A recombinant form of Pseudomonas exotoxin directed at the epidermal growth factor receptor that is cytotoxic without requiring proteolytic processing

Abstract

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.