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.
Highlights
The C3 exoenzyme from Clostridium botulinum ADP-ribosylates RhoA, B, and C at amino acid 41 thereby inactivating it [1]
ADP‐ribosylation of In incells cell C3 lysates wasADP-ribosylation detected by altered of RhoA in SDS-PAGE/Western blotting
The altered migration behavior of RhoA in gel‐shift assay often ambiguous in Chinese hamster ovary (CHO) cells, so that a different approach is necessary for a clear interpretation
Summary
The C3 exoenzyme from Clostridium botulinum ADP-ribosylates RhoA, B, and C at amino acid 41 (asparagine) thereby inactivating it [1]. RhoA belongs to a family of small GTPases that are active in GTP-bound and inactive in GDP-bound form and are involved in regulation of cell division, growth, migration, and neural development. C3 is a simple exoenzyme that lacks specific binding and translocation domain of intracellular acting bacterial protein toxins. C3 is efficiently internalized into different cell types [4] as indicated by ADP-ribosylation of intracellular RhoA. The widely used methods for detection of ADP-ribosylated RhoA are (i) the analysis of the migration behavior of modified RhoA in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blot for RhoA detection, or (ii) the conventional
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