4. Discussion
The selected residues Glu-385 and Glu-387 of the EXE-motif of rCDTa were mutated, as this motif is known to play a strong role in ligand binding and catalysis [3]. They directly correspond to residues Glu-378 and Glu-380 which were shown to play key roles in the cytotoxicity of Ia [17]. In addition Ser-345 was also selected for mutagenesis studies due to its key role in binding Glu-387 and NAD. CDT ADP-ribosylates monomeric actin, which leads to cytoskeletal collapse and eventually to cell death. Cytotoxicity cell assays confirmed that rCDTa-CDTb′ induces cell death and Western blot analysis confirmed that rCDTa ADP-ribosylates G-actin. In the absence of actin, rCDTa binds lower levels of biotin-NAD as expected. SDS-PAGE reveals that the -actin sample does not correlate with the other samples although reactions were handled in the same manner. This is likely a result of gel loading. Consequently the lower amount of protein present is reflected by the almost undetectable biotin-NAD signal in Western analysis. Notably, in the absence of actin, biotin-NAD is still be hydrolysed and released from the rCDTa active site, which given its molecular weight (244.31) is likely to run off SDS-PAGE before Western transfer. In this case, the signal will be even lower and it would be impossible to compare samples. To overcome inaccuracies caused by uncontrolled biotin-NAD hydrolysis reactions contained actin. In the presence of actin, the biotin-NAD signal can be monitored either whilst bound to CDTa constructs or actin. Western blot analysis of CDTa mutants which had diminished or no cytotoxic effect, con- firmed that majority of mutants had lost ADP-ribosyl transferase activity but not the ability to bind biotin-NAD. In cytotoxicity assays S345A is the only mutant inducing significant cell death, albeit less than rCDTa-CDTb′, there is still evidence that this mutation has not inhibited the activity of CDTa but may have slightly reduced its efficacy. In Western blot analysis S345A activity matches that of rCDTa.
