Figure 1

An enzymatic pathway is involved in auto-ADP-ribosylation of CTc. (A) Biotin signals are detected in the periplasmic fractions of E.coli lysate expressing wild type and mutant cholix toxin catalytic fragments (SA). The same blot was re-blotted with rabbit anti-CTc antibody to detect protein expression in each sample (IB). Similar results are obtained by incubating purified protein with either biotinyl-NAD⁺ (B, SA) or ³²P-NAD⁺(C). The blots shown are representative of multiple independent experiments. (D) The structures of these mutants were analyzed by circular dichroism (CD) spectrometry. (E) NAD⁺ glycohydrolase activity (left panel) and auto-ADP-ribosylation activity (right panel) were quantified by 96-well plate based assays. Data are summarized from three independent experiments. Error bars show the standard deviation of the composite data. Asterisks indicate significant reduction of enzyme activity as compared to the wild type CTc with *, p-value < 0.004; **, p-value < 0.0001. (F) Various concentrations of free ADP-ribose were added to the auto-ADP-ribosylation reaction. As a control, the ³²P-ADP-ribosylation signals on CTc were removed by phosphodiesterase I treatment. Asterisk indicates significant reduction of ³²P-auto-ADP-ribosylation signal of the treated over the untreated samples with p-value < 0.01. Data shown is representative of three independent experiments. Error-bars show the standard error of mean (SEM). Statistic analysis was done by student t-test. (G) Excess amount of free ADP-ribose was added to the ADP-ribosylation buffer containing 3 μM purified wild type CTc and 50 μM biotinyl-NAD⁺ at 37°C for 1 hour. The biotinyl-ADP-ribosylation signal was detected by IRDye800CW-SA. In all panels, arrowheads indicate the auto-ADP-ribosylated CTc and arrows indicate the detection of CTc by anti-CTc antibody.