The interleukin-6 (IL-6) partial antagonist (Q159E,T162P)IL-6 interacts with the IL-6 receptor and gp130 but fails to induce a stable hexameric receptor complex.
The extracellular "soluble" domains of the IL-6 receptor (sIL-6R) and gp130 (sgp130) form a hexameric ternary receptor complex together with IL-6, consisting of two molecules of each component. In this report we have investigated the interactions of the partial IL-6 antagonist (Q159E,T162P)IL-6 ((QT)IL-6), with the sIL-6R and sgp130. The kinetic rate constants of the binding of sIL-6R to immobilized monomeric (QT)IL-6 or IL-6 were obtained using an optical biosensor with analysis of the primary data by linear and nonlinear regression. Both methods of analysis showed that, due to a higher off-rate, sIL-6R has lower apparent affinity for (QT)IL-6 than IL-6. The lower affinity of (QT)IL-6 was further confirmed by equilibrium binding measurements at the sensor surface and in solution. Using the biosensor it was also shown that the (QT)IL-6 complex interacts with sgp130, supporting the notion that the biological activity of (QT)IL-6 is mediated via gp130. However, the IL-6 mutant, when incubated with sIL-6R and sgp130, failed to induce a stable hexameric receptor complex, as shown by narrowbore size exclusion chromatography.
Hammacher
A
A
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research and Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia.
Simpson
R J
RJ
Nice
E C
EC
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D006801:Humans; D015850:Interleukin-6 / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D018123:Receptors, Interleukin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012044:Regression Analysis; D000093242:Interleukin-6 Inhibitors
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D017931:DNA Primers; D015850:Interleukin-6; D046911:Macromolecular Substances; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D000093242:Interleukin-6 Inhibitors