Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP: fused receptor domains act as high affinity cytokine-binding proteins.
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.
Metz
Silke
S
Institut für Biochemie, Pauwelsstrasse 30, Universitätsklinikum RWTH Aachen, Germany.
Wiesinger
Monique
M
Vogt
Michael
M
Lauks
Heike
H
Schmalzing
Günther
G
Heinrich
Peter C
PC
Müller-Newen
Gerhard
G
D000210:Acute-Phase Reaction / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D019556:COS Cells; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D050822:Cytokine Receptor gp130 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015195:Drug Design; D015870:Gene Expression / Q000276:immunology; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism / Q000494:pharmacology; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D008113:Liver Neoplasms; D008164:Luminescent Proteins / Q000235:genetics; D053683:Oncostatin M / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D019947:Receptors, Interleukin-6 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D000093242:Interleukin-6 Inhibitors
D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D050822:Cytokine Receptor gp130; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008164:Luminescent Proteins; D053683:Oncostatin M; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; D000093242:Interleukin-6 Inhibitors; C507837:LIF protein, human; C054562:yellow fluorescent protein, Bacteria