PMID: 2503515Aug 25, 1989Paper

Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin.

The Journal of Biological Chemistry
C B SiegallI Pastan

Abstract

Pseudomonas exotoxin is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation. The substitution of the cell recognition domain (domain Ia) with a growth factor such as transforming growth factor alpha (TGF alpha), creates a cell-specific cytotoxic agent, TGF alpha-PE40, which kills cells bearing epidermal growth factor (EGF) receptors. We have used TGF alpha-PE40 to define the role of sequences in domains II, Ib, and III. Various mutations were made in these domains and mutant forms of TGF alpha-PE40 expressed in Escherichia coli. Mutant proteins were then tested for their ADP-ribosylation, EGF receptor-binding, and cell-killing activities. Additionally, the amino boundary of domain III, which contains the ADP-ribosylation activity, was determined by deletion analysis. Data indicate that (i) the functional amino terminus of domain III is near amino acid 400; (ii) deletion of various regions in domain II or conversion of cysteines 265 and 268 to serines results in a loss of cytotoxicity which ranged from 10-fold to more than 150-fold, indicating that domain II is essential for full expression of cytotoxicity; (iii) deletion of the amino terminus of domain Ib ...Continue Reading

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