Structure-guided optimization of the interleukin-6 trans-signaling antagonist sgp130.
Binding of interleukin-6 (IL-6) to its specific receptor IL-6R is a prerequisite for the activation of the signal-transducing receptor glycoprotein 130 (gp130). A soluble form of the IL-6R (sIL-6R) in complex with IL-6 can activate cells lacking membrane-bound IL-6R (trans-signaling). IL-6-trans-signaling is counterbalanced by a naturally occurring, soluble form of gp130 (sgp130), whereby signaling via the membrane-bound IL-6R is not affected. Many inflammatory and neoplastic disorders are driven by IL-6 trans-signaling. By analysis of the three-dimensional structure of gp130 in complex with IL-6 and sIL-6R, we identified amino acid side chains in gp130 as candidates for the generation of sgp130 muteins with increased binding affinity to IL-6/sIL-6R. In addition, with information from modeling and NMR analysis of the membrane proximal domain of gp130, we generated a more stable variant of sgp130Fc. Proteins were tested for binding to the IL-6/sIL-6R-complex, for inhibition of IL-6/sIL-6R-induced cell proliferation and of acute phase gene expression. Several mutations showed an additive effect in improving the binding affinity of human sgp130 toward human IL-6/sIL-6R. Finally, we demonstrate the species specificity of these mutations in the optimal triple mutein (T102Y/Q113F/N114L) both in vitro and in a mouse model of acute inflammation.
Tenhumberg
Stephanie
S
Institute of Biochemistry, Christian-Albrechts-University Kiel, D-24098 Kiel, Germany.
Waetzig
Georg H
GH
Chalaris
Athena
A
Rabe
Björn
B
Seegert
Dirk
D
Scheller
Jürgen
J
Rose-John
Stefan
S
Grötzinger
Joachim
J
D000209:Acute-Phase Proteins / Q000235:genetics / Q000276:immunology*; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D049109:Cell Proliferation / Q000187:drug effects; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D050822:Cytokine Receptor gp130 / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D004195:Disease Models, Animal; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000276:immunology; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D051379:Mice; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D019947:Receptors, Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D012995:Solubility
D000209:Acute-Phase Proteins; C508600:IL6 protein, human; C508603:IL6R protein, human; C494199:IL6ST protein, human; D015850:Interleukin-6; D019947:Receptors, Interleukin-6; D050822:Cytokine Receptor gp130