Human tumor necrosis factor alpha (TNF alpha) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF receptors.
To probe the ligand receptor interface, a number of point mutations were introduced in selected regions of human tumor necrosis factor (TNF) alpha by site-directed mutagenesis. The mutated proteins were expressed in Escherichia coli and analyzed for selective binding to recombinant 55- and 75-kDa TNF receptors in competition with radiolabeled wild-type TNF alpha. Generally, mutations in the loop from position 29 to 34 and at positions 86 and 146 preferentially impaired binding to the 75-kDa TNF receptor, whereas mutations in the region from 143 to 145 mainly affected binding to the 55-kDa TNF receptor. Mutation of the conserved Tyr87 resulted in a dramatic loss of binding activity to both receptors. The selectivity for one or the other receptor type was found to be enhanced by combining two or three point mutations, the effects of the single mutations with respect to receptor selectivity being at least additive. A combination of the mutations Arg32-->Trp and Ser86-->Thr yielded a double mutant (R32W-S86T) with wild-type binding to the 55 kDa, but no measurable binding to the 75-kDa TNF receptor. In contrast, combining the Asp143-->Asn and Ala145-->Arg mutations (D143N-A145R) resulted in a complete loss of binding to the 55-kDa TNF receptor, whereas binding to the 75-kDa TNF receptor was impaired by only 5-10-fold. In functional assays, selective activation of the 55-kDa TNF receptor by the R32W-S86T mutant elicited a full cytotoxic response in human KYM-1 cells and secretion of interleukin 6 and granulocyte-macrophate colony-stimulating factor in human umbilical vein endothelial cells. In contrast, stimulation of the 75-kDa TNF receptor with the D143N-A145R mutant as well as with agonistic antibodies failed to induce these responses.
Loetscher
H
H
Pharmaceutical Research-New Technologies, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Stueber
D
D
Banner
D
D
Mackay
F
F
Lesslauer
W
W
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha