Abstract
Interleukin (IL)-18 induces T cells and natural killer cells to produce not only interferon-gamma but also other cytokines by binding to the IL-18 receptor (IL-18R) alpha and beta subunits. However, little is known about how IL-18, IL-18Ralpha, and IL-18Rbeta form a high-affinity complex on the cell surface and transduce the signal. We found that IL-18 and IL-18Ralpha bind to glycosylphosphatidylinositol (GPI) glycan via the third mannose 6-phosphate diester and the second beta-GlcNAc-deleted mannose 6-phosphate of GPI glycan, respectively. To determine which GPI-anchored glycoprotein is involved in the complex of IL-18 and IL-18Ralpha, IL-18Ralpha of IL-18-stimulated KG-1 cells was immunoprecipitated together with CD48 by anti-IL-18Ralpha antibody. More than 90% of CD48 was detected as beta-GlcNAc-deleted GPI-anchored glycoprotein, and soluble recombinant human CD48 without GPI glycan bound to IL-18Ralpha, indicating that CD48 is associated with IL-18Ralpha via both the peptide portion and the GPI glycan. To investigate whether the carbohydrate recognition of IL-18 is involved in physiological activities, KG-1 cells were digested with phosphatidylinositol-specific phospholipase C before IL-18 stimulation. Phosphatidylinositol-specific phospholipase C treatment inhibited the phosphorylation of tyrosine kinases and the following IL-18-dependent interferon-gamma production. These observations suggest that the complex formation of IL-18.IL-18Ralpha. CD48 via both the peptide portion and GPI glycan triggers the binding to IL-18Rbeta, and the IL-18.IL-18Ralpha.CD48.IL-18Rbeta complex induces cellular signaling.
Highlights
Interleukin (IL)1-18 is a cytokine that induces T cells and natural killer cells to produce interferon (IFN)-␥ [1]
We clearly demonstrated in this study that both IL-18 and IL-18 receptor (IL-18R)␣ have carbohydrate recognition abilities and that each of them separately recognizes a different site in the GPI anchor glycan of CD48
We showed that mannose 6-phosphate or phosphatidylinositol-specific phospholipase C (PI-PLC) treatment inhibits the response of KG-1 cells to IL-18 stimulation in terms of IFN-␥ secretion and intracellular tyrosine phosphorylation and that this may inhibit the binding of CD48 to IL-18R␣ and IL-18
Summary
Materials and Chemicals—rhIL-18 was purchased from Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan). A SalI-EcoRI fragment corresponding to the human IL-18 gene was inserted between the SalI and EcoRI sites of pET3a to produce the expression plasmid pET3a-IL-18 This was used as a template for in vitro transcription and translation in Puresystem® (Post Genome Institute Co., Ltd., Tokyo, Japan) in the presence of [35S]methionine. After SDS-PAGE using a 10.5% acrylamide gel and blotting onto a nitrocellulose filter, the GPI-anchored protein involved in the high-affinity complex of IL181⁄7IL-18R␣1⁄7IL-18R was probed by proaerolysin, mouse anti-proaerolysin antibody (Protox Biotech), and HRP-conjugated goat anti-mouse IgG antibody [12] and visualized by using the ECL system (Amersham Biosciences). Separation of GPI-anchored Proteins by PVL Column Chromatography—GPI-anchored glycoproteins on KG-1 cells were released by digestion with PI-PLC (5 milliunits/ml PBS) at 37 °C for 1 h. Total protein was stained with SYPRO® Orange (Bio-Rad)
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