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Toxins 2016, 8(4), 100; doi:10.3390/toxins8040100

Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody

1
Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
2
Biotechnology and Bioinformatics, Department of Biotechnology, Institute for Biochemistry, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Holger Barth
Received: 17 February 2016 / Revised: 20 March 2016 / Accepted: 29 March 2016 / Published: 1 April 2016
(This article belongs to the Section Bacterial Toxins)
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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. View Full-Text
Keywords: C3-transferase; ADP-Ribosyltransferase; ADP-ribosylated RhoA C3-transferase; ADP-Ribosyltransferase; ADP-ribosylated RhoA
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Rohrbeck, A.; Fühner, V.; Schröder, A.; Hagemann, S.; Vu, X.-K.; Berndt, S.; Hust, M.; Pich, A.; Just, I. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody. Toxins 2016, 8, 100.

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