Interleukin-2 Carbohydrate Recognition Modulates CTLL-2 Cell Proliferation*

Keiko Fukushima and Katsuko YamashitaDagger

From the Department of Biochemistry, Sasaki Institute, 2-2 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 and CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation, 2-3 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 Japan

Received for publication, September 26, 2000, and in revised form, November 10, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha , beta , gamma  subunits, and tyrosine kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the components of this complex, only the IL-2 receptor alpha  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-beta -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 alpha  subunit. Our findings indicate that IL-2 binds the IL-2 receptor alpha  subunit through Man5GlcNAc2 and a specific peptide sequence on the surface of CTLL-2 cells. When IL-2 binds to the IL-2Ralpha subunit, this may trigger formation of the high affinity complex of IL-2-IL-2Ralpha , -beta , and -gamma subunits, leading to cellular signaling.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interleukin-2 (IL-2)1 is a cytokine synthesized by activated T cells (1). 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 (2). IL-2 expresses its physiological functions through interaction with its receptor complex, which consists of three receptor subunits, alpha , beta , and gamma  (IL-2Ralpha , -beta , and -gamma ) (3). Although none of the receptor subunits has intrinsic tyrosine kinase activity, intracellular portions of the IL-2Rbeta and -gamma subunits associate with intracellular tyrosine kinases including Lck (4), Jak1 and Jak3 (5-7) in IL-2-stimulated T-cells, and cellular signaling occurs through tyrosine phosphorylation of several proteins (3). From these observations, it is suggested that a complex consisting of at least IL-2, IL-2Ralpha , IL-2Rbeta , IL-2Rgamma , 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-2Ralpha binds IL-2 with low affinity (Kd ~10 nM), IL-2Rbeta binds IL-2 with very low affinity (Kd ~100 nM), and IL-2Rgamma has no measurable affinity for IL-2 (8-10). Accordingly, the mechanism by which IL-2 stimulates the formation of a high affinity IL-2-IL-2Ralpha , -beta , or -gamma 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. (11) and Zanetta et al. (12) reported that IL-2 recognizes high-mannose type glycans with five or six mannosyl residues as determined by the plate method. Later, Najjam et al. (13) 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. (12) 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 (12). This tentative model was proposed on the basis of the results of analysis of immunoprecipitates obtained using IL-2Rbeta antibody. However, they did not directly show that phosphorylation of Lck kinase co-immunoprecipitated with IL-2Rbeta 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 (5-7).

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-2Ralpha , -beta , -gamma 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-2Ralpha subunit was stained with Galanthus nivalis agglutinin (GNA) which specifically recognizes high-mannose type glycans (14) and the staining was diminished after digestion of the glycans with Man5GlcNAc2-specific endo-beta -N-acetylglucosaminidase D (Endo D) (15). Our findings suggest that dual binding of IL-2 to both a Man5GlcNAc2 moiety and a specific peptide sequence in the IL-2 receptor alpha  subunit serves to trigger the formation of a high-affinity complex of IL-2- IL-2Ralpha , -beta , and -gamma subunits, leading to cellular signaling.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials and Chemicals-- Endo-beta -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 protophormiae endo-beta -N-acetylglucosaminidase (16) was kindly provided by Dr. K. Takegawa, Faculty of Agriculture, Kagawa University, Japan. Fmoc-conjugated Asn-GlcNAc (17) was kindly provided by Dr. T. Inazu, Noguchi Institute, Tokyo, Japan.

Preparation of RhIL-2-- cDNA encoding human IL-2 (RandD Systems Europe Ltd., Abingdon, UK) was used to produce rhIL-2 in Escherichia coli. Plasmid pET3a (Novagen Inc., Madison, WI) containing a T7 promoter was used as the rhIL-2 expression plasmid. A NdeI-HindIII fragment corresponding to a synthetic human IL-2 gene was inserted between the NdeI 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 beta -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 (18), 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.

Cell Culture-- 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 × 106 cells/ml and the culture was then split.

Bioassay of rhIL-2-- 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).

Oligosaccharides-- Manalpha 1right-arrow6(Manalpha 1right-arrow3)Manalpha 1right-arrow6(Manalpha 1right-arrow3) Manbeta 1right-arrow4GlcNAcbeta 1right-arrow4GlcNAc-Asn(Man5GlcNAc2Asn) and Manalpha 1right-arrow6(Manalpha 1right-arrow3)Manalpha 1right-arrow6(Manalpha 1right-arrow2Manalpha 1right-arrow3)Manbeta 1right-arrow4GlcNAcbeta 1right-arrow4GlcNAc-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 Tai et al. (15). (Manalpha 1right-arrow2)2-4[Manalpha 1right-arrow6- (Manalpha 1right-arrow3)Manalpha 1right-arrow6(Manalpha 1right-arrow3)]Manbeta 1right-arrow4GlcNAcbeta 1right-arrow4GlcNAc (Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2) and Manalpha 1right-arrow6 (Manalpha 1right-arrow3)Manbeta 1right-arrow4GlcNAcbeta 1right-arrow4GlcNAc (Man3GlcNAc2) were prepared from 3 g of porcine thyroglobulin glycopeptides by hydrazinolysis followed by re-N-acetylation, and Man7-9GlcNAc2 were 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. protophormiae endo-beta -N-acetylglucosaminidase according to the method described by Kuge et al. (19). The structures of these different glycoasparagines and oligosaccharides were determined through a combination of methylation analysis (20), alpha -mannosidase digestion, partial acetolysis (21), and matrix-assisted laser desorption-time of flight mass spectrometry (Shimadzu Corp., Kyoto, Japan).

Cell Lysis and Immunoprecipitation-- 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 mM phenylmethylsulfonyl 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 beta -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-2Ralpha , anti-IL-2Rbeta , or anti-IL-2Rgamma 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-2Ralpha , -beta , -gamma , 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-2Ralpha subunit antibody and with the appropriate second antibody and visualized.

Preparation of Biotinylated G. nivalis Agglutinin-- 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.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The IL-2 Lectin Activity Is Required to Induce IL-2-dependent Cell Proliferation-- It is known that IL-2 specifically recognizes high-mannose type glycans with 5 or 6 mannosyl residues (11, 12). 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. 1A). 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.



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Fig. 1.   rhIL-2-dependent proliferation of CTLL-2 cells (A) and the effects of high-mannose type glycans on the proliferative response (B). A, rhIL-2 at increasing concentrations was added to 5 × 104 cells/well. After 2 days, the extent of cell proliferation was determined using Cell Titer 96TM Aqueous one solution reagent. B, rhIL-2 (5 units/ml) and various high-mannose type glycans were mixed, and each mixture was kept at 37 °C for 2 h before being added to the culture of CTLL-2 cells. The inhibition curves obtained using Man7GlcNAc2Asn or Man8GlcNAc2Asn were the same as that obtained using Man3GlcNAc2 (data not shown). Results are means of three experiments (standard deviations were less than 5%). M5, M6, M9, and M3 indicate Man5GlcNAc2Asn, Man6GlcNAc2Asn, Man9GlcNAc2Asn, and Man3 GlcNAc2, respectively.

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. 1B). These results suggested that the lectin activity of IL-2 is required for stimulation of IL-2-dependent T-cell proliferation.

Inhibitory Effects of Man5GlcNAc2Asn on Phosphorylation of Tyrosine Kinases Activated by IL-2-- 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 (4), Jak1, and Jak3 (5-7). 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-2Rbeta or -gamma remains to be determined (22). 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, and 10), 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, and 12) 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 beta  and gamma .



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Fig. 2.   The inhibitory effect of Man5GlcNAc2Asn on the phosphorylation of Jak1, Jak3, Lck, and Lyn kinases. CTLL-2 cells were incubated in the absence of IL-2 for 6 h and then further incubated without stimulation (lanes 1, 4, 7, 10), or stimulated with 10 units/ml IL-2 (lanes 3, 6, 9, and 12) or 10 units/ml IL-2 in the presence of 10 µM Man5GlcNAc2Asn (lanes 2, 5, 8, and 11) for 20 min. Immunoprecipitation was performed using anti-Jak1 antibody (lanes 1-3), anti-Jak3 antibody (lanes 4-6), anti-Lck antibody (lanes 7-9), and anti-Lyn antibody (lanes 10-12). Following SDS-PAGE and transfer to a nitrocellulose membrane, the immunoblot was probed with the antiphosphotyrosine (Tyr(P)) monoclonal antibody 4G10. The blot was stripped and reprobed with anti-Jak1 antibody (lanes 1-3), anti-Jak3 antibody (lanes 4-6), anti-Lck antibody (lanes 7-9), and anti-Lyn antibody (lanes 10-12). M5 indicates Man5GlcNAc2Asn.

GNA Staining of the IL-2 Receptor Complex Including Tyrosine Kinases-- 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 alpha , beta , and gamma  (IL-2Ralpha , -beta , and -gamma ) (3), the soluble IL-2Ralpha , -beta , and -gamma independently show low affinity binding to IL-2. That is, the alpha -subunit binds IL-2 with low affinity (Kd ~ 10 nM), the beta -subunit binds IL-2 with very low affinity (Kd ~ 100 nM), and the gamma -subunit has no measurable affinity for IL-2 (8-10). However, as soon as IL-2 forms the high affinity complex with the IL-2Ralpha , -beta , and -gamma 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-2Ralpha , -beta , or -gamma subunit. To detect such a glycoprotein containing Man5-6GlcNAc2 in these immunoprecipitates, we used G. nivalis agglutinin which specifically recognizes high-mannose type glycans (14). CTLL-2 cells (1 × 107 cells/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-2Ralpha , anti-IL-2Rbeta , or anti-IL-2Rgamma 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-2Ralpha , anti-IL-2Rbeta , anti-IL-2Rgamma , 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-2Ralpha upon staining with anti-IL-2Ralpha , a 75-kDa band corresponding to IL-2Rbeta upon staining with anti-IL-2Rbeta , a 64-kDa band corresponding to IL-2Rgamma upon staining with anti-IL-2Rgamma , 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 (3) (Fig. 3A). These results indicated that all of the immunoprecipitates obtained with anti-IL-2Ralpha , -beta , or -gamma antibody in analysis of CTLL-2 cells exposed to IL-2 consisted of the IL-2R complex which at least included IL-2Ralpha , -beta , and -gamma , 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).



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Fig. 3.   GNA staining of the IL-2R complex. CTLL-2 cells (1 × 107 cells/lane) were solubilized and proteins were immunoprecipitated with anti-IL-2Ralpha subunit antibody (lanes 1, 4, 7, and 10-12), anti-IL-2Rbeta subunit antibody (lanes 2, 5, and 8) or anti-IL-2Rgamma subunit antibody (lanes 3, 6, and 9). Each immunoprecipitate was fractionated by polyacrylamide gel electrophoresis on a 10% acrylamide gel, and blotted onto a nitrocellulose membrane. A, each immunoblot was treated with antibodies against IL-2Ralpha , IL-2Rbeta , IL-2Rgamma , Jak1, Jak3, Lyn, or Lck kinase. B, each immunoblot was stained with biotinylated GNA (lanes 4-6) and reprobed with anti-IL-2Ralpha subunit antibody (lanes 7-9). C, immunoblots treated with anti-IL-2Ralpha subunit antibody digested with Endo H (lane 11), Endo D (lane 12), or nondigested (lane 10) were stained with biotinylated GNA.

In view of these results, after the immunoprecipitates obtained with anti-IL-2Ralpha , -beta , and -gamma 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 (14), 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-2Ralpha subunit was stained in each instance, the membranes were reprobed with anti-IL-2Ralpha subunit antibody. As shown in Fig. 3B, a protein band in the same position as the GNA-stained protein band was positively stained with anti-IL-2Ralpha subunit antibody. Furthermore, although only the IL-2Ralpha subunit was immunoprecipitated with anti-IL-2Ralpha 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. 3C). Since Endo H hydrolyzes high-mannose type glycans including Man4-9GlcNAc2 and hybrid-type glycans (23), whereas Endo D hydrolyzes Man3-5GlcNAc2 (15, 24) and since IL-2-dependent proliferation of CTLL-2 cells was inhibited by the addition of Man5GlcNAc2Asn or Man6GlcNAc2Asn, the carbohydrate structure of IL-2Ralpha to which IL-2 binds appears to include Man5GlcNAc2. These results suggest that only the IL-2Ralpha 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-2Ralpha , although all of the subunits of IL-2R have several potential N-glycosylation sites (25-27).

Since the nonglycosylated rhIL-2Ralpha subunit recognizes Lys35, Lys38, Thr42, and Lys43 residues in IL-2 (28), 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-2Ralpha (25). As soon as IL-2 bifunctionally binds to the IL-2Ralpha subunit, formation of the IL-2·IL-2Ralpha complex might occur resulting in a change in conformation of IL-2 which increases the accessibility to the IL-2Rbeta and IL-2Rgamma subunits. This high-affinity complex of IL-2Ralpha , IL-2Rbeta , and IL-2Rgamma subunits may stimulate cellular signaling through tyrosine kinases including Jak1, Jak3, Lck, and Lyn.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-2Ralpha molecule is required to trigger the formation of a high-affinity complex of IL-2-IL-2Ralpha , -beta , -gamma , 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-2Ralpha , -beta , and -gamma complex immunoprecipitated with anti-IL-2Ralpha , -beta , or -gamma antibody contains GNA-stainable IL-2Ralpha , suggesting that bifunctional binding of IL-2 to Man5GlcNAc2 and a specific peptide sequence in the IL-2Ralpha molecule immediately leads to formation of the high-affinity complex of IL-2-IL-2Ralpha , -beta , and -gamma , which subsequently induces tyrosine phosphorylation of IL-2Rbeta and gamma  linked to Jak1, Jak3, Lck, and Lyn. Our results indicate that IL-2Ralpha is a candidate glycoprotein for IL-2 lectin-like binding in vivo, and as soon as the tetramer including IL-2-IL-2Ralpha , -beta , and -gamma is tightly formed, the tyrosine kinases linked to intracellular domains of IL-2Rbeta and -gamma 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-2Rbeta or -gamma subunit.



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Fig. 4.   A tentative schematic model of the formation of the high-affinity IL-2·IL-2Ralpha , -beta , or -gamma complex triggered by recognition of Man5GlcNAc2 in the IL-2Ralpha 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-2Ralpha subunit binds to IL-2, it seems that IL-2 binds a specific region of the IL-2Ralpha 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-2Ralpha and formation of the IL-2·IL-2Ralpha 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-2Ralpha , and the sequential binding to IL-2Rbeta and -gamma 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 (29). 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 (30, 31). Specific glycosyltransferases need to bind not only carbohydrate epitopes but also a specific peptide sequence, as follows. For example, UDP-GlcNAc:lysosomal enzyme N-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 of N-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 (32). GalNAc transferase, responsible for the formation of SO4-GalNAcbeta 1,4GlcNAcbeta 1,2Man on the glycoprotein hormones lutropin and thyrotropin, etc., recognizes both N-acetylglucosaminyl residues and the peptide motif Pro-Xaa-(Arg/Lys) present in each of these glycoproteins (33). 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 (34) and bovine fibroblast growth factor (35) recognize glycosaminoglycans, interleukin-1beta binds the mannose 6-phosphodiester in glycosylphosphatidylinositol-anchored glycoprotein (36), 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 (11). 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. (12) 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 alpha beta subunit when we immunostained the IL-2-IL-2Ralpha , -beta , -gamma , Lck, Lyn, Jak1, and Jak3 complex, which was immunoprecipitated with anti-IL-2Ralpha , -beta , or -gamma antibody in lysates of CTLL-2 cells exposed to IL-2, using anti-TCR alpha beta subunit antibody (data not shown). In contrast, the immunoprecipitates obtained with anti-TCR alpha beta subunit antibody from lysates of CTLL-2 cells exposed to IL-2 did not include any IL-2Ralpha , -beta , or -gamma as determined by immunostaining (data not shown). These results also support the view that the IL-2Ralpha 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-2Ralpha 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. (12) in the case of human peripheral lymphocytes and whether the human IL-2Ralpha subunit has high-mannose type N-glycans to which IL-2 can bind.


    ACKNOWLEDGEMENTS

We thank H. Ideo and Y. Kanaya for technical assistance.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Sasaki Institute, 2-2 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel.: 81-3-3294-3286; Fax: 81-3-3294-2656; E-mail: yamashita@sasaki.or.jp.

Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M008781200


    ABBREVIATIONS

The abbreviations used are: IL-2, interleukin-2; Man, D-mannose; GlcNAc, D-N-acetylglucosamine; Asn, asparagine; IL-2R, IL-2 receptor; Endo H, endo-beta -N-acetylglucosaminidase H; Endo D, endo-beta -N-acetylglucosaminidase D; GNA, Galanthus nivalis agglutinin; rhIL-2, recombinant human interleukin-2; PAGE, polyacrylamide gel electrophoresis; TCR, T-cell receptor; Fmoc, N-(9-fluorenyl)methoxycarbonyl.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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