IL-6 Variants Research Articles

Development of human IL-6 receptor antagonists.

We have shown that through mutagenesis of IL-6 it is possible to separate receptor binding from signal transduction of the cytokine. Mutations in residues important for signal transduction via gp130 result in IL-6 variants that can competitively inhibit wtIL-6 activity in vitro. The differential effects of these signaling deficient mutants on various cell lines of human origin suggest that receptor composition and/or signal transduction pathways may vary between cells of different origin. The observations that three sites have been identified which are important for gp130 interaction raises the question what the role of each region is in the stepwise formation of the active IL-6 receptor complex. The overall tertiary conformation of the beta-site mutants is intact, as judged from their binding characteristics to conformation specific mAbs and IL-6R alpha. As can be deduced from Figure 1, beta-site mutations may therefore affect a direct interaction with gp130, dimerization of IL-6, or maybe a conformational change in IL-6R alpha, important for gp130 interaction. A future challenge will therefore be to determine the function of each of the beta-sites in IL-6 receptor interaction.

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.

Analysis of human IL-6 mutants expressed in Escherichia coli. Biologic activities are not affected by deletion of amino acids 1-28.

We have constructed and analyzed amino terminally deleted analogs of IL-6. Progressively shortened variants of mature IL-6 were constructed at the cDNA level and expressed in Escherichia coli. Mutant proteins were recovered from refractile bodies by solubilizing in 6 M guanidine-HCl. The mutant protein concentration in these preparations was estimated by Western blotting by using an IL-6-specific mAb and the biologic activity was measured in the B9 (hybridoma growth factor) assay. The first 28 amino acids of mature IL-6 could be removed without significantly affecting biologic activity. A further removal of amino acids 29 and 30 resulted in an approximately 50-fold decrease, whereas removal of amino acids 31 to 34 virtually abolished the activity. The mutants showed the same reaction pattern in three other IL-6 assays: induction of murine thymocyte proliferation, induction of fibrinogen synthesis by a human hepatoma cell line (HepG2), and the induction of IgM synthesis by an EBV-transformed B cell line. This suggests that a single functional domain might be responsible for all four activities of IL-6.