Functional role played by the glycosylphosphatidylinositol anchor glycan of CD48 in interleukin-18-induced interferon-gamma production.

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.

Interleukin (IL) 1 -18 is a cytokine that induces T cells and natural killer cells to produce interferon (IFN)-␥ (1). It also has some IFN-␥-independent pro-inflammatory activities because it induces T cells and natural killer cells to synthesize tumor necrosis factor-␣, granulocyte macrophage colony-stimulating factor, nitric oxide, and chemokines (2). It has been reported that IL-18 binds to IL-18 receptor (IL-18R) ␣ and ␤ and that this induces signal transduction pathways that may involve nuclear factor-B (3), p56(lck) (4), and the mitogen-activated protein kinase (MAPK) (4). However, the mechanism by which the formation of IL-18⅐IL-18R␣⅐IL-18R␤ complex leads to intracellular signal transduction remains unclear.
Our laboratory has been interested in the carbohydrate recognition activities of various cytokines because we speculate that sugar chains recognized by these cytokines may function as immunomodulators (5). We previously reported that IL-1␤ specifically recognizes GPI anchor glycans (6). IL-18 has been classified as a member of the IL-1 family on the basis of its structural similarity to IL-1␤ (7). Thus, here we investigated whether IL-18 also has carbohydrate binding ability. We found that like IL-1␤, IL-18 recognizes GPI anchor glycans and that it specifically binds to the third mannose 6-phosphate diester, and this binding is inhibited by the addition of mannose 6-phosphate. Furthermore, it was found that IL-18R␣ also recognizes GPI anchor glycans and that IL-18R␣ specifically binds to the exposed second mannose 6-phosphate. To elucidate the physiological significance of the GPI anchor glycan binding ability of both IL-18 and IL-18R␣, we investigated the effect of phosphatidylinositol-specific phospholipase C (PI-PLC) treatment on the IL-18-stimulated tyrosine-phosphorylation of KG-1 cells. We found that PI-PLC treatment diminished intracellular signaling and IFN-␥ production. It seems that GPI anchor glycan recognition by IL-18 and IL-18R␣ induces formation of the IL-18⅐IL-18R␣⅐GPI anchor glycan complex, and this complex immediately binds to IL-18R␤ and induces intracellular signal transduction. Analysis of the IL-18R␣ or IL-18R␤ immunoprecipitates of IL-18-treated KG-1 cells revealed the presence of a single 47-kDa GPI-anchored protein, CD48, in the complex, and CD48 bound to IL-18R␣ via both GPI anchor glycan and the specific peptide sequence. Thus, it appears that IL-18, IL-18R␣, and CD48 complex formation via GPI glycan and the specific peptide sequences triggers binding to IL-18R␤ and thereby induces intracellular signal transduction and IFN-␥ production.
Preparation of 35 S-rhIL-18 -cDNA encoding human IL-18 was kindly provided by Hayashibara Co. Ltd. The pET3a plasmid (Novagen, Inc., Madison, WI) was used as the T7 expression plasmid. 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 [ 35 S]methionine. The in vitro transcription and translation was accomplished as described in the manufacturer's instructions. An aliquot of the translation products was subjected to SDS-PAGE using 15% polyacrylamide gels and autoradiographed. The translation products were separated from free [ 35 S]methionine using a PD-10 column (Amersham Biosciences) with PBS and used immediately. Reactions using 1 pmol of plasmid DNA template and 30 Ci of [ 35 S]methionine reproducibly provided 150 pmol of 35 S-rhIL-18.
Binding Assays of IL-18 or IL-18R to Glycoproteins-The binding of 35 S-rhIL-18 to various glycoproteins was measured by solid-phase binding assays. Thus, enzyme-linked immunosorbent assay plates (Corning, Inc., Corning, NY) were coated with glycoproteins at 10 g/ml in PBS at 4°C overnight. The plates were washed with 0.05% Tween 20 in PBS, blocked with PBS containing 0.05% Tween 20 and 1% human serum albumin, and then treated with 1-5 ϫ 10 4 dpm of 35 S-IL-18 (1-5 pmol) in PBS containing 0.05% Tween 20 and 0.1% human serum albumin at 37°C for 2 h. After washing with 0.05% Tween 20 in PBS, the bound 35 S-rhIL-18 was released by treatment with 100 l of 1% SDS, and the radioactivity was measured by means of a liquid scintillation counter. The protein concentration used to coat the plates was determined by experiments using various concentrations up to 1 mg/ml.
To hydrolyze the GlcNAc-phosphodiester linkage and the Neu5Ac␣233Gal linkages of shAP, shAP was treated with acid as follows (11): 10 g of shAP was incubated in 50 l of 0.01 N HCl at 100°C for 30 min, after which the pH was adjusted to 7, and de-GlcNAc-shAP was prepared. An aliquot of de-GlcNAc-shAP was also treated with phosphatase. To do this, the pH of the de-GlcNAc-shAP solution was first adjusted to pH 8 followed by incubation with a 20-l slurry of bovine intestine alkaline phosphatase-immobilized beads (Sigma) at 37°C for 1 h. The beads were then removed by centrifugation (1000 ϫ g, 30 s) to generate de-GlcNAc-phosphate-shAP.
Cell Culture-KG-1 human leukemia cells (RCB1166) were obtained from the RIKEN Cell Bank (Ibaraki, Japan) and maintained in complete RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, 50 IU/ml penicillin, and 50 g/ml streptomycin at 37°C under a 5% CO 2 atmosphere. The cells were cultured until the cell density reached 1 ϫ 10 6 cells/ml, after which the culture was split.
IFN-␥ Production of KG-1 Cells Stimulated by IL-18 -For the bioassay, cells were resuspended in complete medium at a cell density of 1 ϫ 10 6 cells/ml and plated out in microtiter plates at 100 l/well. Thereafter, 100 l of rhIL-18 at various concentrations diluted in complete RPMI 1640 medium was added. The cells were incubated overnight at 37°C in a 5% CO 2 atmosphere, after which an IFN-␥ enzyme-linked immunosorbent assay was performed by using a human IFN-␥ enzyme immunoassay kit (Beckman Coulter Co.). To investigate the efffect of mannose 6-phosphate, rhIL-18 and mannose 6-phosphate at various concentrations were mixed and preincubated at 37°C for 30 min before being added to cells. To investigate the effect of PI-PLC, KG-1 cells (1 ϫ 10 6 cells/100 l PBS) were incubated with 5 milliunits/ml PI-PLC in PBS at 37°C for 60 min before IL-18 stimulation.
Detection of Tyrosine-phosphorylated Proteins in IL-18-stimulated KG-1 Cells-KG-1 cells (1 ϫ 10 7 cells) were incubated in the presence or absence of rhIL-18 (10 ng/ml) at 37°C for 20 min. To investigate the effect of PI-PLC, cells were treated with PI-PLC (5 milliunits/ml, 37°C for 60 min) before IL-18 stimulation. After incubation, the cells were harvested, washed twice in PBS, and boiled for 10 min in the sample buffer for SDS-PAGE. After transfer onto nitrocellulose membrane, the blots were incubated with a biotinylated anti-phosphotyrosine monoclonal antibody (Upstate Inc.) followed by avidin-conjugated peroxidase and visualized by means of the ECL system (Amersham Biosciences).
Preparation of Soluble CD48 without GPI Glycan-A cDNA encoding the full open reading frame of CD48 was amplified by polymerase chain reaction from KG-1 cell cDNAs, which were prepared using the Super-Script™ preamplification system for first-strand cDNA synthesis (Invitrogen). The primers used were 5Ј-atggatccCACTTGGTACATAT-GACC (forward primer) and 5Ј-tgaagcttTCAGGTAAGTAACAGGCC (reverse primer) for CD48. The sequences shown in lowercase letters reveal appropriate restriction sites. The amplified cDNA was digested with BamHI and HindIII and cloned into pColdI (Takara Bio Inc., Tokyo, Japan). The sequence of the resulting plasmid was confirmed using an Applied Biosystems Prism 310 Genetic Analyzer (PE Biosystems). The plasmid was transformed with Escherichia. coli BL21 strain. Luria-Bertani broth with 100 g/ml ampicillin was inoculated with the overnight culture of the transformed E. coli. When the absorbance at 600 nm reached a value of 0.5, isopropyl ␤-D-thiogalactopyranoside was added to a final concentration of 1 mM, and the cultures were allowed to stand at 15°C for 24 h. The bacteria were harvested by centrifugation at 5000 ϫ g for 15 min. Cell pellets were resuspended and disrupted using a sonicator in PBS. After centrifugation at 12,000 ϫ g for 20 min, the supernatant was applied to a nickel-nitrilotriacetic acid-agarose column (Amersham Biosciences). After washing the column with 50 mM NaH 2 PO 4 (pH 8.0) containing 20 mM imidazole, CD48 was eluted with 50 mM NaH 2 PO 4 (pH 8.0) containing 250 mM imidazole. The purified CD48 was dialyzed against PBS and checked using SDS-PAGE. Protein concentration was determined using a Bio-Rad Protein Assay dye reagent and bovine serum albumin as a standard.
Binding Assay of Soluble CD48 to IL-18R␣ or IL-18R␤-The binding of soluble CD48 to IL-18R␣ or IL-18R␤ was measured by a solid-phase binding assay. Enzyme-linked immunosorbent assay plates were coated with CD48 (10 g/ml) in PBS at 4°C overnight. The plates were washed with 0.05% Tween 20 in PBS, blocked with PBS containing 0.05% Tween 20 and 3% BSA, and then incubated with various concentrations of the IL-18R␣-Fc chimera or IL-18R␤-Fc chimera in PBS containing 0.05% Tween 20 and 0.1% BSA. Bound IL-18R␣ or IL-18R␤ was treated with goat anti-IL-18R␣ antibody or goat anti-IL-18R␤ antibody (R&D Systems) and HRP-conjugated anti-goat IgG antibody (Amersham Biosciences) and detected with TMB solution. To investigate the inhibitory effect of anti-CD48 antibody, the plates were treated with mouse anti-CD48 antibody after blocking with BSA.
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. Released GPI-anchored proteins were applied to a PVL-Sepharose column. PVL was obtained from Wako Chemicals. PVL-Sepharose (3 mg/ml) was prepared from PVL and CNBr-Sepharose (Amersham Biosciences). After flow-through components were collected, bound components were eluted with PBS containing 0.3 M GlcNAc. Both components were applied to a PD-10 column (Amersham Biosciences) for desalting and freeze-dried. After SDS-PAGE using 10.5% acrylamide gel and blotting onto nitrocellulose membrane, CD48 was detected using mouse anti-CD48 antibody and HRP-conjugated goat anti-mouse IgG antibody. Before blotting, total protein was stained with SYPRO® Orange (Bio-Rad).

IL-18
Binds to the GPI-anchored Protein shAP-We previously reported that IL-1␤ is able to bind to carbohydrates and that it recognizes the GPI anchor glycan (6). Because IL-1␤ shares amino acid sequence homology with IL-18, we investigated whether IL-18 also has carbohydrate binding activity. First, we performed binding assays using plates coated with 10 IL-18 Binding to GPI Anchor Glycan of CD48 g/ml ribonuclease B (13), thyroglobulin (14), ovalbumin (15), transferrin (16), fetuin (17), T-H glycoprotein (18), CEA (19), and shAP (11,20), which involve the respective glycan structures as cited references. Because BSA resulted in relatively high nonspecific binding, we used human serum albumin as the blocking reagent. We prepared 35 S-rhIL-18 by in vitro translation in the presence of [ 35 S]methionine followed by separation from excess [ 35 S]methionine by PD-10 column chromatography. The resulting 35 S-rhIL-18 protein was immediately used in the binding assays. We found that 35 S-rhIL-18 bound to shAP ( Fig. 1A; Fig. 2B, E), T-H glycoprotein (Fig. 1B), and CEA (Fig. 1C) in a dose-dependent manner. The binding of 35 S-rhIL-18 to plates coated with 10 g/ml CEA, shAP, or T-H glycoprotein was concentration-dependent up to 2 ϫ 10 4 dpm/ 100 l. These bindings did not change in the presence of 1 mM EDTA, which shows that the binding of IL-18 to shAP does not require divalent cations. Thus, IL-18 appears, like IL-1␤ (6), to bind to GPI-anchored glycoproteins.
Inhibitory Effects of Haptenic Sugars Derived from the GPI Anchor Glycan of shAP on the Carbohydrate Binding Activity of 35 S-rhIL-18 -The N-glycans and GPI anchor glycan structures of shAP have been determined, as summarized in Fig. 2A (11). Consequently, we used this protein to more precisely determine the carbohydrate binding specificity of IL-18. To do this, we investigated the inhibitory effects on the binding of 35 S-rhIL-18 to shAP of haptenic sugars derived from the GPI anchor glycan of shAP. The saccharides tested and their inhibitory effects are summarized in Table I. The biantennary sugar chain of shAP, which is an asialo-N-linked sugar chain, was not inhibitory at concentrations of up to 1 mM. In contrast, mannose 6-phosphate, which is a constituent of the GPI anchor glycan of shAP, was an effective inhibitor. However, other constituents of the GPI anchor glycan of shAP, such as ethanolamine phosphate, inositol phosphate, and GlcNAc␤1-phosphate, were not inhibitory up to 1 mM. In addition, mannose 6-sulfate, mannose 1-phosphate, glucose 6-phosphate, and mannitol-6-phosphate were not inhibitory at concentrations up to 1 mM, which indicate that mannose substituted with phosphate at the C-6 position is necessary for the carbohydrate binding of IL-18.
These observations led us to speculate that IL-18 recognizes a mannose 6-phosphate diester moiety in GPI anchor glycans. To test this, we removed the second mannose 6-phosphate diester of shAP by mild acid and phosphatase treatment and performed the IL-18-binding assay again. Because de-GlcNAc shAP and de-GlcNAc␤13phosphate shAP maintained their ability to bind to 35 S-rhIL-18 (Fig. 2B, • and Ⅺ), it appears that IL-18 specifically binds to the third mannose 6-phosphate diester in the GPI anchor glycan.
The Binding of IL-18 to the GPI Anchor Glycan Modulates IFN-␥ Production and Intracellular Tyrosine Phosphorylation of IL-18-stimulated KG-1 Cells-It is known that human leukemia KG-1 cells produce IFN-␥ when they are stimulated with IL-18 (1). To determine whether the recognition of the GPI

IL-18 Binding to GPI Anchor Glycan of CD48
anchor glycan by IL-18 is important for the expression of its physiological functions, we assessed the ability of mannose 6-phosphate and PI-PLC to block the stimulatory effects of IL-18 on the production of IFN-␥ production by KG-1 cells. First, we confirmed that incubation of KG-1 cells (1 ϫ 10 5 cells/well) for 24 h in the presence of rhIL-18 indeed induced them to produce IFN-␥ in a rhIL-18-dependent manner (Fig.  3A). Because 10 ng/ml IL-18 was sufficient to stimulate IL-18dependent IFN-␥ production, the following experiments were performed at this concentration. As shown in Fig. 3B, mannose 6-phosphate inhibited the IFN-␥ production of KG-1 cells, although 10 Ϫ5 M mannose 6-phosphate was not sufficient to inhibit all the activity. These results suggest that mannose 6-phosphate competes for IL-18 binding to the GPI anchor glycans on the cell surface and thereby suppresses the IL-18induced physiological activity of KG-1 cells. Furthermore, removal of the GPI-anchored proteins on KG-1 cells by PI-PLC treatment inhibited the rhIL-18-stimulated IFN-␥ production (Fig. 3B) and tyrosine phosphorylation (Fig. 4). These results indicate that IL-18 binding of the third mannose 6-phosphate diester in the GPI-anchor glycan is at least required to enhance IL-18-dependent intracellular signal transduction and IFN-␥ production.
IL-18R␣ Binds to Both IL-18 and the GPI Anchor Glycan-Because it is known that the first step in the signal transduction cascade induced by IL-18 involves its binding to IL-18R␣ (21), we speculated that IL-18R␣ may interact with the second mannose 6-phosphate diester of the GPI anchor glycan. To test this notion, we coated plates with intact shAP, de-␤-GlcNAc-shAP, and de-GlcNAc␤13phosphate-shAP and assessed the binding of the IL-18R␣-Fc chimera by using a mouse anti-human IgG Fc antibody and HRP-conjugated anti-mouse IgG (Fig. 5). We found that IL-18R␣ only bound to de-␤-GlcNAc-shAP in a dose-dependent manner (Fig. 5A, •) and did not recognize intact shAP or de-GlcNAc␤13phosphate-shAP (Fig.  5A, E and Ⅺ); on the other hand, IL-18R␤ did not show any binding ability to these shAP derivatives (Fig. 5B). These results suggest that both IL-18 and IL-18R␣ can recognize the GPI anchor glycan, although they have different binding specificities. Thus, IL-18 binds to the third mannose 6-phosphate diester, whereas IL-18R␣ binds to the second mannose 6-phosphate.
Identification of the GPI-anchored Protein Involved in the Complex of IL-18⅐IL-18R␣⅐IL-18R␤-If IL-18 and IL-18R␣ binding to a GPI-anchored glycoprotein triggers the formation of the IL-18⅐IL-18R␣⅐IL-18R␤ complex, a specific GPI-anchored glycoprotein should be co-immunoprecipitated with IL-18R␣ or IL-18R␤ in lysates of IL-18-stimulated KG-1 cells. To identify the GPI-anchored protein recognized by IL-18 and IL-18R␣, the IL-18⅐IL-18R␣⅐IL-18R␤ complex was immunoprecipitated by using anti-IL-18R␣ or IL-18R␤ antibody, and the co-immunoprecipitated GPI-anchored protein was detected by using proaerolysin, which specifically binds to GPI-anchor glycans (11). Only a single 47-kDa protein was detected as a GPI- anchored protein in the anti-IL-18R␣ antibody or anti-IL-18R␤ antibody immunoprecipitates in lysates of IL-18-stimulated KG-1 cells (Fig. 6A, lanes 3 and 5). However, the immunoprecipitates using rabbit IgG instead of anti-IL-18R␣ or anti-IL-18R␤ antibody were not stained by proaerolysin (Fig. 6A, lanes  6 and 7). Because whole GPI-anchored proteins in KG-1 cells were detected as shown in lane 1 of Fig. 6A, it appears that this 47-kDa protein is specifically involved in the IL-18⅐IL-18R␣⅐IL-18R␤ complex. We also confirmed that IL-18R␤ was co-immunoprecipitated with IL-18R␣ by staining the immunoprecipitates with anti-IL-18R␤ antibody in IL-18-stimulated KG-1 cells; moreover, IL-18R␣ was co-immunoprecipitated with IL-18R␤ (data not shown).
It has been reported that CD48 is one of the GPI-anchored proteins on T cells and associates with protein kinase p56lck (22). We found that an anti-CD48 antibody stained the immunoprecipitates that were generated by using anti-IL-18R␣ or anti-IL-18R␤ antibodies (Fig. 6B, lanes 8 -11); on the other hand, the immunoprecipitates using rabbit IgG were not stained by anti-CD48 antibody (Fig. 6B, lanes 12 and 13). In the absence of IL-18, CD48 was weakly detected in the immunoprecipitate by using anti-IL-18R␣ antibody or anti-IL-18R␤ antibody (Fig. 6B, lanes 8 and 10). Therefore, CD48 seems to be a GPI-anchored protein that weakly associates with IL-18R␣.
GPI Anchor Glycan of CD48 Lacks ␤GlcNAc-CD48 was specifically immunoprecipitated with IL-18R␣, although many GPI-anchored glycoproteins exist on the cell surface. In order to determine why CD48 specifically binds to IL-18R␣, we investigated whether CD48 has ␤GlcNAc-deleted GPI glycan or whether the CD48 protein portion interacts with IL-18R␣. After KG-1 cells were treated with PI-PLC, released GPI-an-chored glycoproteins were subjected to PVL-Sepharose column chromatography, which specifically recognizes the ␤GlcNAc residue (23). Approximately half of GPI-anchored proteins flowed through the column (PVL Ϫ component), and the remainder was eluted with 0.3 M GlcNAc (PVL ϩ component) (Fig. 7,  lanes 1 and 2). Both PVL Ϫ and PVL ϩ components were applied to SDS-PAGE, blotted, and immunostained with anti-CD48 antibody. More than 90% of CD48 molecules were PVL Ϫ components (Fig. 7, lanes 3 and 4), suggesting that most CD48 molecules have ␤GlcNAc-deleted GPI glycan and can bind to IL-18R␣.
sequence and GPI glycan. We also showed that mannose 6-phosphate or 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. These results also suggested that CD48 binding to IL-18R␣ via the peptide portion was not strong enough to retain the association after inhibition of binding via GPI glycan. As previously reported, IL-18 induces signal transduction pathways that may involve nuclear factor-B (3), p56lck (4), and MAPK (4). It has not been shown that p56lck and MAPK associate with IL-18R␣ and IL-18R␤, but p56lck has been reported to associate with CD48 (22,24). This in turn suggests that CD48 is an integral com-ponent of the signal-transducing IL-18 complex. Because it is known that the IL-18⅐IL-18R␣ complex binds to IL-18R␤ (21), it is likely that after IL-18R␣, IL-18, and CD48 form a complex on the cell surface, this complex immediately binds to IL-18R␤, as presented in the model shown in Fig. 9, and that this leads to signal transduction and IFN-␥ production. This is the first report showing that a GPI anchor glycan of CD48 may actually be essential for the delivery of the IL-18 signal.
We found that IL-18 binds to the third mannose 6-phosphate diester, whereas IL-18R␣ binds to the second exposed mannose 6-phosphate of GPI glycan. On the other hand, Ͼ90% of CD48 does not have GlcNAc␤13phosphate36 mannose residue (Fig.  8), although GlcNAc␤13phosphate residue is a common epitope of GPI glycan (11). Because a ␤-N-acetylglucosamine residue of the GlcNAc␤13phosphate36 mannose residue must be removed to be recognized by IL-18R␣, the ␤-N-acetylglucosamine residue that protects the exposure of the second mannose 6-phosphate diester in the GPI anchor glycan may regulate the IL-18-dependent immune response. As a result, we are currently determining whether a GlcNAc␤13phosphate-specific ␤-N-acetylglucosaminidase is secreted together with IL-18 by stimulated macrophages.
The inhibitory effect of mannose 6-phosphate on the IL-18stimulated production of IFN-␥ by KG-1 cells was very weak (its 50% inhibitory concentration was 10 Ϫ5 M). However, we could not use more than 10 Ϫ5 M mannose 6-phosphate because it changed the pH of the medium. It is likely that mannose 6-phosphate does not efficiently inhibit the recognition by IL-18 or IL-18R␣ of the endogenous GPI anchor glycan in CD48 because the endogenous GPI anchor glycan is a rather strong ligand compared with exogenous mannose 6-phosphate.
Although both tumor necrosis factor-␣ and IL-18 recognize the GPI anchor glycan, they bind to different sites because tumor necrosis factor-␣ binds the second mannose 6-phosphate diester (5), whereas IL-18 binds the third mannose 6-phosphate diester. These observations indicate that different cytokines may have precise and unique carbohydrate binding specificities. IL-1␤ and IL-18 have moderate sequence similarity (7), and the receptors for IL-1␤ and IL-18 belong to the IL-1 receptor family (25). The intracellular signaling pathways of IL-1␤ and IL-18 also share the same downstream mediators (3). Because IL-1␤ also recognizes GPI anchor glycans (6), we are now investigating whether the mechanism by which IL-1␤ recognizes the GPI anchor glycan is equivalent to that of IL-18.
GPI-anchored proteins are widely distributed on the cell surface (26). GPI anchor glycans are essential for embryogenesis and skin development in mice (27), and GPI deficiencies cause paroxysmal nocturnal hemoglobinuria in humans (28). Moreover, GPI-anchored proteins are receptors for bacterial toxins, clostridial ␣-toxin (29), aerolysin (30), and the plant toxin enterolobin (31). Here we show a novel putative function of the GPI anchor glycan of CD48, namely, as an immunomodulator of the response to IL-18. Our future studies will analyze FIG. 7. Detection of ␤-GlcNAc residues of CD48. GPI-anchored glycoproteins on KG-1 cells were released from the cells by digestion with PI-PLC (5 milliunits/ml, 37°C, 1 h) and subjected to PVL-Sepharose column chromatography. Flow-through components and bound components were subjected to SDS-PAGE and blotting and stained with anti-CD48 antibody. Lanes 1 and 2, protein staining by SYPRO® Orange; lanes 3 and 4, immunostaining with anti-CD48 antibody.