ALTERATION OF A PROTEASE-SENSITIVE REGION OF PSEUDOMONAS EXOTOXIN PROLONGS ITS SURVIVAL IN THE CIRCULATION OF MICE

Pseudomonas exotoxin A (PE) is a single-chain 66-kDa polypeptide that kills eukaryotic cells by ADP-ribosylation of translational elongation factor 2. PE is composed of three major structural domains whose func tions are binding of cells (I), translocation (II), and ADP-ribosylati on (III). Here we describe a protease cleavage target that is located near arginine-490 on the surface of domain III. We made several differ ent types of mutations near arginine-490. Deletion of arginine-490 or replacement of arginine-490 and -492 with serine and lysine or with tw o lysines resulted in protease-resistant molecules that were fully cyt otoxic and had normal ADP-ribosylation activity. However, the half-lif e in mouse blood of the PE-DELTA-490 mutant was 24 min whereas that of PE was 13 min. Furthermore, two PE mutants that were protease-hyperse nsitive, PEGlu246,247,249 and PEGlu57,246,247,249 (in which glutamate residues replace basic residues at the indicated positions), had very short half-lives. These data indicate that protease sensitivity is an important determinant in the half-life of PE in the circulation and su ggest that the half-life of other proteins may be prolonged by removal of protease sites. Deletion of arginine-492 or the replacement of ami no acids 486-491 with three glycines markedly diminished ADP-ribosylat ion activity and cytotoxicity, indicating that this region of domain I II is also important for catalytic activity.