Identification of the carboxyl-terminal amino acids important for the ADP-ribosylation activity of Pseudomonas exotoxin A

J. T. Chow, M. S. Chen, H. C P Wu, J. Hwang

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15 Citations (Scopus)

Abstract

The ADP-ribosylation domain of Pseudomonas exotoxin A (PE) has been identified to reside in structural domain III (residues 405-613) and a portion of domain Ib (residues 385-404) of the molecule (Hwang, J., FitzGerald, D.J., Adhya, S., and Pastan, I., (1987) Cell 48, 128-136). To further determine the carboxyl end region essential for ADP-ribosylation activity, we constructed sequential deletions at the carboxyl-terminal of PE. Our results show that a clone with a deletion of the carboxyl-terminal amino acid residues from Arg-609 to Lys-613 and replaced with Arg-Asn retained wild-type PE ADP-ribosylation activity. Deletion of the terminal amino acid residues from Ala-596 to Lys-613 and replaced with Val-Ile-Asn reduced ADP-ribosylation activity by 75%, while deletions of 36 or more amino acids from the carboxyl terminus completely lose their ADP-ribosylation activity. These modified PEs were also examined for their ability to block PE cytotoxicity. Our results show that modified PEs which lost their ADP-ribosylation activity correspondingly lost their cytotoxicity. Furthermore, extracts containing PE fragments without ADP-ribosylation activty were able to block the cytotoxic activity of intact PE. Our results thus indicate that carboxyl-terminal amino acids in the Ser-595 region are crucial for ADP-ribosylation activity and, consequently, cytotoxicity of PE. The modified PEs which have lost their ADP-ribosylation activity may also be a route to new PE vaccines.

Original languageEnglish
Pages (from-to)18818-18823
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number31
Publication statusPublished - 1989
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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