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Klaus Aktories - One of the best experts on this subject based on the ideXlab platform.
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exchange of glutamine 217 to glutamate of clostridium limosum Exoenzyme C3 turns the asparagine specific adp ribosyltransferase into an arginine modifying enzyme
Biochemistry, 2006Co-Authors: Martin Vogelsgesang, Klaus AktoriesAbstract:C3-like ADP-ribosyltransferaseses are produced by Clostridium species, Bacillus cereus, and various Staphylococcus aureus strains. The Exoenzymes modify the low-molecular-mass GTPases RhoA, B, and C. In structural studies of C3-like Exoenzymes, an ARTT-motif (ADP-ribosylating turn−turn motif) was identified that appears to be involved in substrate specificity and recognition (Han, S., Arvai, A. S., Clancy, S. B., Tainer, J. A. (2001) J. Mol. Biol. 305, 95−107). Exchange of Gln217, which is a key residue of the ARTT-motif, to Glu in C3 from Clostridium limosum results in inhibition of ADP-ribosyltransferase activity toward RhoA. The mutant protein is still capable of NAD-binding and possesses NAD+ glycohydrolase activity. Whereas recombinant wild-type C3 modifies Rho proteins specifically at an asparagine residue (Asn41), Gln217Glu-C3 is capable of ADP-ribosylation of poly-arginine but not poly-asparagine. Soybean trypsin inhibitor, a model substrate for many arginine-specific ADP-ribosyltransferases, is m...
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Crystal structure of the C3bot–RalA complex reveals a novel type of action of a bacterial Exoenzyme
The EMBO Journal, 2005Co-Authors: Alexander Pautsch, Martin Vogelsgesang, Jens Tränkle, Christian Herrmann, Klaus AktoriesAbstract:C3 Exoenzymes from bacterial pathogens ADP-ribosylate and inactivate low-molecular-mass GTPases of the Rho subfamily. Ral, a Ras subfamily GTPase, binds the C3 Exoenzymes from Clostridium botulinum and C. limosum with high affinity without being a substrate for ADP ribosylation. In the complex, the ADP-ribosyltransferase activity of C3 is blocked, while binding of NAD and NAD-glycohydrolase activity remain. Here we report the crystal structure of C3 from C. botulinum in a complex with GDP-bound RalA at 1.8 A resolution. C3 binds RalA with a helix–loop–helix motif that is adjacent to the active site. A quaternary complex with NAD suggests a mode for ADP-ribosyltransferase inhibition. Interaction of C3 with RalA occurs at a unique interface formed by the switch-II region, helix α3 and the P loop of the GTPase. C3-binding stabilizes the GDP-bound conformation of RalA and blocks nucleotide release. Our data indicate that C. botulinum Exoenzyme C3 is a single-domain toxin with bifunctional properties targeting Rho GTPases by ADP ribosylation and Ral by a guanine nucleotide dissociation inhibitor-like effect, which blocks nucleotide exchange.
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entrapment of rho adp ribosylated by clostridium botulinum C3 Exoenzyme in the rho guanine nucleotide dissociation inhibitor 1 complex
Journal of Biological Chemistry, 2003Co-Authors: Harald Genth, Ralf Gerhard, Klaus Aktories, Akio Maeda, Mutsuki Amano, Kozo Kaibuchi, Ingo JustAbstract:Abstract RhoA, -B, and -C are ADP-ribosylated by Clostridium botulinum Exoenzyme C3 to induce redistribution of the actin filaments in intact cells, a finding that has led to the notion that the ADP-ribosylation blocks coupling of Rho to the downstream effectors. ADP-ribosylation, however, does not alter nucleotide binding, intrinsic, and GTPase-activating protein-stimulated GTPase activity. ADP-ribosylated Rho is even capable of activating the effector protein ROK in a recombinant system. Treatment of cells with a cell-permeable chimeric C3 toxin led to complete localization of modified Rho to the cytosolic fraction based on the complexation of ADP-ribosylated Rho with the guanine-nucleotide dissociation inhibitor-1 (GDI-1). The modified complex turned out to be resistant to phosphatidylinositol 4,5-bisphosphate- and GTPγS-induced release of Rho from GDI-1. Thus, ADP-ribosylation leads to entrapment of Rho in the GDI-1 complex. The increased stability of the GDI complex prevented binding of Rho to membrane-associated players of the GTPase cycle such as the activating guanine nucleotide exchange factors and effector proteins.
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Activation of phospholipase D1 by ADP-ribosylated RhoA.
Biochemical and Biophysical Research Communications, 2003Co-Authors: Harald Genth, Ralf Gerhard, Klaus Aktories, Martina Schmidt, Ingo JustAbstract:Abstract Clostridium botulinum Exoenzyme C3 exclusively ADP-ribosylates RhoA, B, and C to inactivate them, resulting in disaggregation of the actin filaments in intact cells. The ADP-ribose resides at Asn-41 in the effector binding region, leading to the notion that ADP-ribosylation inactivates Rho by blocking coupling of Rho to its downstream effectors. In a recombinant system, however, ADP-ribosylated Rho bound to effector proteins such as phospholipase D-1 (PLD1), Rho-kinase (ROK), and rhotekin. The ADP-ribose rather mediated binding of Rho-GDP to PLD1. ADP-ribosylation of Rho-GDP followed by GTP-γ-S loading resulted in binding but not in PLD activation. On the other hand, ADP-ribosylation of Rho previously activated by binding to GTP-γ-S resulted in full PLD activation. This finding indicates that ADP-ribosylation seems to prevent GTP-induced change to the active conformation of switch I, the prerequisite of Rho–PLD interaction. In contrast to recombinant systems, ADP-ribosylation in intact cells results in functional inactivation of Rho, indicating other mechanisms of inactivation than blocking effector coupling.
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Studies on the active-site structure of C3-like Exoenzymes: Involvement of glutamic acid in catalysis of ADP-ribosylation
Biochimie, 2000Co-Authors: Klaus Aktories, Gerhard Fritz, Martin Jung, J Bohmer, Joël Vandekerckhove, Ingo JustAbstract:Abstract Various C3-like ADP-ribosyltransferases like Clostridium botulinum Exoenzyme C3, C limosum transferase, B cereus transferase and a transferase from Staphylococcus aureus (EDIN) selectively modify the low-molecular mass GTP-binding proteins RhoA,B,C. UV-irradiation of C limosum transferase in the presence of [carbonyl- 14 C]NAD resulted in radiolabeling of Glu-174. Concomitantly, ADP-ribosyltransferase and NAD glycohydrolase activities were inhibited. Site-directed mutagenesis of Glu-174 (E174D, E174Q) which resulted in more than 1000-fold reduction of enzyme activity, suggests that the glutamic acid residue is essentially involved in the catalytic action of C3-like transferases. These findings support the view that all bacterial ADP-ribosyltransferases share a similar active-site structure.
Ingo Just - One of the best experts on this subject based on the ideXlab platform.
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Improvement of Nitric Oxide–Dependent Vasodilatation by HMG-CoA Reductase Inhibitors Through Attenuation of Endothelial Superoxide Anion Formation
Arteriosclerosis Thrombosis and Vascular Biology, 2020Co-Authors: Andreas H. Wagner, Ingo Just, Thomas Köhler, Uwe Rückschloss, Markus HeckerAbstract:Abstract—Three 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester–stimulated superoxide anion (O2−) formation in endothelium-intact segments of the rat aorta in a time- and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 μmol/L). The HMG-CoA reductase product mevalonic acid (400 μmol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac with Clostridium sordellii lethal toxin but not by inactivation of p21 Rho with Clostridium botulinum Exoenzyme (C3). A mevalonate-sensitive inhibition of phorbol ester–stimulated O2− formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (SOD) activity and expression was detected, whereas that of endothelial nitric oxide (NO) s...
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detection and quantification of adp ribosylated rhoa b by monoclonal antibody
Toxins, 2016Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Xuan Khang Vu, Sarah Berndt, Viola Fühner, Michael Hust, Andreas Pich, Ingo JustAbstract:Clostridium botulinum Exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
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Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody
Toxins, 2016Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Xuan Khang Vu, Sarah Berndt, Viola Fühner, Michael Hust, Andreas Pich, Ingo JustAbstract:Clostridium botulinum Exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
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Detection and quantification of ADP-ribosylated RhoA/B by monoclonal antibody
Toxins, 2016Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Xuan Khang Vu, Sarah Berndt, Viola Fühner, Michael Hust, Andreas Pich, Ingo JustAbstract:© 2016 by the authors; licensee MDPI, Basel, Switzerland. Clostridium botulinum Exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
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vimentin mediates uptake of C3 Exoenzyme
PLOS ONE, 2014Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Andreas Pich, Markus Holtje, Gudrun Ahnerthilger, Ingo JustAbstract:: Clostridium botulinum C3 Exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 Exoenzyme.
Patrice Boquet - One of the best experts on this subject based on the ideXlab platform.
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12 Modifications of small GTP-binding proteins by bacterial protein toxins
Methods in Microbiology, 2004Co-Authors: Patrice BoquetAbstract:Publisher Summary This chapter focuses on the modifications of small GTP-binding proteins by bacterial protein toxins. Rho GTP-binding proteins belong to the p21 Ras superfamily which is divided into five main branches: Ras, Rho, Rab, Sar/Arf and Ran. Such as other small GTPases Rho are molecular switches playing the role of intracellular timers in signal transduction cascades. Bound to GTP they are in their active form and upon hydrolysis of the nucleotide into GDP they return back to their inactive state. Their ability to hydrolyze GTP into GDP, in conjunction with a helper protein (GTPase-activating-protein GAP), has led to them being named small GTPases. Members of the Rho subfamily of GTPases are clearly preferred targets of several bacterial toxins and virulence factors that can manipulate them to either activate or inhibit these proteins permanently. The discovery of Clostridium botulinurn Exoenzyme C3 has led to the elucidation of the role of the Rho GTPases in the control of the cytoskeleton organization therefore allowing one to understand the molecular mechanism of large clostridial cytotoxins together with the discovery of a new covalent modification of target proteins by bacterial toxins.
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Inhibition of p21 Rho in intact cells by C3 diphtheria toxin chimera proteins.
Methods in Enzymology, 2004Co-Authors: Patrice Boquet, Michel R Popoff, M Giry, Emmanuel Lemichez, Patricia Bergez-aulloAbstract:Publisher Summary This chapter describes the realization of a fusion protein between Exoenzyme C3 and diphtheria toxin (DT) fragment B and discusses the use of this molecule on cultured cells to inactivate the p21 Rho, thereby producing an alteration of the actin microfilament network. Clostridium botulinum ( C . botulinum ) C and D strains produce an ADP-ribosyltransferase called “Exoenzyme C3,” which selectively modifies the p21 GTP-binding protein Rho on its asparagine-41. The inactivation of the Rho protein by ADP-ribosylation on introduction (by microinjection or forced pinocytosis) of Exoenzyme C3 into the cytosol induces the loss of actin filaments in cultured cells. Thus, the use of Exoenzyme C3 is of great help in understanding the functional role of the p21 Rho protein that belongs to the p21 Ras superfamily of small GTP-binding proteins. DT is synthesized by toxigenic Corynebacterium diphtheriae as a single-chain protein that is subsequently cleaved into two fragments linked by a disulfide bridge.
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Bacterial toxins inhibiting or activating small GTP-binding proteins.
Annals of the New York Academy of Sciences, 1999Co-Authors: Patrice BoquetAbstract:: Amino acids located on the switch 1 or switch 2 domains of small GTPases of the Ras and Rho family are targets of several bacterial toxins. Exoenzyme C3 from Clostridium botulinum ADP-ribosylates specifically Rho at R43 and prevents the recruitment of Rho on the cell membrane. This blocks the downstream effects of the Rho GTPase. However, Exoenzyme C3 is not a toxin, and chimeric proteins fusing C3 with the B moiety of either diphtheria toxin or Pseudomonas aeruginosa exotoxin A have been produced to intoxicate cells with low concentration of C3. C. difficile toxin B modifies by glucosylation Rho on T37 and Rac and Cdc42 on T35. Glucosylation of Rho, Rac, and Cdc42 blocks the binding of these GTPases on their downstream effectors. C. sordellii lethal toxin modifies Ras, Rap, and Rac on T35 by glucosylation. Cytotoxic necrotizing factor 1 (CNF1), from uropathogenic Escherichia coli strains, deamidates Q63 of Rho into E63, thereby blocking the intrinsic or GAP-mediated GTPase of Rho. This allows permanent activation of Rho. Thus, Rho GTPases are targets for three different toxin activities. Molecular mechanisms of these toxins are discussed.
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Bacterial toxins and the Rho GTP-binding protein: what microbes teach us about cell regulation
Cell Death & Differentiation, 1998Co-Authors: Carla Fiorentini, Michel Gauthier, Gianfranco Donelli, Patrice BoquetAbstract:In the present review activities of two bacterial toxins, Clostridium botulinum Exoenzyme C3 and Escherichia coli CNF1, both acting on the GTP-binding protein Rho are analyzed. Proteins belonging to the Rho family regulate the actin cytoskeleton and act as molecular switches in a number of signal transduction pathways. C3 and CNF1 have opposite effects on Rho thus representing useful tools for studies on cell division, cell differentiation and apoptosis.
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A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly.
The EMBO Journal, 1993Co-Authors: P. Aullo, Michel R Popoff, M Giry, S. Olsnes, Christine Kocks, Patrice BoquetAbstract:Abstract We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum Exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L. monocytogenes utilizes a cellular rho dependent mechanism in this process.
Astrid Rohrbeck - One of the best experts on this subject based on the ideXlab platform.
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detection and quantification of adp ribosylated rhoa b by monoclonal antibody
Toxins, 2016Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Xuan Khang Vu, Sarah Berndt, Viola Fühner, Michael Hust, Andreas Pich, Ingo JustAbstract:Clostridium botulinum Exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
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Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody
Toxins, 2016Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Xuan Khang Vu, Sarah Berndt, Viola Fühner, Michael Hust, Andreas Pich, Ingo JustAbstract:Clostridium botulinum Exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
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Detection and quantification of ADP-ribosylated RhoA/B by monoclonal antibody
Toxins, 2016Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Xuan Khang Vu, Sarah Berndt, Viola Fühner, Michael Hust, Andreas Pich, Ingo JustAbstract:© 2016 by the authors; licensee MDPI, Basel, Switzerland. Clostridium botulinum Exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
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vimentin mediates uptake of C3 Exoenzyme
PLOS ONE, 2014Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Andreas Pich, Markus Holtje, Gudrun Ahnerthilger, Ingo JustAbstract:: Clostridium botulinum C3 Exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 Exoenzyme.
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inhibition of macrophage migration by c botulinum Exoenzyme C3
Naunyn-schmiedebergs Archives of Pharmacology, 2012Co-Authors: Jacqueline Rotsch, Astrid Rohrbeck, Sandra Hagemann, Ingo Just, Harald Genth, Tanja Kolbe, Ilona Schelle, Stefanie C HuelsenbeckAbstract:C3-like Exoenzymes are produced by various microorganism including Clostridium botulinum (C3bot), Bacillus cereus and Staphylococcus aureus. C3bot is the prototype of C3-like Exoenzymes that specifically ADP-ribosylates and thereby inactivates Rho(A/B/C). C3-like Exoenzymes are not yet regarded as virulence factors, as the lack of cell entry domains results in a poor accessibility of the C3-like Exoenzymes to cells. In this study, the sensitivity of various cell lines to C3bot has been reinvestigated. Primary monocytes as well as cultured macrophage-like cells including J774A.1 cells and RAW macrophages exhibit a tenfold higher sensitivity to C3bot than fibroblasts and epithelial cells. RhoA ADP-ribosylation by C3bot resulted in the formation of pronounced bipolar protrusions based on defective tail retraction. The formation of bipolar protrusion resulted in inhibited macrophage migration. These findings suggested that macrophages appear to be target cells of C3bot. Migration of macrophage is a prerequiste for their recruitment to the site of pathogen invasion or tissue damage. Inhibition of macrophage migration likely preserves the survival of C3-producing microorganisms. The observations of this study reinforce the paradigm of a role of C3-like Exoenzymes as virulence factors.
Sandra Hagemann - One of the best experts on this subject based on the ideXlab platform.
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detection and quantification of adp ribosylated rhoa b by monoclonal antibody
Toxins, 2016Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Xuan Khang Vu, Sarah Berndt, Viola Fühner, Michael Hust, Andreas Pich, Ingo JustAbstract:Clostridium botulinum Exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
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Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody
Toxins, 2016Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Xuan Khang Vu, Sarah Berndt, Viola Fühner, Michael Hust, Andreas Pich, Ingo JustAbstract:Clostridium botulinum Exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
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Detection and quantification of ADP-ribosylated RhoA/B by monoclonal antibody
Toxins, 2016Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Xuan Khang Vu, Sarah Berndt, Viola Fühner, Michael Hust, Andreas Pich, Ingo JustAbstract:© 2016 by the authors; licensee MDPI, Basel, Switzerland. Clostridium botulinum Exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
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vimentin mediates uptake of C3 Exoenzyme
PLOS ONE, 2014Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Andreas Pich, Markus Holtje, Gudrun Ahnerthilger, Ingo JustAbstract:: Clostridium botulinum C3 Exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 Exoenzyme.
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inhibition of macrophage migration by c botulinum Exoenzyme C3
Naunyn-schmiedebergs Archives of Pharmacology, 2012Co-Authors: Jacqueline Rotsch, Astrid Rohrbeck, Sandra Hagemann, Ingo Just, Harald Genth, Tanja Kolbe, Ilona Schelle, Stefanie C HuelsenbeckAbstract:C3-like Exoenzymes are produced by various microorganism including Clostridium botulinum (C3bot), Bacillus cereus and Staphylococcus aureus. C3bot is the prototype of C3-like Exoenzymes that specifically ADP-ribosylates and thereby inactivates Rho(A/B/C). C3-like Exoenzymes are not yet regarded as virulence factors, as the lack of cell entry domains results in a poor accessibility of the C3-like Exoenzymes to cells. In this study, the sensitivity of various cell lines to C3bot has been reinvestigated. Primary monocytes as well as cultured macrophage-like cells including J774A.1 cells and RAW macrophages exhibit a tenfold higher sensitivity to C3bot than fibroblasts and epithelial cells. RhoA ADP-ribosylation by C3bot resulted in the formation of pronounced bipolar protrusions based on defective tail retraction. The formation of bipolar protrusion resulted in inhibited macrophage migration. These findings suggested that macrophages appear to be target cells of C3bot. Migration of macrophage is a prerequiste for their recruitment to the site of pathogen invasion or tissue damage. Inhibition of macrophage migration likely preserves the survival of C3-producing microorganisms. The observations of this study reinforce the paradigm of a role of C3-like Exoenzymes as virulence factors.