Structure and function relationship of Pseudomonas exotoxin A. An immunochemical study

We have raised antisera against Pseudomonas exotoxin A (PE) and domains Ia and III to study the structure-function relationships of PE. Anti-PE antibody (Ab(PE)) was shown to abolish the ADP-ribosylation activity of PE. However, either antidomain Ia antibody nor antidomain III antibody inhibited the ADP-ribosylation activity of PE. This suggests that the inhibition of ADP-ribosylation by Ab(PE) results from the binding of Ab(PE) to the region between domains Ia and III. Since the binding of Ab(PE) to PE did not inhibit NAD hydrolysis in the absence of elongation factor 2, the inhibitory effect of Ab(PE) on ADP-ribosylation may be due to steric hindrance rather than a direct action on the catalytic function. Thus, the interface between domain Ia and III may be the site of entry of elongation factor 2 during ADP-ribosylation. The antibodies were also used to study both the inhibitory effects of PE on protein synthesis and its cytotoxic activity. Either Ab(PE) or antidomain Ia antibody, but not antidomain III antibody, was able to reverse the inhibition of protein synthesis by PE and to bloock its cytotoxicity. In addition, rabbits immunized with domain Ia acquired tolerance against 100 μg of PE injected subcutaneously. These results suggest that domain Ia is the cell-binding domain of PE and may be used for vaccination against PE-mediated diseases.