Interleukin-2 Carbohydrate Recognition Modulates CTLL-2 Cell Proliferation
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m008781200
ISSN1083-351X
AutoresKeiko Fukushima, Katsuko Yamashita,
Tópico(s)Fungal Infections and Studies
ResumoInterleukin-2 (IL-2) specifically recognizes high-mannose type glycans with five or six mannosyl residues. To determine whether the carbohydrate recognition activity of IL-2 contributes to its physiological activity, the inhibitory effects of high-mannose type glycans on IL-2-dependent CTLL-2 cell proliferation were investigated. Man5GlcNAc2Asn added to CTLL-2 cell cultures inhibited not only phosphorylation of tyrosine kinases but also IL-2-dependent cell proliferation. We found that a complex of IL-2, IL-2 receptor α, β, γ subunits, and tyrosine kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the components of this complex, only the IL-2 receptor α subunit was stained with Galanthus nivalis agglutinin which specifically recognizes high-mannose type glycans. This staining was diminished after digestion of the glycans with endo-β-N-acetylglucosaminidase H or D, suggesting that at least a N-glycan containing Man5GlcNAc2 is linked to the extracellular portion of the IL-2 receptor α subunit. Our findings indicate that IL-2 binds the IL-2 receptor α subunit through Man5GlcNAc2 and a specific peptide sequence on the surface of CTLL-2 cells. When IL-2 binds to the IL-2Rα subunit, this may trigger formation of the high affinity complex of IL-2-IL-2Rα, -β, and -γ subunits, leading to cellular signaling. Interleukin-2 (IL-2) specifically recognizes high-mannose type glycans with five or six mannosyl residues. To determine whether the carbohydrate recognition activity of IL-2 contributes to its physiological activity, the inhibitory effects of high-mannose type glycans on IL-2-dependent CTLL-2 cell proliferation were investigated. Man5GlcNAc2Asn added to CTLL-2 cell cultures inhibited not only phosphorylation of tyrosine kinases but also IL-2-dependent cell proliferation. We found that a complex of IL-2, IL-2 receptor α, β, γ subunits, and tyrosine kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the components of this complex, only the IL-2 receptor α subunit was stained with Galanthus nivalis agglutinin which specifically recognizes high-mannose type glycans. This staining was diminished after digestion of the glycans with endo-β-N-acetylglucosaminidase H or D, suggesting that at least a N-glycan containing Man5GlcNAc2 is linked to the extracellular portion of the IL-2 receptor α subunit. Our findings indicate that IL-2 binds the IL-2 receptor α subunit through Man5GlcNAc2 and a specific peptide sequence on the surface of CTLL-2 cells. When IL-2 binds to the IL-2Rα subunit, this may trigger formation of the high affinity complex of IL-2-IL-2Rα, -β, and -γ subunits, leading to cellular signaling. interleukin-2 d-mannose d-N-acetylglucosamine asparagine IL-2 receptor endo-β-N-acetylglucosaminidase H endo-β-N-acetylglucosaminidase D Galanthus nivalis agglutinin recombinant human interleukin-2 polyacrylamide gel electrophoresis T-cell receptor N-(9-fluorenyl)methoxycarbonyl Interleukin-2 (IL-2)1 is a cytokine synthesized by activated T cells (1Gillis S. Ferm M.M. Ou W. Smith K.A. J. Immunol. 1978; 120: 2027-2032PubMed Google Scholar). IL-2 promotes the proliferation of IL-2-dependent T cells and functions as an immunomodulator of activated B cells, macrophages, and natural killer cells (2Taniguchi T. Matsui H. Fujita T. Hatakeyama M. Kashima N. Fuse A. Hamuro J. Nishi-Takaoka C. Yamada G. Immunol. Rev. 1986; 92: 121-133Crossref PubMed Scopus (67) Google Scholar). IL-2 expresses its physiological functions through interaction with its receptor complex, which consists of three receptor subunits, α, β, and γ (IL-2Rα, -β, and -γ) (3Nelson B.H. Willerford D.M. Adv. Immunol. 1998; 70: 1-81Crossref PubMed Google Scholar). Although none of the receptor subunits has intrinsic tyrosine kinase activity, intracellular portions of the IL-2Rβ and -γ subunits associate with intracellular tyrosine kinases including Lck (4Hatakeyama M. Kono T. Kobayashi N. Kawahara A. Levin S.D. Perlmutter R.M. Taniguchi T. Science. 1991; 252: 1523-1528Crossref PubMed Scopus (508) Google Scholar), Jak1 and Jak3 (5Tanaka N. Asao H. Ohbo K. Ishii N. Takeshita T. Nakamura M. Sasaki H. Sugamura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7271-7275Crossref PubMed Scopus (65) Google Scholar, 6Johnston J.A. Kawamura M. Kirken R.A. Chen Y.Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (507) Google Scholar, 7Witthuhn B.A. Silvennoinen O. Miura O. Lai K.S. Cwik C. Liu E.T. Ihle J.N. Nature. 1994; 370: 153-157Crossref PubMed Scopus (535) Google Scholar) in IL-2-stimulated T-cells, and cellular signaling occurs through tyrosine phosphorylation of several proteins (3Nelson B.H. Willerford D.M. Adv. Immunol. 1998; 70: 1-81Crossref PubMed Google Scholar). From these observations, it is suggested that a complex consisting of at least IL-2, IL-2Rα, IL-2Rβ, IL-2Rγ, and tyrosine kinases including Lck, Jak1, and Jak3 might be formed in CTLL-2 cells stimulated by IL-2. However, it has been reported that each IL-2 receptor subunit alone shows only weak binding to IL-2. IL-2Rα binds IL-2 with low affinity (K d ∼10 nm), IL-2Rβ binds IL-2 with very low affinity (K d ∼100 nm), and IL-2Rγ has no measurable affinity for IL-2 (8Arima N. Kamio M. Imada K. Hori T. Hattori T. Tsudo M. Okuma M. Uchiyama T. J. Exp. Med. 1992; 176: 1265-1272Crossref PubMed Scopus (84) Google Scholar, 9Takeshita T. Ohtani K. Asao H. Kumaki S. Nakamura M. Sugamura K. J. Immunol. 1992; 148: 2154-2158PubMed Google Scholar, 10Anderson D.M. Kumaki S. Ahdieh M. Bertles J. Tometsko M. Loomis A. Giri J. Copeland N.G. Gilbert D.J. Jenkins N.A. Valentine V. Shapiro D. Morris S. Park L. Cosman D. J. Biol. Chem. 1995; 270: 29862-29869Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). Accordingly, the mechanism by which IL-2 stimulates the formation of a high affinity IL-2-IL-2Rα, -β, or -γ complex remains unclear. Although research on the carbohydrate recognition of IL-2 has a long history, its physiological function has not been clearly determined. Sherblom et al. (11Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar) and Zanetta et al. (12Zanetta J.-P. Alonso C. Michalski J.-C. Biochem. J. 1996; 318: 49-53Crossref PubMed Scopus (26) Google Scholar) reported that IL-2 recognizes high-mannose type glycans with five or six mannosyl residues as determined by the plate method. Later, Najjamet al. (13Najjam S. Mulloy B. Theze J. Gordon M. Gibbs R. Rider C.C. Glycobiology. 1998; 8: 509-516Crossref PubMed Scopus (53) Google Scholar) found that rhIL-2 binds to heparin specifically. However, since the addition of heparin did not show any inhibitory effect on IL-2-dependent cell proliferation, it was suggested that the interaction between IL-2 and heparin is not related to such activity. Zanetta et al. (12Zanetta J.-P. Alonso C. Michalski J.-C. Biochem. J. 1996; 318: 49-53Crossref PubMed Scopus (26) Google Scholar) presented a cross-linking model in which it was hypothesized that, in the case of human peripheral lymphocytes, IL-2 binds to not only the IL-2 receptor via the IL-2 receptor-binding sites but also the TCR complex containing glycosylated CD3 (12Zanetta J.-P. Alonso C. Michalski J.-C. Biochem. J. 1996; 318: 49-53Crossref PubMed Scopus (26) Google Scholar). This tentative model was proposed on the basis of the results of analysis of immunoprecipitates obtained using IL-2Rβ antibody. However, they did not directly show that phosphorylation of Lck kinase co-immunoprecipitated with IL-2Rβ subunit occurs, or that high-mannose type glycan has an inhibitory effect on IL-2-dependent cell proliferation. Accordingly, whether the carbohydrate recognition activity of IL-2 contributes to the physiological function of IL-2 still remains unclear. Moreover, it has been reported recently that the catalytic activation of Jak1 and Jak3 kinases is induced within minutes after formation of a IL-2·IL-2 receptor high-affinity complex (5Tanaka N. Asao H. Ohbo K. Ishii N. Takeshita T. Nakamura M. Sasaki H. Sugamura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7271-7275Crossref PubMed Scopus (65) Google Scholar, 6Johnston J.A. Kawamura M. Kirken R.A. Chen Y.Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (507) Google Scholar, 7Witthuhn B.A. Silvennoinen O. Miura O. Lai K.S. Cwik C. Liu E.T. Ihle J.N. Nature. 1994; 370: 153-157Crossref PubMed Scopus (535) Google Scholar). In this paper, we report that addition of high-mannose type glycans inhibits not only IL-2-dependent CTLL-2 cell proliferation but also the phosphorylation of the related tyrosine kinases including Jak1, Jak3, Lck, and Lyn. Furthermore, a high affinity complex including IL-2Rα, -β, -γ subunits, and Jak1, Jak3, Lck, Lyn tyrosine kinases is formed in IL-2-stimulated CTLL-2 cells. Among the co-immunoprecipitated components of the complex, only the IL-2Rα subunit was stained with Galanthus nivalis agglutinin (GNA) which specifically recognizes high-mannose type glycans (14Shibuya N. Goldstein I.J. Van Damme E.J.M. Peumans W.J. J. Biol. Chem. 1988; 263: 728-734Abstract Full Text PDF PubMed Google Scholar) and the staining was diminished after digestion of the glycans with Man5GlcNAc2-specific endo-β-N-acetylglucosaminidase D (Endo D) (15Tai T. Yamashita K. Ogata-Arakawa M. Koide N. Muramatsu T. Iwashita S. Inoue Y. Kobata A. J. Biol. Chem. 1975; 250: 8569-8575Abstract Full Text PDF PubMed Google Scholar). Our findings suggest that dual binding of IL-2 to both a Man5GlcNAc2 moiety and a specific peptide sequence in the IL-2 receptor α subunit serves to trigger the formation of a high-affinity complex of IL-2- IL-2Rα, -β, and -γ subunits, leading to cellular signaling. Endo-β-N-acetylglucosaminidase H (Endo H) and Endo D were obtained from Seikagaku Kogyo Co. (Tokyo, Japan). Prestained protein markers used as molecular weight markers for SDS-PAGE were obtained from BioLabs Inc. (Hertfordshire, United Kingdom). Arthrobacter protophormiaeendo-β-N-acetylglucosaminidase (16Fan J.-Q. Takegawa K. Iwahara S. Kondo A. Kato I. Abeygunawardana C. Lee Y.C. J. Biol. Chem. 1995; 270: 17723-17729Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) was kindly provided by Dr. K. Takegawa, Faculty of Agriculture, Kagawa University, Japan. Fmoc-conjugated Asn-GlcNAc (17Mizuno M. Muramoto I. Kobayashi K. Yaginuma H. Inazu T. Synthesis. 1999; 1: 162-165Crossref Scopus (66) Google Scholar) was kindly provided by Dr. T. Inazu, Noguchi Institute, Tokyo, Japan. cDNA encoding human IL-2 (RandD Systems Europe Ltd., Abingdon, UK) was used to produce rhIL-2 inEscherichia coli. Plasmid pET3a (Novagen Inc., Madison, WI) containing a T7 promoter was used as the rhIL-2 expression plasmid. ANdeI-HindIII fragment corresponding to a synthetic human IL-2 gene was inserted between theNdeI and HindIII sites of pET3a to produce the expression plasmid. The rhIL-2 gene was expressed in E. coli strain BL21(DE3) under the control of the T7 promoter. A 15-ml culture of E. coli BL21(DE3) cells containing the IL-2 plasmid was incubated overnight until the cells reached the stationary phase of growth and this culture was used to inoculate 500 ml of L broth containing 100 μg/ml ampicillin. After incubation for 2.5 h at 37 °C, IL-2 production was induced by addition of 0.5 mm isopropyl β-thiogalactoside, and the cells were grown for 2.5 h. IL-2 was produced mainly in inclusion bodies. The inclusion bodies were solubilized and IL-2 was refolded by a method described previously (18Tsuji T. Nakagawa R. Sugimoto N. Fukuhara K. Biochemistry. 1987; 26: 3129-3134Crossref PubMed Scopus (54) Google Scholar), with slight modification, as follows. The cells were collected by centrifugation and homogenized by lysozyme treatment and sonication at 4 °C. The lysate was centrifuged at 10,000 rpm for 10 min, and the precipitate was collected. The pellet was dissolved in 20 mm Tris-HCl buffer (pH 8.3) containing 10 mm EDTA and 6 m guanidine hydrochloride. Then, the solution was treated with 10 mm reduced glutathione and 1 mm oxidized glutathione in the presence of 2 m guanidine hydrochloride at pH 8.0. The solution was kept for 16 h at room temperature, then it was dialyzed against phosphate-buffered saline. An aliquot of the dialysate was subjected to SDS-PAGE using a 15% acrylamide gel to check the purity of the rhIL-2. The biological activity of the recovered soluble rhIL-2 protein was determined in a proliferation assay using CTLL-2 cells. Human IL-2 purchased from Sigma-Aldrich Co. was used as the standard for the units of activity. Protein concentration was estimated using the Bio-Rad Protein Assay dye reagent with bovine serum albumin as the standard. The amount of activity displayed by the rhIL-2 used in this study was 1–10 units/ng. Mouse T cell line CTLL-2 (RCB0637) was obtained from the RIKEN Cell Bank (Ibaraki, Japan). CTLL-2 cells were maintained in complete RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 100 units/ml rhIL-2 at 37 °C under a 5% CO2 atmosphere. The cells were cultured until the cell density reached 1.5 × 106cells/ml and the culture was then split. For the bioassay, 2 days after the last addition of rhIL-2, the cells were washed three times in RPMI 1640 medium. The cells were then resuspended in complete medium at a cell density of 1 × 105 cells/ml and plated out in microtiter plates, 100 μl/well. Then 100 μl of rhIL-2 at various concentrations, diluted in complete RPMI 1640 medium, was added. The cells were incubated at 37 °C in a 5% CO2 atmosphere for 2 days, then 20 μl of Cell Titer 96TM Aqueous one solution reagent was added to each well. After incubating the mixture for 2 h, the absorbance at 525 nm was read using a dual wavelength flying spot scanning densitometer CS-9300PC (Shimazu Corp. Kyoto, Japan). Cell Titer 96TM Aqueous one solution reagent used to measure cell proliferation activity was obtained from Promega Corp.The solution is composed of a novel tetrazolium compound and an electron coupling reagent, phenazine ethosulfate in Dulbecco's phosphate-buffered saline (pH 6.0). Manα1→6(Manα1→3)Manα1→6(Manα1→3) Manβ1→4GlcNAcβ1→4GlcNAc-Asn(Man5GlcNAc2Asn) and Manα1→6(Manα1→3)Manα1→6(Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc-Asn (Man6GlcNAc2Asn) were prepared by exhaustive Pronase digestion of ovalbumin followed by Dowex 50 × 2 (H+ form) column chromatography (200–400 mesh, 1.5 × 150 cm) according to the method described by Taiet al. (15Tai T. Yamashita K. Ogata-Arakawa M. Koide N. Muramatsu T. Iwashita S. Inoue Y. Kobata A. J. Biol. Chem. 1975; 250: 8569-8575Abstract Full Text PDF PubMed Google Scholar). (Manα1→2)2–4[Manα1→6- (Manα1→3)Manα1→6(Manα1→3)]Manβ1→4GlcNAcβ1→4GlcNAc (Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2) and Manα1→6 (Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (Man3GlcNAc2) were prepared from 3 g of porcine thyroglobulin glycopeptides by hydrazinolysis followed by re-N-acetylation, and Man7–9GlcNAc2were each isolated by Bio-Gel P-4 (under 400 mesh, 2.0 × 100 cm) column chromatography. Each oligosaccharide was converted to the asparaginyl oligosaccharide from Man7–9GlcNAc2 and Fmoc-conjugated Asn-GlcNAc by treatment with A. protophormiaeendo-β-N-acetylglucosaminidase according to the method described by Kuge et al. (19Hara-Kuge S. Ohkura T. Seko A. Yamashita K. Glycobiology. 1999; 9: 833-839Crossref PubMed Scopus (69) Google Scholar). The structures of these different glycoasparagines and oligosaccharides were determined through a combination of methylation analysis (20Yamashita K. Hitoi A. Matsuda Y. Tsuji A. Katumuna N. Kobata A. J. Biol. Chem. 1983; 258: 1098-1107Abstract Full Text PDF PubMed Google Scholar), α-mannosidase digestion, partial acetolysis (21Tai T. Yamashita K. Ito S. Kobata A. J. Biol. Chem. 1977; 252: 6687-6694Abstract Full Text PDF PubMed Google Scholar), and matrix-assisted laser desorption-time of flight mass spectrometry (Shimadzu Corp., Kyoto, Japan). To investigate the phosphorylation of kinases in the presence and absence of Man5GlcNAc2Asn, the following experiments were performed. CTLL-2 cells were washed twice with RPMI 1640 medium containing 10% fetal calf serum, suspended at 2 × 106 cells/ml and incubated for 6 h at 37 °C in the absence of IL-2. The cells were then stimulated with rhIL-2 (5 units/ml) for 20 min at 37 °C in the presence or absence of 10 μm Man5GlcNAc2Asn, followed by centrifugation at 3000 rpm for 5 min at 4 °C. Cells (1 × 107 cells/lane) were lysed for 60 min on ice by adding 1 ml of lysis buffer containing 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mm sodium fluoride, 1 mm sodium orthovanadate, 10 μm pepstatin A, 1 μg/ml leupeptin, 100 kallikrein units/ml aprotinin, and 1 mmphenylmethylsulfonyl fluoride. Cell lysates were cleared by centrifugation for 15 min at 1.5 × 104 rpm and used for the immunoprecipitation. The tyrosine kinases were individually immunoprecipitated from cell lysates with anti-Jak1, anti-Jak3 (Upstate Biotechnology, NY), anti-Lyn, or anti-Lck antibody (Santa Cruz Biotechnology, Inc., CA) according to the manufacturer's protocol. After fractionation of the immunoprecipitates by SDS-PAGE, the proteins were transferred to a nitrocellulose membrane. The blots were then probed with anti-phosphotyrosine monoclonal antibody (4G10, UBI) and with the appropriate second antibody and visualized by means of the ECL system (Amersham Pharmacia Biotech). The blots were stripped with 62.5 mm Tris/HCl (pH 6.7) containing 2% SDS and 100 mm β-mercaptoethanol at 50 °C for 30 min and reprobed with anti-Jak1, anti-Jak3, anti-Lyn, or anti-Lck antibody to evaluate the amount of the corresponding tyrosine kinase. To detect any glycoprotein with high-mannose type glycans among the constituents of the IL-2 receptor complex, the following experiments were performed. Cells (1 × 107 cells/lane) which had been cultured continuously in the presence of IL-2 were lysed by adding 1 ml of lysis buffer containing 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10 μm pepstatin A, 1 μg/ml leupeptin, 100 kallikrein units/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, and 1 mm mannolactone (Sigma-Aldrich) and incubating the mixture for 60 min on ice. Cell lysates were cleared by centrifugation for 15 min at 1.5 × 104 rpm and used for the immunoprecipitation. The supernatants of the cell lysates were treated with rabbit anti-IL-2Rα, anti-IL-2Rβ, or anti-IL-2Rγ antibody (Santa Cruz Biotechnology, Inc., CA) according to the manufacturer's protocol and the immunoprecipitates were fractionated by SDS-PAGE. The immunoprecipitates were then probed with anti-IL-2Rα, -β, -γ, anti-Lck, anti-Jak1, anti-Jak3, or anti-Lyn antibody and with the appropriate second antibody and visualized by means of the ECL system (Amersham Pharmacia Biotech). Otherwise, membranes were incubated at 37 °C for 18 h in the presence or absence of Endo H (10 milliunits/100 μl of citrate-phosphate buffer (pH 6.5)/cm2) or Endo D (10 milliunits/100 μl of citrate-phosphate buffer (pH 6.5)/cm2) and stained with biotinylated GNA (80 μg/ml), followed by treatment with avidin peroxidase, and visualized by means of the ECL system. Then, the blot was reprobed with anti-IL-2Rα subunit antibody and with the appropriate second antibody and visualized. G. nivalis agglutinin was obtained from Sigma-Aldrich Co. and sulfo-NHS-biotin was obtained from Pierce. 1 mg of GNA was dissolved in 500 μl of phosphate-buffered saline and 1 mg of sulfo-NHS-biotin was dissolved in 1 ml of distilled water. The GNA solution was mixed with 30 μl of sulfo-NHS-biotin solution and left to stand on ice for 2 h. After dialysis against phosphate-buffered saline, the biotinylated GNA was used as a probe. It is known that IL-2 specifically recognizes high-mannose type glycans with 5 or 6 mannosyl residues (11Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar, 12Zanetta J.-P. Alonso C. Michalski J.-C. Biochem. J. 1996; 318: 49-53Crossref PubMed Scopus (26) Google Scholar). We studied whether the lectin activity is indispensable for induction of IL-2-dependent cell proliferation. As the first step, we investigated whether this process is inhibited by addition of high-mannose type glycans. It is known that CTLL-2 cells, a mouse T-cell line, proliferate in a manner dependent on IL-2. Upon incubating the cells (1 × 104/well) in the presence of rhIL-2 at 5 units/ml for 48 h, the cells showed a proliferative response which was dependent on the concentration of rhIL-2 (Fig. 1 A). The extent of cell proliferation was determined colorimetrically (see "Experimental Procedures"). Since the concentration of rhIL-2 required to stimulate maximum IL-2-dependent cell proliferation was found to be 5 units/ml, the following experiments were performed at this concentration. Mixtures were prepared containing 5 units/ml rhIL-2 and high-mannose type glycans at various concentrations and, after being left standing for 2 h at 37 °C, the mixtures were added to the wells containing the cultured cells. In this experiment, Man5GlcNAc2Asn and Man6GlcNAc2Asn were found to dose dependently inhibit the proliferative response of these cells to rhIL-2 in vitro, whereas Man7GlcNAc2Asn, Man8GlcNAc2Asn, Man9GlcNAc2Asn, and Man3GlcNAc2 did not show any inhibitory effect (Fig. 1 B). These results suggested that the lectin activity of IL-2 is required for stimulation of IL-2-dependent T-cell proliferation. It has been reported that, in the case of IL-2-induced proliferation of CTLL-2 cells, signal transduction occurs via tyrosine kinases including Lck (4Hatakeyama M. Kono T. Kobayashi N. Kawahara A. Levin S.D. Perlmutter R.M. Taniguchi T. Science. 1991; 252: 1523-1528Crossref PubMed Scopus (508) Google Scholar), Jak1, and Jak3 (5Tanaka N. Asao H. Ohbo K. Ishii N. Takeshita T. Nakamura M. Sasaki H. Sugamura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7271-7275Crossref PubMed Scopus (65) Google Scholar, 6Johnston J.A. Kawamura M. Kirken R.A. Chen Y.Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (507) Google Scholar, 7Witthuhn B.A. Silvennoinen O. Miura O. Lai K.S. Cwik C. Liu E.T. Ihle J.N. Nature. 1994; 370: 153-157Crossref PubMed Scopus (535) Google Scholar). In preliminary experiments, we found that Lyn is also phosphorylated as a result of IL-2 stimulation in CTLL-2 cells. Although Lyn was originally reported to be phosphorylated as a result of IL-2 stimulation in a B-cell line, whether the association site is IL-2Rβ or -γ remains to be determined (22Torigoe T. Saragovi H.U. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2674-2678Crossref PubMed Scopus (86) Google Scholar). To further confirm whether the lectin activity of IL-2 modulates the cellular signal transduction mechanism, phosphorylation of Jak1, Jak3, Lck, and Lyn were comparatively studied in the presence and absence of Man5GlcNAc2Asn in the medium. After culturing the cells in the absence of IL-2 for 6 h, CTLL-2 cells in G0 phase were stimulated with rhIL2 (10 units/ml) at 37 °C for 30 min in the presence or absence of Man5GlcNAc2Asn (10 μm). Then, the cells (1 × 107 cells/lane) were solubilized and proteins in the lysates were immunoprecipitated with anti-Jak1, anti-Jak3, anti-Lck, or anti-Lyn antibody. Tyrosine-phosphorylated proteins were identified by immunoblotting with an antiphosphotyrosine monoclonal antibody, 4G10 (anti-Tyr(P)). As shown in Fig.2, proteins in lysates of CTLL-2 cells in G0 phase (lanes 1, 4, 7, and10), lysates of IL-2-treated cells (lanes 3, 6, 9, and 12), and lysates of cells incubated with IL-2 in the presence of Man5GlcNAc2Asn (lanes 2, 5, 8, and 11) were immunoprecipitated with anti-Jak1 (lanes 1–3), anti-Jak3 (lanes 4–6), anti-Lck (lanes 7–9), and anti-Lyn antibody (lanes 10–12). The levels of phosphorylated Jak1, Jak3, Lck, and Lyn, which were detected by the phosphotyrosine-specific 4G10 antibody, were increased in IL-2-induced cells (lanes 3, 6, 9, and12) as compared with cells in G0 phase (lanes 1, 4, 7, and 10). In contrast, phosphorylation of these tyrosine kinases in cells stimulated with IL-2 in the presence of Man5GlcNAc2Asn was exclusively reduced (lanes 2, 5, 8, and 11). These results indicate that the carbohydrate recognition function of IL-2 modulates signal transduction through Jak1, Jak3, Lck, and Lyn linked to IL-2 receptor subunits β and γ. The results described above indicated that the carbohydrate recognition function of IL-2 was involved in the cellular signaling system. Although it is known that IL-2 induces the formation of an IL-2·IL-2 receptor complex which includes the three receptor subunits α, β, and γ (IL-2Rα, -β, and -γ) (3Nelson B.H. Willerford D.M. Adv. Immunol. 1998; 70: 1-81Crossref PubMed Google Scholar), the soluble IL-2Rα, -β, and -γ independently show low affinity binding to IL-2. That is, the α-subunit binds IL-2 with low affinity (K d ∼ 10 nm), the β-subunit binds IL-2 with very low affinity (K d ∼ 100 nm), and the γ-subunit has no measurable affinity for IL-2 (8Arima N. Kamio M. Imada K. Hori T. Hattori T. Tsudo M. Okuma M. Uchiyama T. J. Exp. Med. 1992; 176: 1265-1272Crossref PubMed Scopus (84) Google Scholar, 9Takeshita T. Ohtani K. Asao H. Kumaki S. Nakamura M. Sugamura K. J. Immunol. 1992; 148: 2154-2158PubMed Google Scholar, 10Anderson D.M. Kumaki S. Ahdieh M. Bertles J. Tometsko M. Loomis A. Giri J. Copeland N.G. Gilbert D.J. Jenkins N.A. Valentine V. Shapiro D. Morris S. Park L. Cosman D. J. Biol. Chem. 1995; 270: 29862-29869Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). However, as soon as IL-2 forms the high affinity complex with the IL-2Rα, -β, and -γ subunits, cellular signaling is triggered. If a lectin-like interaction between IL-2 and a specific glycoprotein is the trigger for formation of the high-affinity receptor complex, a specific glycoprotein having Man5–6GlcNAc2 should be co-immunoprecipitated with the IL-2 receptor complex in the lysates of IL-2-stimulated CTLL-2 cells using antibody against the IL-2Rα, -β, or -γ subunit. To detect such a glycoprotein containing Man5–6GlcNAc2 in these immunoprecipitates, we used G. nivalis agglutinin which specifically recognizes high-mannose type glycans (14Shibuya N. Goldstein I.J. Van Damme E.J.M. Peumans W.J. J. Biol. Chem. 1988; 263: 728-734Abstract Full Text PDF PubMed Google Scholar). CTLL-2 cells (1 × 107cells/lane) which had been continuously cultured in the presence of IL-2 were solubilized with the lysis buffer containing 0.1% SDS, 0.5% deoxycholate, and 1% Nonidet P-40, the proteins were immunoprecipitated with anti-IL-2Rα, anti-IL-2Rβ, or anti-IL-2Rγ antibody, and each of the immunoprecipitates was fractionated by polyacrylamide gel electrophoresis on a 10% acrylamide gel and blotted onto nitrocellulose membranes. The membranes were then treated with anti-IL-2Rα, anti-IL-2Rβ, anti-IL-2Rγ, anti-Lck, anti-Lyn, anti-Jak1, or anti-Jak3 antibody. Although antibody against each subunit of IL-2R was used for immunoprecipitation, all immunoprecipitates showed a 55-kDa band corresponding to IL-2Rα upon staining with anti-IL-2Rα, a 75-kDa band corresponding to IL-2Rβ upon staining with anti-IL-2Rβ, a 64-kDa band corresponding to IL-2Rγ upon staining with anti-IL-2Rγ, a 56-kDa band corresponding to Lck upon staining with anti-Lck, a 115-kDa band corresponding to Jak1 upon staining with anti-Jak1 kinase, a 115-kDa band corresponding to Jak3 upon staining with anti-Jak3, and a 56-kDa band corresponding to Lyn upon staining with anti-Lyn antibody (3Nelson B.H. Willerford D.M. Adv. Immunol. 1998; 70: 1-81Crossref PubMed Google Scholar) (Fig.3 A). These results indicated that all of the immunoprecipitates obtained with anti-IL-2Rα, -β, or -γ antibody in analysis of CTLL-2 cells exposed to IL-2 consisted of the IL-2R complex which at least included IL-2Rα, -β, and -γ, and the kinases Lck, Lyn, Jak1, and Jak3. In contrast, this complex could not be observed in CTLL-2 cells incubated in the absence of IL-2 (data not shown). In view of these results, after the immunoprecipitates obtained with anti-IL-2Rα, -β, and -γ had been fractionated by polyacrylamide gel electrophoresis on a 10% acrylamide gel and blotted onto nitrocellulose membranes, the membranes were stained with biotinylated GNA which rather specifically recognizes Man5GlcNAc2Asn (14Shibuya N. Goldstein I.J. Van Damme E.J.M. Peumans W.J. J. Biol. Chem. 1988; 263: 728-734Abstract Full Text PDF PubMed Google Scholar), to detect the constituent to which IL-2 can bind through its carbohydrate recognition site. Since only a single 55-kDa band corresponding to the IL-2Rα subunit was stained in each instance, the membranes were reprobed with anti-IL-2Rα subunit antibody. As shown in Fig. 3 B, a protein band in the same position as the GNA-stained protein band was positively stained with anti-IL-2Rα subunit antibody. Furthermore, although only the IL-2Rα subunit was immunoprecipitated with anti-IL-2Rα subunit antibody in the case of CTLL-2 cells incubated in the absence of IL-2, the same constituent of the immunoprecipitate was positively stained with GNA (data not shown). When each blot was treated with Endo H (Fig. 3, lane 11) or Endo D (Fig. 3,lane 12), the band positively stained with GNA (Fig. 3,lane 10) was diminished to 3% (Endo H) or 15% (Endo D), calculated on the basis of the intensity of chemiluminescence (Fig.3 C). Since Endo H hydrolyzes high-mannose type glycans including Man4–9GlcNAc2 and hybrid-type glycans (23Tai T. Yamashita K. Kobata A. Biochem. Biophys. Res. Commun. 1977; 78: 434-441Crossref PubMed Scopus (119) Google Scholar), whereas Endo D hydrolyzes Man3–5GlcNAc2 (15Tai T. Yamashita K. Ogata-Arakawa M. Koide N. Muramatsu T. Iwashita S. Inoue Y. Kobata A. J. Biol. Chem. 1975; 250: 8569-8575Abstract Full Text PDF PubMed Google Scholar, 24Tai T. Ito S. Yamashita K. Muramatsu T. Kobata A. Biochem. Biophys. Res. Commun. 1975; 65: 968-974Crossref PubMed Scopus (64) Google Scholar) and since IL-2-dependent proliferation of CTLL-2 cells was inhibited by the addition of Man5GlcNAc2Asn or Man6GlcNAc2Asn, the carbohydrate structure of IL-2Rα to which IL-2 binds appears to include Man5GlcNAc2. These results suggest that only the IL-2Rα subunit has the high-mannose type glycan with Man5GlcNAc2, among the components of the IL-2R complex in CTLL-2 cells, and that IL-2 bifunctionally binds a high-mannose type glycan and a specific peptide sequence of IL-2Rα, although all of the subunits of IL-2R have several potentialN-glycosylation sites (25Shimizu A. Kondo S. Takeda S.-I. Yodoi J. Ishida N. Sabe H. Osawa H. Diamantstein T. Nikaido T. Honjo T. Nucleic Acids Res. 1985; 13: 1505-1516Crossref PubMed Scopus (71) Google Scholar, 26Kono T. Doi T. Yamada G. Hatakeyama M. Minamoto S. Tsudo M. Miyasaka M. Miyata T. Taniguchi T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1806-1810Crossref PubMed Scopus (64) Google Scholar, 27Cao X. Kozak C.A. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8464-8468Crossref PubMed Scopus (63) Google Scholar). Since the nonglycosylated rhIL-2Rα subunit recognizes Lys35, Lys38, Thr42, and Lys43 residues in IL-2 (28Sauve K. Nachman M. Spence C. Bailon P. Campbell E. Tsien W.H. Kondas J.A. Hakimi J. Ju G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4636-4640Crossref PubMed Scopus (102) Google Scholar), another peptide sequence of IL-2 may bind a high-mannose type glycan with Man5GlcNAc2 which is linked to Asn33, Asn43, or Asn200 of mouse IL-2Rα (25Shimizu A. Kondo S. Takeda S.-I. Yodoi J. Ishida N. Sabe H. Osawa H. Diamantstein T. Nikaido T. Honjo T. Nucleic Acids Res. 1985; 13: 1505-1516Crossref PubMed Scopus (71) Google Scholar). As soon as IL-2 bifunctionally binds to the IL-2Rα subunit, formation of the IL-2·IL-2Rα complex might occur resulting in a change in conformation of IL-2 which increases the accessibility to the IL-2Rβ and IL-2Rγ subunits. This high-affinity complex of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits may stimulate cellular signaling through tyrosine kinases including Jak1, Jak3, Lck, and Lyn. Our findings presented in this paper clearly demonstrate that the dual recognition by IL-2 of a specific peptide sequence and a carbohydrate epitope in the IL-2Rα molecule is required to trigger the formation of a high-affinity complex of IL-2-IL-2Rα, -β, -γ, and that Man5GlcNAc2Asn or Man6GlcNAc2Asn in the medium inhibits not only IL-2-dependent CTLL-2 proliferation but also tyrosine phosphorylation of Jak1, Jak3, Lck, and Lyn. Furthermore, the IL-2-IL-2Rα, -β, and -γ complex immunoprecipitated with anti-IL-2Rα, -β, or -γ antibody contains GNA-stainable IL-2Rα, suggesting that bifunctional binding of IL-2 to Man5GlcNAc2 and a specific peptide sequence in the IL-2Rα molecule immediately leads to formation of the high-affinity complex of IL-2-IL-2Rα, -β, and -γ, which subsequently induces tyrosine phosphorylation of IL-2Rβ and γ linked to Jak1, Jak3, Lck, and Lyn. Our results indicate that IL-2Rα is a candidate glycoprotein for IL-2 lectin-like binding in vivo, and as soon as the tetramer including IL-2-IL-2Rα, -β, and -γ is tightly formed, the tyrosine kinases linked to intracellular domains of IL-2Rβ and -γ are immediately phosphorylated and induce cellular signaling. On the basis of the results described, we propose a tentative schematic model as shown in Fig. 4, although the binding site of Lyn has not been determined to be the IL-2Rβ or -γ subunit. In our investigation of the inhibitory effects of Man5GlcNAc2Asn and Man6GlcNAc2Asn on IL-2-dependent cell proliferation, the 2-h preincubation time before addition of the mixture to the cells was found to be critical and exogenous Man5GlcNAc2Asn added to the mixture could not replace the glycan bound to IL-2. As soon as the high-mannose type glycan linked to the extracellular domain of the IL-2Rα subunit binds to IL-2, it seems that IL-2 binds a specific region of the IL-2Rα subunit and this dual recognition is too strong to be replaced by exogenous Man5GlcNAc2Asn. On the basis of the experimental results, we speculate that the conformation of carbohydrate-bound IL-2 may immediately change to fit with a specific peptide sequence in IL-2Rα and formation of the IL-2·IL-2Rα complex may be a trigger to form the high-affinity complex which consists of all constituents required for the cell signaling to occur. This may be the reason why large amounts of exogenous Man5GlcNAc2Asn cannot substitute for the endogenous glycoprotein, and why inhibitory effects of oligomannosides on the T-cell proliferative response to IL-2 have not been reported until today. To our knowledge, this is the first report to directly demonstrate that carbohydrate recognition activity is essential for stimulation of IL-2-dependent T-cell proliferation and cellular signaling. Anyway, it indicates that IL-2 bifunctionally recognizes both a high-mannose type glycan and a specific peptide sequence in IL-2Rα, and the sequential binding to IL-2Rβ and -γ subunits is necessary for expression of IL-2-induced cellular signal transduction. Similar dual recognition of protein and carbohydrate epitopes has been reported in the case of several proteins. P-selectin is one of the members of the selectin family which can mediate the initial rolling interaction between leukocytes and vascular endothelium. As all members of the selectin family can bind to related fucosylated or sialylated tetrasaccharide structures, such as sialyl-Lewisx or sialyl-Lewisa, P-selectin can bind to P-selectin glycoprotein ligand 1 which has sialyl Lewisx-type structures on the O-linked glycan (29Foxall C. Watson S.R. Dowbenko D. Fennie C. Lasky L.A. Kiso M. Hasegawa A. Asa D. Brandley B.K. J. Cell Biol. 1992; 117: 895-902Crossref PubMed Scopus (655) Google Scholar). Additionally, P-selectin glycoprotein ligand 1-P-selectin binding requires the sulfotyrosine residues located within the region consisting of the first 19 amino acids, although E-selectin can bind to P-selectin glycoprotein ligand 1 without the sulfotyrosine residues (30Sako D. Comess K.M. Barone K.M. Camphausen R.T. Cumming D.A. Shaw G.D. Cell. 1995; 83: 323-331Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 31Pouyani T. Seed B. Cell. 1995; 83: 333-343Abstract Full Text PDF PubMed Scopus (357) Google Scholar). Specific glycosyltransferases need to bind not only carbohydrate epitopes but also a specific peptide sequence, as follows. For example, UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase is indispensable for the biosynthesis of phosphomannosyl residues on lysosomal enzymes which mediate their binding to mannose 6-phosphate receptors and which mediate targeting to an endosomal compartment where the hydrolases are subsequently packaged into lysosomes. Selective transfer ofN-acetylglucosamine-1-phosphate to mannose residues on lysosomal enzymes by this enzyme involves the dual recognition of mannosyl residues and the carboxyl lobe of the lysosomal hydrolase cathepsin D which is shared among lysosomal hydrolases (32Baranski T.J. Cantor A.B. Kornfeld S. J. Biol. Chem. 1992; 267: 23342-23348Abstract Full Text PDF PubMed Google Scholar). GalNAc transferase, responsible for the formation of SO4-GalNAcβ1,4GlcNAcβ1,2Man on the glycoprotein hormones lutropin and thyrotropin, etc., recognizes bothN-acetylglucosaminyl residues and the peptide motif Pro-Xaa-(Arg/Lys) present in each of these glycoproteins (33Dharmesh S.M. Skelton T.P. Baenziger J.U. J. Biol. Chem. 1993; 268: 17096-17102Abstract Full Text PDF PubMed Google Scholar). These reports suggest that these are members of an emerging family of binding proteins with specificity for both protein and carbohydrate epitopes. On the basis of the mechanisms of carbohydrate recognition involved, cytokines have been grouped into three types to date. It has been determined that growth factors including granulocyte-macrophage colony-stimulating factor (34Gordon M.Y. Riley G.P. Watt S.M. Greaves M.F. Nature. 1987; 326: 403-405Crossref PubMed Scopus (457) Google Scholar) and bovine fibroblast growth factor (35Bashkin P. Doctrow S. Klagsbrun M. Svahn C. Folkman J. Vlodavsky I. Biochemistry. 1989; 28: 1737-1743Crossref PubMed Scopus (516) Google Scholar) recognize glycosaminoglycans, interleukin-1β binds the mannose 6-phosphodiester in glycosylphosphatidylinositol-anchored glycoprotein (36Fukushima K. Hara-Kuge S. Ohkura T. Seko A. Ideo H. Inazu T. Yamashita K. J. Biol. Chem. 1997; 272: 10579-10584Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), and IL-2 recognizes high-mannose type glycans. However, whether other cytokines have strict carbohydrate recognition ability should be more precisely investigated. Cytokines have not been reported to have a common carbohydrate recognition domain. However, IL-2 has a limited degree of sequence homology in the amino-terminal portion compared with the COOH-terminal domains of three C-type mannose binding lectins (11Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar). In preliminary experiments, since we found that at least one of the conserved amino acid residues was involved in carbohydrate recognition by IL-2, the further confirmation will be required in the near future. Zanetta et al. (12Zanetta J.-P. Alonso C. Michalski J.-C. Biochem. J. 1996; 318: 49-53Crossref PubMed Scopus (26) Google Scholar) previously reported that IL-2 binds a glycosylated CD3 of TCR which is linked to the Lck kinase in human peripheral lymphocytes. However, we could not find any TCR αβ subunit when we immunostained the IL-2-IL-2Rα, -β, -γ, Lck, Lyn, Jak1, and Jak3 complex, which was immunoprecipitated with anti-IL-2Rα, -β, or -γ antibody in lysates of CTLL-2 cells exposed to IL-2, using anti-TCR αβ subunit antibody (data not shown). In contrast, the immunoprecipitates obtained with anti-TCR αβ subunit antibody from lysates of CTLL-2 cells exposed to IL-2 did not include any IL-2Rα, -β, or -γ as determined by immunostaining (data not shown). These results also support the view that the IL-2Rα subunit itself is a glycoprotein containing the carbohydrate recognition site of IL-2 in murine CTLL-2 cells. Which of the three N-glycosylation sites in murine IL-2Rα has the carbohydrate to which IL-2 binds will be determined in the near future. We are further investigating whether IL-2 has another carbohydrate recognition mechanism as proposed by Zanetta et al. (12Zanetta J.-P. Alonso C. Michalski J.-C. Biochem. J. 1996; 318: 49-53Crossref PubMed Scopus (26) Google Scholar) in the case of human peripheral lymphocytes and whether the human IL-2Rα subunit has high-mannose type N-glycans to which IL-2 can bind. We thank H. Ideo and Y. Kanaya for technical assistance.
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