A proper amino terminus of diphtheria toxin is important for cytotoxicity.
A series of deletions and substitutions were made at the 5' end of the gene fusion between the first 388 codons of diphtheria toxin (DT) and a cDNA encoding human IL2. The chimeric protein (DT388-IL2) was expressed and purified from E. coli and found to be very cytotoxic to a human T cell line, HUT 102, that expresses a large number of IL2 receptors. Deletion of the first five amino acids of DT resulted in a non-cytotoxic chimeric protein that had both ADP-ribosylation activity and IL2 receptor binding activity. Deletion of the first two amino acids of DT had little effect on cytotoxicity, while deletion of the first four amino acids or of two acidic residues at positions 3 and 4 greatly reduced cytotoxicity. Unexpectedly, a mutant containing a single leucine in place of the first two amino acids (gly, ala) was 2-3 fold more active. The amino terminus of DT may participate in the translocation of the A chain to the cytosol in a manner similar to Pseudomonas exotoxin (PE) in which a specific C-terminal sequence has been proposed to be involved in its cytotoxicity.
Chaudhary
V K
VK
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
FitzGerald
D J
DJ
Pastan
I
I
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D002678:Chimera; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004167:Diphtheria Toxin / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015375:Receptors, Interleukin-2 / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping
D004167:Diphtheria Toxin; D007376:Interleukin-2; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D000246:Adenosine Diphosphate Ribose