Analysis of sequences in domain II of Pseudomonas exotoxin A which mediate translocation.
Pseudomonas exotoxin (PE) contains 613 amino acids that are arranged into 3 structural domains. PE exerts its cell-killing effects in a series of steps initiated by binding to the cell surface and internalization into endocytic vesicles. The toxin is then cleaved within domain II near arginine-279, generating a C-terminal 37-kDa fragment that is translocated into the cytosol where it ADP-ribosylates elongation factor 2 and arrests protein synthesis. In this study, we have focused on the functions of PE which are encoded by domain II. We have used the chimeric toxin TGF alpha-PE40 to deliver the toxin's ADP-ribosylating activity to the cell cytosol. Deletion analysis revealed that sequences from 253 to 345 were essential for toxicity but sequences from 346 to 364 were dispensable. Additional point mutants were constructed which identified amino acids 339 and 343 as important residues while amino acids 344 and 345 could be altered without loss of cytotoxic activity. Our data support the idea that domain II functions by first allowing PE to be processed to a 37-kDa fragment and then key sequences such as those identified in this study mediate the translocation of ADP-ribosylation activity to the cytosol.
Siegall
C B
CB
Division of Cancer Biology Diagnosis Centers, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Ogata
M
M
Pastan
I
I
FitzGerald
D J
DJ
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D001693:Biological Transport, Active; D002678:Chimera; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D016211:Transforming Growth Factor alpha / Q000235:genetics; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D001427:Bacterial Toxins; D005098:Exotoxins; D016211:Transforming Growth Factor alpha; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A