Reference : Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP: fused receptor domains...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP: fused receptor domains act as high affinity cytokine-binding proteins.
Metz, Silke [> >]
Wiesinger, Monique mailto [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >]
Vogt, Michael [> >]
Lauks, Heike [> >]
Schmalzing, Gunther [> >]
Heinrich, Peter C. [> >]
Muller-Newen, Gerhard [> >]
Journal of Biological Chemistry
Yes (verified by ORBilu)
United States
[en] Acute-Phase Reaction/genetics ; Animals ; Antineoplastic Agents/pharmacology ; Bacterial Proteins/genetics ; Baculoviridae/genetics ; COS Cells ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Cercopithecus aethiops ; Cytokine Receptor gp130/chemistry/genetics/metabolism ; Drug Design ; Gene Expression/immunology ; Humans ; Interleukin-6/antagonists & inhibitors/metabolism/pharmacology ; Leukemia Inhibitory Factor/pharmacology ; Liver Neoplasms ; Luminescent Proteins/genetics ; Oncostatin M/pharmacology ; Protein Structure, Tertiary ; Receptors, Interleukin-6/chemistry/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; STAT3 Transcription Factor/metabolism
[en] Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6Ralpha and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6Ralpha, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6.IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.

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