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Article (Scientific journals)
Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP: fused receptor domains act as high affinity cytokine-binding proteins.
Metz, Silke; Wiesinger, Monique; Vogt, Michael et al.
2007In Journal of Biological Chemistry, 282 (2), p. 1238-48
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Keywords :
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
Abstract :
[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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Metz, Silke
Wiesinger, Monique ;  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
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Title :
Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP: fused receptor domains act as high affinity cytokine-binding proteins.
Publication date :
Journal title :
Journal of Biological Chemistry
Publisher :
American Society for Biochemistry and Molecular Biology, United States - Maryland
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Issue :
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Peer reviewed :
Peer Reviewed verified by ORBi
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