title: Cellular processing of the interleukin-2 fusion toxin DAB486-IL-2 and efficient delivery of diphtheria fragment A to the cytosol of target cells requires Arg194.
abstract: We have used site-directed mutagenesis to examine the role played by Arg191, Arg193, and Arg194 of the fusion toxin DAB486-IL-2 in the intoxication of high affinity interleukin-2 receptor-bearing T-lymphocytes. These arginine residues are positioned in the proteolytically sensitive 14-amino acid loop subtended by the disulfide bond between Cys187 and Cys202 in this fusion toxin. DAB486-IL-2 was formed by the genetic substitution of the native diphtheria toxin receptor binding domain with human interleukin-2 (Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B., and Murphy, J.R. (1987) Protein Eng. 1, 493-498). We demonstrate that substitution of Arg194 with Gly results in a 1000-fold loss of DAB486-IL-2 potency. Since trypsin "nicking" of the Gly194 mutant restores biologic activity, we conclude that Arg194 is required for the cellular processing of the fusion toxin which results in the release of fragment A into the cytosol.
authors.affiliation: Evans Department of Clinical Research, Boston University Medical Center, Massachusetts 92118.
authors.forename: D P
authors.initials: DP
authors.lastname: Williams
authors.forename: Z
authors.initials: Z
authors.lastname: Wen
authors.forename: R S
authors.initials: RS
authors.lastname: Watson
authors.forename: J
authors.initials: J
authors.lastname: Boyd
authors.forename: T B
authors.initials: TB
authors.lastname: Strom
authors.forename: J R
authors.initials: JR
authors.lastname: Murphy
mesh_terms: D000818:Animals; D001120:Arginine; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D007376:Interleukin-2 / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010957:Plasmids; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D013601:T-Lymphocytes / Q000276:immunology; D014357:Trypsin
chemical_list: C054780:DAB(486)-interleukin 2; D004167:Diphtheria Toxin; D007376:Interleukin-2; D015345:Oligonucleotide Probes; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D001120:Arginine; D014357:Trypsin