Immunologic Function and Molecular Insight of Recombinant Interleukin-18.

In recent years, cytokine-mediated therapy has emerged as further advance alternative in cancer therapy. Interleukin-18 (IL-18) has exhibited interesting anti-cancer properties especially when combined with IL-12. We engineered IL-18 in order to improve its activity using single point mutagenesis. IL-18 mutants were constructed according to binding residues and polarity which we tried to increase polarity in M33Q and M60Q, enhanced cationicity in E6K, and flexibility in T63A. All IL-18 proteins were expressed in Pichia pastoris, purified, and then measured the activity by treating with the NK-92MI cell line to evaluate interferon-γ (IFN-γ) stimulation. The E6K and T63A mutant forms showed higher activity with respect to native proteins at the concentration of 200 ng mL-1 by inducing the expression of IFN-γ, about factors of 9 and 4, respectively. Meanwhile, M33Q and M60Q had no significant activity to induce IFN-γ. Interestingly, the combination of E6K and T63A mutations could synergize the induction activity of IL-18 to be 16 times at 200 ng mL-1. Furthermore, molecular dynamics studies have elucidated the effect due to mutation on conformation of the binding site of IL-18. The results turn out that E6K provides structural perseverance against mutation, while M33Q and M60Q promote vivid overall change in protein conformation, especially at the binding site. For T63A, mutation yields small difference in structure but clearly increases structural flexibility. However, a small structural change was observed when T63A was combined with E6K. Our research resulted in a novel version of IL-18 which could be a new key candidate for cytokine-mediated therapy.

The structural basis for receptor recognition of human interleukin-18.

Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and ß (Rß) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors' recognition mode for IL-18 is similar to IL-1ß; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity.

Purification, crystallization and preliminary X-ray crystallographic analysis of human IL-18 and its extracellular complexes.

Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the interleukin-1 (IL-1) family, is involved in the pathogenesis of autoimmune/autoinflammatory and allergic diseases such as juvenile idiopathic arthritis and bronchial asthma. IL-18 forms a signalling complex with the IL-18 receptor α (IL-18Rα) and ß (IL-18Rß) chains; however, the detailed activation mechanism remains unclear. Here, the IL-18-IL-18Rα binary and IL-18-IL-18Rα-IL-18Rß ternary complexes were purified and crystallized as well as IL-18 alone. An X-ray diffraction data set for IL-18 was collected to 2.33 Šresolution from a crystal belonging to space group P21, with unit-cell parameters a = 68.15, b = 79.51, c = 73.46 Å, ß = 100.97°. Crystals of both the IL-18 binary and ternary complexes belonging to the orthorhombic space groups P21212 and P212121, respectively, diffracted to 3.10 Šresolution. Unit-cell parameters were determined as a = 135.49, b = 174.81, c = 183.40 Šfor the binary complex and a = 72.56, b = 111.56, c = 134.57 Šfor the ternary complex.

Expression, purification and structural analysis of human IL-18 binding protein: a potent therapeutic molecule for allergy.

BACKGROUND: While interleukin-18 (IL-18) plays an important role in the innate and adaptive immune responses, it can also cause severe allergic inflammatory reactions. Thus it is a molecule currently being targeted for therapy. The natural intrinsic inhibitor of IL-18 receptor activation, IL-18 binding protein (IL-18BP), shows a great potential for the treatment of allergy. METHODS: Expression and purification of recombinant human IL-18BP (rhIL-18BP) were performed using the baculovirus system to develop a therapeutic molecule for the treatment of IL-18-related diseases and to investigate the structural basis of its inhibitory mechanism. RESULTS: Purified rhIL-18BP potently inhibited the production of interferon-gamma by peripheral blood mononuclear cells in the presence of lipopolysaccharide and by human myelomonocytic KG-1 cells in the presence of IL-18 (IC50 = 0.4 nM). Surface plasmon resonance showed a high affinity (Kd = 0.46 nM) for rhIL-18BP in binding hIL-18. Structural analysis indicated that the stoichiometry between IL-18 and IL-18BP is 1 : 1 in solution and the model structure of the complex suggests that the key residues on IL-18 (L5, K53, S55) and the estimated key residues on IL-18BP (F93,Y97, F104) could have interactions. The structural mechanism of IL-18BP inhibition might be a competition for Site 2 on rIL-18 so that IL-18BP can prevent IL-18 receptor alpha from binding to Site 2 and inhibit IL-18 receptor activation. CONCLUSIONS: IL-18BP has unique features with respect to its structure, binding mode and inhibitory mechanism. It is a molecule that has a great potential for the therapy of allergy.