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: 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 (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. 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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 by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support... peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS . Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated a subunit of the heterotrimeric GTPase. deamidated by the toxin. The deamidated GTPases were found to lose their GTPase activity and, as a result, stimulate downstream signal- ing pathways. Taken