title: Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation.
abstract: Interleukin-6 (IL-6) drives the sequential assembly of a receptor complex formed by the IL-6 receptor (IL-6R alpha) and the signal transducing subunit, gp130. A model of human IL-6 (hIL-6) was constructed by homology using the structure of bovine granulocyte colony stimulating factor. The modeled cytokine was predicted to interact sequentially with the cytokine binding domains of IL-6R alpha and gp130 bridging them in a way similar to that of the interaction between growth hormone and its homodimeric receptor. Several residues on helices A and C which were predicted as contact points between IL-6 and gp130 and therefore essential for IL-6 signal transduction, were subjected to site-directed mutagenesis individually or in combined form. Interestingly, while single amino acid changes never produced major alterations in IL-6 bioactivity, a subset of double mutants of Y31 and G35 showed a considerable reduction of biological activity and were selectively impaired from associating with gp130 in binding assays in vitro, while they maintained wild-type affinity towards hIL-6-R alpha. More importantly, we demonstrated the antagonistic effect of mutant Y31D/G35F versus wild-type IL-6.
authors.affiliation: Department of Genetics, Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Pomezia, Rome, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Savino
authors.forename: A
authors.initials: A
authors.lastname: Lahm
authors.forename: A L
authors.initials: AL
authors.lastname: Salvati
authors.forename: L
authors.initials: L
authors.lastname: Ciapponi
authors.forename: E
authors.initials: E
authors.lastname: Sporeno
authors.forename: S
authors.initials: S
authors.lastname: Altamura
authors.forename: G
authors.initials: G
authors.lastname: Paonessa
authors.forename: C
authors.initials: C
authors.lastname: Toniatti
authors.forename: G
authors.initials: G
authors.lastname: Ciliberto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D018360:Crystallography, X-Ray; D050822:Cytokine Receptor gp130; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry; D013006:Growth Hormone / Q000737:chemistry; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018123:Receptors, Interleukin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019947:Receptors, Interleukin-6; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
chemical_list: D015703:Antigens, CD; C494199:IL6ST protein, human; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D050822:Cytokine Receptor gp130; D016179:Granulocyte Colony-Stimulating Factor; D013006:Growth Hormone