Search

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.

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitous in eukaryotes. The minimum conserved GPI core structure of all GPI-anchored glycans has been determined as EtN-PO4-6Manalpha1-2Manalpha1-6Manalpha1-4GlcN-myo-inositol-PO3H. Human placental alkaline phosphatase (AP) has been reported to be a GPI-anchored membrane protein. AP carries one N-glycan, (NeuAcalpha2-->3)2Gal2GlcNAc2Man3GlcNAc(+/-Fuc)GlcNAc, and a GPI anchor, which contains an ethanolamine phosphate diester group, as a side chain. However, we found that both sialidase-treated soluble AP (sAP) and its GPI-anchored glycan bound to a Psathyrella velutina lectin (PVL)-Sepharose column, which binds beta-GlcNAc residues. PVL binding of asialo-sAP and its GPI-anchored glycan was diminished by digestion with diplococcal beta-N-acetylhexosaminidase or by mild acid treatment. After sequential digestion of asialo-sAP with beta-N-acetylhexosaminidase and acid phosphatase, the elution patterns on chromatofocusing gels were changed in accordance with the negative charges of phosphate residues. Trypsin-digested sAP was analyzed by liquid chromatography/electrospray ionization mass spectrometry, and the structures of two glycopeptides with GPI-anchored glycans were confirmed as peptide-EtN-PO4-6Manalpha1-->2(GlcNAcbeta1-PO4-->6)Manalpha1-6(+/-EtN-PO4-->)Manalpha1-->4GlcN, which may be produced by endo-alpha-glucosaminidase. In addition to AP, GPI-anchored carcinoembryonic antigen, cholinesterase, and Tamm-Horsfall glycoprotein also bound to a PVL-Sepharose column, suggesting that the beta-N-acetylglucosaminyl phosphate diester residue is widely distributed in human GPI-anchored glycans. Furthermore, we found that the beta-N-acetylglucosaminyl phosphate diester residue is important for GPI anchor recognition of aerolysin, a channel-forming toxin derived from Aeromonas hydrophila.