deficient MEF cells expressing either Ga11or the chimeric Gaq⁄ 11 105–113(Fig 1B) and examined their sensitivity to PMT As shown in Fig 4A, both Ga11and Gaq⁄ 11 105–113were 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 Gaq,
Ga11and the chimeric Gaq ⁄ 11 105–113conferred sensitivity
to PMT on Gaq ⁄ 11-deficient MEF cells, although the magnitude of the response to PMT was small in the cells expressing Ga11 or Gaq ⁄ 11 105–113 compared to those expressing Gaq We also found that a weak band appeared on the western blot of Gaq⁄ 11-deficient MEF cells treated with PMT, suggesting an additional sub-strate besides Gaqand Ga11(Fig 4A) According to the previous study [16], PMT-induced deamidation causes pI shift of native Ga proteins We analyzed the Ga11from MEF Gaq⁄ 11-deficient cells expressing Ga11with or with-out treatment of PMT by native gel electrophoresis The results obtained confirmed that PMT increased the migration of Ga11, as well as Gaq, in native gel electro-phoresis, as detected by Gaq⁄ 11-specific immunoblot analysis, and the Gaq Q209E-specific antibody only recognized the migration-increased Ga11 (Fig S2A) Furthermore, using immunoprecipitation of Ga11 and western blotting, we confirmed that PMT deamidated
Ga11expressed in MEF Gaq⁄ 11-deficient cells (Fig S2B)
Application of GaqQ209E-specific monoclonal antibodies to detect PMT activity
The GaqQ209E 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 Gaq and western blotting, we con-firmed that the intracellular Gaq was deamidated (Fig S3A) Furthermore, we examined whether the
Gaq Q209E antibodies detect the deamidated Ga in PMT-treated cells The combination of Gaq Q209E-antibody, 3G3, and fluorescent-labeled anti-rat IgG in immunofluorescent microscopy recognized the cells affected by PMT (Fig 4D)
Trang 4Orth et al [15,16] recently reported that PMT activates
heterotrimeric GTPase-dependent signaling pathways
by deamidating Gai2, Gai1 and Gaq Although the
deamidation of Gai2 by PMT was identified by MS
[16], that of Gai1 and Gaqwas 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
Gaq 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)
(μ M ) 0 0.01 0.1 1 0.01 0.1 1
C-PMT WT
1 0.1 0.01
PMT WT PMT C1165S
B
C
50
PMT: Gα i/q β 1 γ s
= 10 n M : 1 μ M
MW (kDa)
37 75
25
DTT
50 37 75
25
D
GST
0.01 0.01
GST-C3 C1165S GST-C3
WT
50 37 75
25
Protein ( μ M )
G α q Q209E
MW (kDa)
MW (kDa)
C-PMT WT Mock
C-PMT C1165S C-PMT C1159S
PMT: G α i/q β 1 γ s
= 100 n M : 1 μ M
Gα i/q β 1 γ s = 1 μ M
Gα i/q β 1 γ s = 1 μ M
A
C-PMT ΔC1 C-PMT
WT
0.01 0.1
C-PMT C1165S
C-PMT C1159S
50 37 75
25
C-PMT (n M )
MW (kDa)
α q
50
WB:
WB:
CBB:
WB:
CBB:
G α q 50
WB:
WB:
Gα q
G α q
Fig 2 In vitro deamidation of Gai⁄ q b1csby
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-GaqQ209E (3F6) (upper
panel) Recombinant Gai⁄ q b1csproteins after
incubation with PMT or PMT variants were
applied at 4.5 lg per each lane The loaded
recombinant Gai⁄ qwas 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 lm Ga 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 Gaq⁄ 11
-deficient cells expressing Ga q Q209E was
used as the positive control (B) In vitro
deamidation of Gai⁄ q by PMT under
reduc-ing conditions Ga i ⁄ q b 1 c s at 1 l M 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 Gaqby the C3 domain The indicated
con-centrations of GST-C3 WT, GST-C3 C1165S
or GST and 1 l M Gai⁄ qb 1 c s were incubated
in the presence of 5 m M dithiotreitol.
Trang 5The in vitro assay with the monoclonal antibodies
provided insights into the enzymatic action of PMT
(a) C-PMT deamidates Gaq 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 Cys1159 and Cys1165 in the C3 domain is essential for formation of the catalytic triad comprising Cys1165, His1205 and Asp1220 (Fig S4) [21] (d) Ga in the heterotrimeric state was a more prefera-ble substrate for PMT than monomeric Gaq 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 Gaq Q209E-specific antibodies also detected deamidated Ga proteins in PMT-treated cells (Fig 4) and, by using them, we found Ga11 to be a substrate for PMT Gaq ⁄ 11 105–113, which has the Gaqbackbone, along with the helix aB of the helical domain of Ga11, was also deamidated by PMT Moreover, MEF()) cells complemented with Gaq or Gaq⁄ 11 105–113 responded
to PMT with an increase in intracellular inositol phos-phates, indicating the activation of PLCb downstream
of Gaq or Ga11 Furthermore, PMT increased the migration of Ga11protein in native gel electrophoresis, probably as a result of PMT-catalyzed deamidation The combination of immunoprecipitation and western blotting by using the GaqQ209E-specific antibody also revealed that Ga11 expressed in MEF()) cells was deamidated by PMT (Fig S2) These results were inconsistent with the previous observation that Ga11
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 Ga11- or Ga11 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 Gaq and Ga11 are sensitive to PMT because they share approximately 90% homology [28] Furthermore, a weak band appeared on the western blot of Gaq ⁄ 11 -deficient MEF cells treated with PMT, suggesting an additional substrate besides Gaq and Ga11 (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
1
0 0.01 0.1 0 0.01 0.1 1 75
37
50
MW (kDa)
C-PMT ( μ M )
B
75
37
50
MW (kDa)
5
G protein (μ M )
Buff er
Buff er
50
WB:
CBB:
WB:
CBB:
Fig 3 Gaqmonomer serves as a substrate for PMT (A) Gai⁄ qb 1 c s
or Gai⁄ q at 1 l M 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) Gai⁄ qb1cs, or Gai⁄ q at 1 and 5 l M 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 Gai⁄ qb1cswere 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-GaqQ209E (3F6) (upper panel) The loaded
recombi-nant Gai⁄ q was visualized by Coomasie Brilliant Blue staining (lower
panel).
Trang 6Gai1, Gai2, Ga12 and Ga13, known as substrates for
PMT, are not linked to PLCb Therefore, the weak
band in PMT-treated MEF())cells may represent them
because the Gaq Q209E antibodies recognized the
deamidated Gai2 and Ga12⁄ 13, although the switch 2
regions comprise distinct amino acid sequences In
addition, Ga14 or Ga15 could comprise candidate because the sequence of the WT Gq-peptide is com-pletely consistent with or highly homologous to the corresponding region of Ga14 or Ga15, and the anti-bodies recognized the Ga14 Q205E mutant protein in the lysate of cells expressing mouse Ga14 Q205E on
A
C
WB:
+PMT
No stimulation
Deamidated G α q Nucleus
+ mG
+ mG
α q/11 105–113 Swiss3T3 MEF (–)
+ mG
MEF (–)
D B
0 10 20 30 40
3 H]-T otal inositol phosphates (×10
3 cpm/well)
PMT (ng/ml) 0 100
MEF (–)
n.s.
n.s.
P = 0.0040
P = 0.0056
P = 0.0174
P = 0.0019
Anti-Gα q Q209E
Anti-Gα q
1000
PMT (ng/ml)
Anti-β-actin
Anti- β-actin
Anti-G α q Q209E Anti-Gα q
Fig 4 Ga11as another target for PMT (A) Swiss3T3 cells and Gaq⁄ 11-deficient MEF cells [MEF())] complemented with Gaqor Ga11were treated with 100 ngÆmL)1PMT 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 Gaqand Ga11 The cells that had been labeled with [ 3 H]myo-inositol for 48 h were treated with 100 ngÆmL)1PMT, 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 deamidevalu-ated Gaqin 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 Gaq⁄ 11-deficient cells expressing GaqQ209E was used as the positive control (D) Immunofluorescent microscopy of Swiss3T3 cells treated with 100 ngÆmL)1PMT 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.
Trang 7western 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
Gq-peptide
Experimental procedures
Construction of plasmids
Plasmids for retroviral transduction
was performed with a sense primer with a HindIII site and
an antisense primer with a KpnI site (Table S1)
the template DNA Consequently, each amplified DNA
(Invitrogen, Carlsbad, CA, USA), and then sequenced
with BamHI and NotI and cloned into the BamHI-NotI
site of pCXbsr [30] The resultant plasmids were
QuikChange II site-directed mutagenesis kit (Stratagene, La
Jolla, CA, USA) with the mutagenized primers listed in
accor-dance with the manufacturer’s instructions
Plasmids for transfection into 293T cells
was constructed by PCR cloning PCR was performed with
a sense primer and an antisense primer as shown in
His-TOPO TA (Invitrogen) and then sequenced Plasmid clones
containing the correct sequence of each Ga subunit were
a QuikChange II site-directed mutagenesis kit (Stratagene)
with the mutagenized primers listed in Table S1 and
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
trans-fected with the plasmids by using Lipofectamin 2000 (Invi-trogen) in accordance with the manufacturer’s instructions
plate The next day, 1.0 lg of the plasmid was transfected After 24 h of incubation, the cells were lysed and subjected
antibodies as described in the Experimental procedures
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
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
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
gener-ated based on the method established by Kishiro et al [35]
Trang 8A 10-week-old female WKY⁄ Izm rat (SLC, Shizuoka,
Japan) was immunized with an emulsion containing a
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
hybridoma supernatants were screened by an ELISA
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
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
15 min After treatment with 0.1% Triton X-100 in
antibody, 3G3, for 1 h at room temperature They were
488-conjugated anti-rat IgG serum (Invitrogen) for 30 min
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 Gai⁄ qb1csand
monomeric Gai⁄ q
Baculovirus amplification for Gai⁄ qb1csand monomeric
Gai⁄ q
followed by a TEV cleavage site, amino acids 1–28 of rat
Gai1, a linker of Arg and Ser, and the 37–359 amino acid
Gcs 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 Gai⁄ qb1csand Gai⁄ q
into High 5 cells (Invitrogen) and the cells harvested after
cell pellet was resuspended in lysis buffer (20 mm Hepes, pH 8.0, 100 mm NaCl, 3 mm MgCl2, 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
NaCl, 1 mm MgCl2, 50 lm 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
with buffer A supplemented with 150 mm imidazole (pH 8.0) The eluate was dialyzed against buffer A in which
2 mm dithiotreitol was substituted for 10 mm b-mercapto-ethanol The protein was concentrated using a VIVASPIN2
bril-liant blue staining (Fig S1A) For preparation of the
High 5 cells and the purification was performed as for
Purification of recombinant PMT and mutants
All the recombinant proteins were produced by E coli
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
with purified recombinant PMT and its mutants at a molar
Trang 9ratio of 100 : 1, 10 : 1 or 1 : 1 in 20 mm Tris-HCl (pH 7.5),
the monoclonal rat antibody 3F6, isolated as above
PLC assay
were washed with inositol-free DMEM (IF-DMEM) twice
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
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
(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,
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
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
from Merck KGaA (Darmstadt, Germany) for anti-Gas, (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 Gai⁄ qb1csfrom Dr T Kozasa (University
of Illinois, Chicago, IL, USA), of plasmids for Gaq,
Ga11, and Gai-2 from Dr M I Simon (California Institute of Technology, CA, USA), of Ga13from Dr
H Itoh (Nara Institute of Science and Technology) and of Gaq ⁄ 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
References
1 Higgins TE, Murphy AC, Staddon JM, Lax AJ & Rozengurt E (1992) Pasteurella multocida toxin is a potent inducer of anchorage-independent cell growth Proc Natl Acad Sci USA 89, 4240–4244
2 Mullan PB & Lax AJ (1996) Pasteurella multocida toxin
is a mitogen for bone cells in primary culture Infect Immun 64, 959–965
3 Rozengurt E, Higgins T, Chanter N, Lax AJ & Staddon
JM (1990) Pasteurella multocida toxin: potent mitogen for cultured fibroblasts Proc Natl Acad Sci USA 87, 123–127
4 Nougayrede JP, Taieb F, De Rycke J & Oswald E (2005) Cyclomodulins: bacterial effectors that modu-late the eukaryotic cell cycle Trends Microbiol 13, 103–110
5 Murphy AC & Rozengurt E (1992) Pasteurella
4,5-bisphosphate hydrolysis by bombesin, vasopressin, and endothelin Requirement for a functional G protein J Biol Chem 267, 25296–25303
6 Baldwin MR, Lakey JH & Lax AJ (2004) Identification and characterization of the Pasteurella multocida toxin translocation domain Mol Microbiol 54, 239–250
Trang 107 Wilson BA, Zhu X, Ho M & Lu L (1997) Pasteurella
phospholipase Cb1 J Biol Chem 272, 1268–1275
8 Orth JH, Lang S & Aktories K (2004) Action of
domain of Gaq J Biol Chem 279, 34150–34155
9 Orth JH, Lang S, Taniguchi M & Aktories K (2005)
10 Zywietz A, Gohla A, Schmelz M, Schultz G &
Offermanns S (2001) Pleiotropic effects of Pasteurella
G11 J Biol Chem 276, 3840–3845
11 Essler M, Hermann K, Amano M, Kaibuchi K,
Heese-mann J, Weber PC & Aepfelbacher M (1998)
via Rho kinase and myosin light chain phosphatase
J Immunol 161, 5640–5646
12 Lacerda HM, Lax AJ & Rozengurt E (1996) Pasteurella
induces p125FAK and paxillin tyrosine
phosphoryla-tion, actin stress fiber formaphosphoryla-tion, and focal contact
assembly in Swiss 3T3 cells J Biol Chem 271, 439–445
13 Seo B, Choy EW, Maudsley S, Miller WE, Wilson BA
& Luttrell LM (2000) Pasteurella multocida toxin
-depen-dent transactivation of the epidermal growth factor
receptor J Biol Chem 275, 2239–2245
14 Staddon JM, Barker CJ, Murphy AC, Chanter N, Lax
AJ, Michell RH & Rozengurt E (1991) Pasteurella
cells J Biol Chem 266, 4840–4847
15 Orth JH, Fester I, Preuss I, Agnoletto L, Wilson BA &
16 Orth JH, Preuss I, Fester I, Schlosser A, Wilson BA &
Aktories K (2009) Pasteurella multocida toxin activation
of heterotrimeric G proteins by deamidation Proc Natl
Acad Sci USA 106, 7179–7184
17 Busch C, Orth J, Djouder N & Aktories K (2001)
Bio-logical activity of a C-terminal fragment of Pasteurella
18 Orth JH, Blocker D & Aktories K (2003) His1205 and
His1223 are essential for the activity of the mitogenic
19 Pullinger GD, Sowdhamini R & Lax AJ (2001)
Locali-zation of functional domains of the mitogenic toxin of
Pasteurella multocida Infect Immun 69, 7839–7850
20 Ward PN, Miles AJ, Sumner IG, Thomas LH &
Lax AJ (1998) Activity of the mitogenic Pasteurella
Infect Immun 66, 5636–5642
21 Falbo V, Pace T, Picci L, Pizzi E & Caprioli A (1993) Isolation and nucleotide sequence of the gene encoding cytotoxic necrotizing factor 1 of Escherichia coli Infect Immun 61, 4909–4914
22 Oswald E, Sugai M, Labigne A, Wu HC, Fiorentini C, Boquet P & O’Brien AD (1994) Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli mod-ifies the small GTP-binding proteins Rho involved in assembly of actin stress fibers Proc Natl Acad Sci USA
91, 3814–3818
23 Kitadokoro K, Kamitani S, Miyazawa M, Hanajima-Ozawa M, Fukui A, Miyake M & Horiguchi Y (2007) Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin Proc Natl Acad Sci USA 104, 5139–5144
24 Kamitani S, Kitadokoro K, Miyazawa M, Toshima H, Fukui A, Abe H, Miyake M & Horiguchi Y (2010) Characterization of the membrane-targeting C1 domain
in Pasteurella multocida toxin J Biol Chem 285, 25467– 25475
25 Geissler B, Tungekar R & Satchell KJ (2010) Identifica-tion of a conserved membrane localizaIdentifica-tion domain within numerous large bacterial protein toxins Proc Natl Acad Sci USA 107, 5581–5586
26 Tesmer VM, Kawano T, Shankaranarayanan A, Kozasa T & Tesmer JJ (2005) Snapshot of activated G proteins at the membrane: the Gaq-GRK2-Gbc complex Science (New York, NY) 310, 1686–1690
27 Horiguchi Y, Inoue N, Masuda M, Kashimoto T, Katahira J, Sugimoto N & Matsuda M (1997)
reor-ganization of actin stress fibers through deamidation of Gln-63 of the GTP-binding protein Rho Proc Natl Acad Sci USA 94, 11623–11626
28 Mizuno N & Itoh H (2009) Functions and regulatory mechanisms of Gq-signaling pathways Neurosignals 17, 42–54
29 Strathmann M & Simon MI (1990) G protein diversity:
a distinct class of alpha subunits is present in verte-brates and inverteverte-brates Proc Natl Acad Sci USA 87, 9113–9117
30 Akagi T, Shishido T, Murata K & Hanafusa H (2000)
pathway in transformation Proc Natl Acad Sci USA
97, 7290–7295
31 Iguchi T, Sakata K, Yoshizaki K, Tago K, Mizuno N
& Itoh H (2008) Orphan G protein-coupled receptor GPR56 regulates neural progenitor cell migration via a
14469–14478
32 Miyazawa M, Kitadokoro K, Kamitani S, Shime H & Horiguchi Y (2006) Crystallization and preliminary crystallographic studies of the Pasteurella multocida