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Analysis of sequences in domain II of Pseudomonas exotoxin A which mediate translocation
C B Siegall1, M Ogata, I Pastan
1Division of Cancer Biology Diagnosis Centers, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Pseudomonas exotoxin (PE) contains 613 amino acids that are arranged into 3 structural domains. PE exerts its cell-killing effects in a series of steps initiated by binding to the cell surface and internalization into endocytic vesicles. The toxin is then cleaved within domain II near arginine-279, generating a C-terminal 37-kDa fragment that is translocated into the cytosol where it ADP-ribosylates elongation factor 2 and arrests protein synthesis. In this study, we have focused on the functions of PE which are encoded by domain II. We have used the chimeric toxin TGF alpha-PE40 to deliver the toxin's ADP-ribosylating activity to the cell cytosol. Deletion analysis revealed that sequences from 253 to 345 were essential for toxicity but sequences from 346 to 364 were dispensable. Additional point mutants were constructed which identified amino acids 339 and 343 as important residues while amino acids 344 and 345 could be altered without loss of cytotoxic activity. Our data support the idea that domain II functions by first allowing PE to be processed to a 37-kDa fragment and then key sequences such as those identified in this study mediate the translocation of ADP-ribosylation activity to the cytosol.
Insights
Researchers identified key amino acid sequences within Pseudomonas exotoxin (PE) domain II essential for its cell-killing activity. These findings clarify how PE translocates its ADP-ribosylation function into the cytosol for protein synthesis inhibition.
Area of Science:
- Molecular Biology
- Toxicology
- Cell Biology
Background:
- Pseudomonas exotoxin (PE) is a potent cytotoxin composed of 613 amino acids organized into three structural domains.
- PE mediates cell killing through a process involving cell surface binding, endocytosis, and translocation of its toxic moiety into the cytosol.
- The C-terminal 37-kDa fragment, generated by cleavage within domain II, inhibits protein synthesis via ADP-ribosylation of elongation factor 2.
Purpose of the Study:
- To elucidate the specific functions of domain II in Pseudomonas exotoxin's cytotoxic mechanism.
- To identify the critical amino acid residues within domain II responsible for toxin processing and translocation.
- To understand the structure-activity relationship governing PE's ADP-ribosylation and translocation capabilities.
Main Methods:
- Construction and analysis of chimeric toxins, such as TGF alpha-PE40, to deliver PE's ADP-ribosylating activity.
- Deletion analysis of PE domain II to identify essential and dispensable sequences for toxicity.
- Site-directed mutagenesis to pinpoint specific amino acid residues critical for cytotoxic activity.
Main Results:
- Deletion analysis indicated that sequences from amino acids 253 to 345 within domain II are crucial for PE's toxicity.
- Sequences from amino acids 346 to 364 were found to be dispensable for cytotoxic activity.
- Point mutations identified amino acids 339 and 343 as vital for toxicity, while alterations at 344 and 345 did not abolish activity.
Conclusions:
- Domain II of Pseudomonas exotoxin plays a critical role in its cytotoxic mechanism, facilitating both processing and translocation of the ADP-ribosylating activity.
- Specific sequences and amino acid residues within domain II are essential for the efficient delivery of the toxin's effector function to the cytosol.
- These findings contribute to a deeper understanding of PE's mechanism of action and may inform the development of targeted therapeutics.