Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6.
We have used site-directed and in-frame deletion mutational analysis in order to explore the structural features of the IL-6 portion of the diphtheria toxin-related interleukin-6 (IL-6) fusion toxin DAB389-IL-6 that are essential for receptor-binding and subsequent inhibition of protein synthesis in target cells. Deletion of the first 14 amino acids of the IL-6 component of the fusion toxin did not alter either receptor binding affinity or cytotoxic potency. In contrast, both receptor binding and cytotoxic activity were abolished when the C-terminal 30 amino acids of the fusion toxin were deleted. In addition, we explored the relative role of the disulfide bridges within the IL-6 portion of DAB389-IL-6 in the stabilization of structure required for receptor-binding. The analysis of mutants in which the substitution of either Cys440, Cys446, Cys469 or Cys479 to Ser respectively, demonstrates that only the disulfide bridge between Cys469 and Cys479 is required to maintain a functional receptor binding domain. In addition, the internal in-frame deletion of residues 435-451, which includes Cys440 and Cys446, was found to reduce, but not abolish receptor binding affinity. These results further demonstrate that the disulfide bridge between Cys440 and Cys446 is not essential for receptor-binding. However, the reduced cytotoxic potency of DAB389-IL6(delta 435-451) suggests that the conformation and/or receptor binding sites associated with this region of the fusion toxin is/are important for maintaining the wild type receptor binding affinity and cytotoxic potency.
Evans Department of Clinical Research, Boston University Medical Center, University Hospital, MA 02118.
L F
LF
Jean
C A
CA
Waters
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D
Keemy
J R
JR
Murphy
D001483:Base Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D004167:Diphtheria Toxin / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers; D017353:Gene Deletion; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016364:Reading Frames; D011971:Receptors, Immunologic / Q000378:metabolism*; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins / Q000235:genetics
C081484:DAB(389)-interleukin 6; D004167:Diphtheria Toxin; D004337:Drug Carriers; D015850:Interleukin-6; D011971:Receptors, Immunologic; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins