Analysis of Pseudomonas exotoxin activation and conformational changes by using monoclonal antibodies as probes.
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.
Ogata
M
M
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Pastan
I
I
FitzGerald
D
D
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001427:Bacterial Toxins; D002460:Cell Line; D004229:Dithiothreitol / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005098:Exotoxins / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004229:Dithiothreitol; D000939:Epitopes; D005098:Exotoxins; D014508:Urea; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A