Abstract
Pseudomonas exotoxin (PE) is a protein toxin composed of three structural domains. In its native form, the toxin is a 66,000-Mr proenzyme that must be activated to express full ADP-ribosylating activity. To study the process of activation and accompanying conformational changes, we have isolated 10 monoclonal antibodies to a 40,000-Mr fragment of the toxin (PE40) that exhibits full enzyme activity but lacks the toxin's cell-binding domain and contains amino acids 253 to 613 (comprising domains II, Ib, and III). By using mutant PE molecules in which all of domain I and portions of domains II, Ib, and III were deleted, the locations of the epitopes for each of the antibodies were determined. Eight of these monoclonal antibodies were further characterized. Of these eight, all reacted with soluble PE40 and an interleukin-2-PE40 conjugate, but only two reacted strongly with native soluble PE. However, all eight reacted with PE after it had been immobilized on nitrocellulose or after it had been activated to express full ADP-ribosylating activity. Antibodies were also assessed for their ability to neutralize the cytotoxic activity of either PE or interleukin-2-PE40. These antibodies should be useful as probes for monitoring the activation and processing of PE that occur during endocytosis and in determining the location of epitopes that are important for toxin activity.