Distinct biological activities of C3 and ADP‐ribosyltransferase‐deficient C3‐E174Q

Abstract

Low-molecular-weight GTP-binding proteins of the Rho family control the organization of the actin cytoskeleton in eukaryotic cells. Dramatic reorganization of the actin cytoskeleton is caused by the C3 exoenzyme derived from Clostridium botulinum (C3), based on ADP-ribosylation of RhoA ⁄ B ⁄ C. In addition, wild-type as well as ADP-ribosyltransferase-deficient C3-E174Q induce axonal outgrowth of primary murine hippocampal neurons and prevent growth cone collapse, indicating a non-enzymatic mode of action. In this study, we compared the effects of C3-E174Q and wild-type C3 in the murine hippocampal cell line HT22. Treatment of HT22 cells with C3 resulted in Rho ADP-ribosylation and cell rounding. The ADP-ribosyltransferase-deficient mutant C3-E174Q did not induce either Rho ADP-ribosylation or morphological changes. C3 as well as C3-E174Q treatment resulted in growth arrest, reduced expression of cyclin D levels, and increased expression of RhoB, a negative regulator of cell-cycle progression. Serum starvation induced apoptosis in HT22 cells, as determined on the basis of increased expression of caspase-9 and Bax. C3 but not C3-E174Q protected serum-starved HT22 cells from apoptosis. This is the first study separating ADP-ribosyltransferase-dependent from ADP-ribosyltransferase-independent effects of C3. While morphological changes and anti-apoptotic activity strictly depend on ADP-ribosyltransferase activity, the anti-proliferative effects are independent of ADP-ribosyltransferase activity. Structured digital abstractl Rhotekin physically interacts with RHOA by pull down (View interaction)

A Recombinant Fusion Toxin Based on Enzymatic Inactive C3bot1 Selectively Targets Macrophages

“…Therefore, the C3bot1E174Q-induced cell cycle arrest which was recently reported for the murine hippocampal cell line HT22 [42] might be a cell-type specific effect.…”

Structural constraints-based evaluation of immunogenic avirulent toxins from Clostridium botulinum C2 and C3 toxins as subunit vaccines

“…Mutants of our study may not able to catalyze ADP-ribosylation reaction owing to their poor ligand efficiency and binding affinity of NAD + to the binding core. Enzyme deficient mutants of these toxins have been used as drug delivery systems and molecular tools for studying many pathophysiological mechanisms, but no one has been evaluated them as vaccine candidates (Dmochewitz et al, 2013;Fahrer et al, 2010aFahrer et al, , 2010bRohrbeck et al, 2012). A single amino acid substitution/mutation reported as an evolutionary constraint that can change antigenicity of many bacterial and viral antigens (Foley, 2015;Koide et al, 2005;Liang et al, 2010;Meyer and Wilke, 2015;Yoo and Deregt, 2001).…”

“…Site-directed mutagenesis was carried out to understand the role of functional residues involving in ADP-ribosyltransferase activity of C2I (Barth et al, 1998;Han and Tainer, 2002) and in channel/pore forming property of C2II on actin (Blöcker et al, 2003a(Blöcker et al, , 2003bLang et al, 2008). Several mutants were developed from C3 by site-directed mutagenesis for inactive NAD-glycohydrolase activity, (Ménétrey et al, 2002) blocking RalA interface, (Pautsch et al, 2005) decreasing ADP-ribosylation activity (Han et al, 2001) and inactive ADP-ribosyltransferase activity (Dmochewitz et al, 2013;Rohrbeck et al, 2012). Thus, site-directed mutagenesis experiments have been supported to define the functional roles of the critical residues in their structures and catalytic mechanisms.…”

Toxins as tools

“…Because Ral functions are also altered by C3 binding (at least, in vitro activation of phospholipase D by Ral is blocked by C3), it is feasible that high concentrations of C3, which are used for microinjection studies, affect Ral signaling. Whether antiapoptotic effects [58] and neuronal growth [59] described for ADP-ribosylationincompetent C3 preparations depend on Ral remains to be clarified.…”