title: Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.
abstract: 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.
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. rohrbeck.astrid@mh-hannover.de.
authors.forename: Astrid
authors.initials: A
authors.lastname: Rohrbeck
authors.affiliation: Biotechnology and Bioinformatics, Department of Biotechnology, Institute for Biochemistry, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany. v.fuehner@tu-braunschweig.de.
authors.forename: Viola
authors.initials: V
authors.lastname: Fühner
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. schroeder.anke@mh-hannover.de.
authors.forename: Anke
authors.initials: A
authors.lastname: Schröder
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. hagemann.sandra@mh-hannover.de.
authors.forename: Sandra
authors.initials: S
authors.lastname: Hagemann
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. xuan-khang.vu@stud.mh-hannover.de.
authors.forename: Xuan-Khang
authors.initials: XK
authors.lastname: Vu
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. berndtsarah@web.de.
authors.forename: Sarah
authors.initials: S
authors.lastname: Berndt
authors.affiliation: Biotechnology and Bioinformatics, Department of Biotechnology, Institute for Biochemistry, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany. m.hust@tu-bs.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. pich.andreas@mh-hannover.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pich
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. just.ingo@mh-hannover.de.
authors.forename: Ingo
authors.initials: I
authors.lastname: Just
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001905:Botulinum Toxins / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein
keywords: ADP-Ribosyltransferase; ADP-ribosylated RhoA; C3-transferase