Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4.
DAB389-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-related fusion protein which has been shown to be selectively toxic to cells expressing the mIL-4 receptor. In this report, we have used site-directed and in-frame deletion mutagenesis to study the role of the putative C-terminal alpha-helix (helix E) of the mIL-4 component of DAB389-mIL-4 in the intoxication process. We demonstrate that deletion of the C-terminal 15 amino acids of the fusion toxin leads to loss of cytotoxicity. The substitution of Phe496 with either Pro, Ala or Tyr, results in a greater than 20-fold decrease in cytotoxic activity of the respective mutant fusion toxins. In addition, substitution of Leu497 with either Ala or Glu results in a similar loss of cytotoxic activity. All of these mutant forms of the mIL-4 fusion toxin demonstrate a significant decrease in binding affinity (Ki) to the mIL-4 receptor in a competitive radioligand binding assay. In marked contrast, however, the substitution of Asp495 with Asn results in a 4-fold increase in cytotoxic potency and binding affinity to mIL-4 receptor bearing cells in vitro.
Lakkis
F
F
Evans Department of Clinical Research, University Hospital, Boston, MA 02118.
Landgraf
B
B
Wen
Z
Z
Strom
T B
TB
Murphy
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015847:Interleukin-4 / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D014407:Tumor Cells, Cultured
D004167:Diphtheria Toxin; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen; D011993:Recombinant Fusion Proteins; C076961:diphtheria toxin-related-murine interleukin-4-fusion toxin; D015847:Interleukin-4