Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor.
It was previously shown that amino acids 609-613 (REDLK) at the C-terminus of Pseudomonas exotoxin (PE) are necessary for cytotoxicity, presumably by directing the toxin to the endoplasmic reticulum (ER) [Chaudhary, Jinno, FitzGerald and Pastan (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 308-312]. Using the anti-[interleukin 2 receptor (IL2R)] immunotoxin anti-Tac(Fv)-PE38 (AT-PE38REDLK), it was found that removing the terminal lysine did not alter the activity, but replacing REDL with KDEL, the most common ER retention sequence, increased activity. To determine which amino acid in KDEL was responsible for the increase in activity, we tested eight C-terminal mutants of AT-PE38REDLK. Using IL2R-bearing MT-1 cells, we found that the glutamate residue of KDEL was required for high activity, as the cytotoxicity of AT-PE38 ending in KDEL, RDEL, KEEL or REEL was much greater than that of AT-PE38 ending in REDL, KEDL, RDDL or KDDL. Using freshly isolated lymphocytic leukaemia cells, AT-PE38 ending in KDEL, REEL or RDEL was more than 100-fold more cytotoxic than AT-PE38 ending in KEDL, REDL, RDDL or the native sequence REDLK. The RDEL sequence also improved the cytotoxic activity of an interleukin 4-PE38 toxin fusion protein. Improved cytotoxic activity correlated with improved binding of the C-termini to the KDEL receptor on rat Golgi membranes. These data indicate that the glutamate residue of KDEL improves the cytotoxicity of PE by increasing binding to a sorting receptor which transports the toxin from the transreticular Golgi apparatus to the ER, where it is translocated to the cytosol and inhibits protein synthesis.
Kreitman
R J
RJ
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Pastan
I
I
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D001483:Base Sequence; D001692:Biological Transport; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005098:Exotoxins / Q000737:chemistry / Q000633:toxicity*; D018698:Glutamic Acid / Q000737:chemistry*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000633:toxicity; D021382:Protein Sorting Signals; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000633:toxicity*; D051381:Rats; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D018000:Receptors, Peptide / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D018698:Glutamic Acid; D000922:Immunotoxins; D009842:Oligopeptides; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011500:Protein Synthesis Inhibitors; D015375:Receptors, Interleukin-2; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C064459:KDEL receptor; C073762:lysyl-aspartyl-glutamyl-leucine