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Mutagenesis of Pseudomonas exotoxin in identification of sequences responsible for the animal toxicity
V K Chaudhary1, Y Jinno, M G Gallo
1Division of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Pseudomonas exotoxin (PE) is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation. The deletion of the cell recognition domain (domain Ia) of PE results in a molecule that does not bind to target cells and has low toxicity in mice (Hwang, J., FitzGerald, D.J.P., Adhya, S., and Pastan, I. (1987) Cell 48, 129-136). To determine the specific sequences required for cell binding as well as cell and animal toxicity, a series of domain I mutants was constructed. Using a T7 promoter-based expression system and an OmpA signal sequence, large amounts of the various mutant toxins were secreted into the periplasm from which they were easily purified in milligram quantities. The data indicate that amino acids at positions 246, 247, and 249 have an important role in the toxicity of PE. Conversion of these amino acids to glutamic acid or glycine but not to lysine or deletion of amino acids 241-250 diminishes the toxicity of PE. When combined with a mutation at position 57 a molecule is created that has very low toxicity against cultured cells or in mice.
Insights
Specific amino acids in Pseudomonas exotoxin (PE) domain I are crucial for cell binding and toxicity. Mutating these sites significantly reduces PE
Area of Science:
- Molecular Biology
- Toxicology
- Protein Engineering
Background:
- Pseudomonas exotoxin (PE) has three domains: cell recognition, membrane translocation, and ADP-ribosylation.
- Deleting PE's cell recognition domain (domain Ia) abolishes cell binding and reduces toxicity.
Purpose of the Study:
- To identify specific amino acid sequences in PE domain I essential for cell binding and toxicity.
- To engineer PE variants with altered toxicological profiles.
Main Methods:
- Construction and expression of a series of PE domain I mutants using a T7 promoter system.
- Purification of mutant toxins from the periplasm.
- Assessment of cell binding and toxicity in cultured cells and animal models.
Main Results:
- Amino acids at positions 246, 247, and 249 in PE domain I are critical for toxicity.
- Mutations at these positions (e.g., to glutamic acid or glycine) significantly diminish PE toxicity.
- A combined mutation at position 57 further reduces toxicity in cells and mice.
Conclusions:
- Specific residues within PE domain I dictate its cytotoxic activity.
- Targeted mutagenesis of PE domain I can modulate its toxicity, offering potential for therapeutic applications.
- Understanding these structure-function relationships is key for developing targeted toxin-based therapies.