Pseudomonas exotoxin A mutants. Replacement of surface-exposed residues in domain III with cysteine residues that can be modified with polyethylene glycol in a site-specific manner.

Pseudomonas exotoxin A (PE) is composed of three structural and functional domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the cell membrane, and domain III for ADP-ribosylation of elongation factor 2. To investigate the role of the amino acids exposed on the surface of domain III, we replaced 15 of these, generating 29 different mutants at positions 412, 416, 418, 490, 513, 516, 522, 551, 576, 590, 599, 604, 606, 607 and 608. All but one mutant retained substantial ADP-ribosylation and cytotoxic activities. Modification of proteins with monomethoxy-polyethylene glycol (mPEG) prolongs their circulation in the blood stream and reduces their immunogenicity. Unlike PEGylated enzymes acting on small molecule substrates, PEGylated toxins may lose those functions that are based on macromolecular interactions. Therefore, we selectively PEGylated mutant PEs at positions 490, 513, 516, 522, 604, and 606. Most PEs modified by a 5-kDa mPEG via a disulfide or a thioether bond retained high cytotoxic activity. However, when a 20-kDa mPEG was used there was a decrease in cytotoxic activity with the disulfide-bonded molecules being more active. Positions 522 and 604 are good sites for PEGylation, but 490 is not. We also found that PEGylation of PE 522C prolonged its in vivo circulation time in mice.