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J. Biol. Chem., Vol. 276, Issue 33, 31202-31208, August 17, 2001

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Carbohydrate Recognition Site of Interleukin-2 in Relation to Cell Proliferation^*

Keiko Fukushima, Sayuri Hara-Kuge, Hiroko Ideo, and Katsuko Yamashita

From the Department of Biochemistry, Sasaki Institute, 2-2 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 and Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation, 2-5 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

Received for publication, March 29, 2001, and in revised form, June 1, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interleukin-2 (IL-2) is a cytokine with important roles in the immune system. IL-2 initially binds a high mannose-type glycan and a specific peptide sequence of the IL-2 receptor -subunit and sequentially forms a high affinity complex of IL-2·IL-2 receptor -, -, and -subunits. This formation induces cellular signaling and cell proliferation (Fukushima, K., and Yamashita, K. (2001) J. Biol. Chem. 276, 7351-7356). To determine the carbohydrate-binding site of IL-2, we prepared wild-type and point-mutated ³⁵S-IL-2 by an in vitro transcription and translation method. We found that wild-type ³⁵S-IL-2 tends to form a dimer spontaneously, and the dimeric form has both carbohydrate recognition activity and cell proliferation activity. Moreover, substitution of Asn-26 in IL-2 with Gln or Asp conserved the dimeric form and affected the carbohydrate recognition activities in correspondence with the cell proliferation activities, suggesting that Asn-26 in IL-2 is involved in the carbohydrate recognition site. These results suggest that the carbohydrate recognition of IL-2 dimer triggers formation of high affinity complex (IL-2·IL-2R, -, -)₂, and the hetero-octamer stimulates IL-2-dependent T-cell proliferation by intensifying cellular signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interleukin-2 (IL-2)¹ has been widely studied as a mediator of cellular signaling in the immune system. The receptor for IL-2 (IL-2R) consists of -, -, and -subunits (IL-2R, -, and -), and the intracellular portions of the - and -subunits are associated with a variety of cytoplasmic proteins including tyrosine kinases Jak1 and Jak3 (1, 2). The phosphorylated cytoplasmic domain of IL-2R especially plays a critical role in attracting downstream signaling molecules including the transcription factors STAT3 and STAT5 into the activated receptor complex (1, 3). IL-2R, -, and - bind to distinct sites of IL-2, and these associations appear to occur in a stepwise manner (4-8). When IL-2R, -, or - was independently expressed, IL-2R bound IL-2 with low affinity (K_d ~10 nM); IL-2R bound IL-2 with very low affinity (K_d ~100 nM), and IL-2R had no measurable affinity for IL-2, whereas co-expressed IL-2R, -, and - bound IL-2 with a very high affinity constant (K_d ~pM) (9-11). We reported previously that IL-2 binds IL-2R through Man₅GlcNAc₂ and a specific peptide sequence in IL-2R on the surface of CTLL-2 cells and that this dual recognition triggers formation of the high affinity complex of IL-2·IL-2R, -, -, leading to downstream signaling (12).

IL-2R binds Lys-35, Arg-38, Phe-42, and Lys-43 residues of IL-2 (8), but there is no evidence as to which amino acids in IL-2 interact with which high mannose-type glycans. A limited degree of sequence homology was identified in the amino-terminal portion of IL-2 compared with the carboxyl-terminal domains of C-type human mannose-binding lectin, MBP(H), and two rat liver mannose-binding proteins, MBP(A) and MBP(C) (13). Glu-15, Asn-26, Gly-27, Asn-30, Cys-58, and Glu-67 are the amino acids conserved among MBP(H), MBP(A), MBP(C), and IL-2. His-16 and Leu-19 are common between MBP(H) and IL-2, whereas Leu-21 is conserved among MBP(C), MBP(H), and IL-2. Accordingly, we introduced point mutations at Glu-15, Leu-19, Leu-21, Asn-26, Gly-27, Asn-30, and Glu-67 into IL-2 cDNA by PCR, and we synthesized ³⁵S-IL-2 muteins by in vitro transcription and translation in the presence of [³⁵S]methionine. Then we investigated the relationship between carbohydrate recognition activities and CTLL-2 cell proliferation activities of these muteins. Furthermore, since IL-2 synthesized in Escherichia coli (rhIL-2) showed heterogeneous molecular forms from various oligomers to the monomer on Superose 12 column chromatography, we investigated the cell proliferation activity of each form, and we discussed the form of the IL-2·IL-2 receptor complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Chemicals-- Redivue^TM L-[³⁵S]methionine (1175 Ci/mmol) was purchased from Amersham Pharmacia Biotech. Ribonuclease B (type III-B from bovine pancreas), ribonuclease A, ovalbumin, thyroglobulin, and human serum albumin were obtained from Sigma. Endo--N-acetylglucosaminidase H (Endo H) was obtained from Seikagaku Kogyo Co., Ltd. (Tokyo, Japan). Blue dextran 2000, chymotrypsinogen A, ribonuclease A, and bovine serum albumin (Gel Filtration LMW Calibration kit) as molecular weight markers for Superose 12 column chromatography were purchased from Amersham Pharmacia Biotech.

Preparation of rhIL-2-- cDNA encoding human IL-2 (R & D Systems Europe Ltd., Abingdon, UK) was used to produce rhIL-2 in E. coli. Plasmid pET3a (Novagen Inc., Madison, WI) was used as the T7 expression plasmid. An NdeI-HindIII fragment corresponding to a synthetic human IL-2 cDNA was inserted between the NdeI and HindIII sites of pET3a to produce the expression plasmid (pET3a-IL-2). The pET3a-IL-2 was expressed in E. coli strain BL21(DE3) under the control of the T7 promoter. A 15-ml culture of E. coli BL21(DE3) containing pET3a-IL-2 was grown overnight to stationary phase and was used to inoculate 500 ml of L broth containing 100 µg/ml ampicillin. After incubation for 2.5 h at 37 °C, cells were induced to produce rhIL-2 with 0.5 mM isopropyl--thiogalactoside and grown for 2.5 h. rhIL-2 was mainly incorporated in inclusion bodies. Therefore, the inclusion bodies were solubilized, and rhIL-2 was refolded as follows (14). 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 allowed to stand for 16 h at room temperature and then dialyzed against PBS. An aliquot of rhIL-2 was subjected to SDS-PAGE using 12.5% polyacrylamide gels to check the purity. The biological activity of the refolded rhIL-2 was determined in a proliferation assay using CTLL-2 cells. The unit of rhIL-2 purchased from Sigma was used as a standard. The protein concentration was determined by Bio-Rad Protein Assay dye reagent using bovine serum albumin as a standard. The unit of rhIL-2 used in this study was 1-10 units/ng.

Preparation of ³⁵S-rhIL-2-- pET3a-IL-2 produced as described above was used as a template for in vitro transcription and translation in TnT®-coupled reticulocyte lysate systems (Promega Corp., Madison, WI) in the presence of [³⁵S]methionine. To remove the endogenous mannose-binding lectins from the reagents, the reagents were mixed with a 20% slurry of ovalbumin-Sepharose (5 mg/ml) (20 µl) and centrifuged before the reagents were used. 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. An aliquot was also subjected to size-exclusion chromatography using a Superose 12 column (300 mm long, 10 mm inner diameter) and then eluted with PBS (flow rate, 0.5 ml/min) (Amersham Pharmacia Biotech). The Superose 12 column was pretreated with 100 µl of 3% human serum albumin in PBS to inhibit nonspecific adsorption. The remaining translation products were separated from free [³⁵S]methionine using a PD-10 column (Amersham Pharmacia Biotech) with PBS and used immediately. One reaction using 1 µg of plasmid DNA template, 25 µl of lysate, and 20 µCi of [³⁵S]methionine provided reproducibly 93 ± 4.3 fmol of ³⁵S-rhIL-2 (1.3 × 10⁴ dpm/fmol). The unit of ³⁵S-rhIL-2 was determined in a proliferation assay using CTLL-cells, as described above. The unit of ³⁵S-rhIL-2 synthesized by in vitro transcription and translation was about 50 units/ng.

Solid-phase Binding Assay-- The binding of ³⁵S-rhIL-2 to ribonuclease B or ribonuclease A was measured by a solid-phase binding assay. Enzyme-linked immunosorbent assay plates (Corning Costar Japan, Tokyo) were coated with 100 µl of ribonuclease B or ribonuclease A at 20 µg/ml in PBS at 4 °C overnight. Ribonuclease B treated with Endo H was also used as a coated glycoprotein. For Endo H treatment, 10 µl of a reaction mixture containing 10 mg/ml ribonuclease B and 0.05 units of Endo H in 0.1 M citrate-phosphate buffer, pH 5.0, was incubated at 37 °C for 18 h. The plates were washed with 0.05% Tween 20 in PBS and blocked with PBS containing 0.05% Tween 20 and 3% human serum albumin. Sequentially, the plates were treated with 2 × 10⁵ dpm of ³⁵S-rhIL-2, which corresponds to 15 fmol, in PBS containing 0.05% Tween 20 and 3% human serum albumin at 37 °C for 2 h. After washing with 0.05% Tween 20 in PBS, the bound ³⁵S-rhIL-2 was released by treatment with 100 µl of 1% SDS. The radioactivity was measured by liquid scintillation counter.

Cell Culture-- The 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 unit/ml rhIL-2 at 37 °C under 5% CO₂ atmosphere. rhIL-2 at 5 unit/ml final concentration was added to the cultures every 2 days. 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. They were then resuspended in complete medium and plated out in microtiter wells at a density of 10⁵ cells/ml with 100 µl added per well. A series of concentrations of ³⁵S-rhIL-2 diluted in 100 µl of complete RPMI 1640 medium was then added. After the cells had been incubated at 37 °C with 5% CO₂ for 2 days, 20 µl of Cell Titer 96® Aqueous one solution reagent was added to each well, and the absorbance at 525 nm was read after incubation for 2 h by a dual wavelength flying spot scanning densitometer CS-9300PC (Shimadzu Corp. Kyoto). The Cell Titer 96® Aqueous one solution reagent used to measure cell proliferation activity was obtained from Promega Corp. The solution was composed of a tetrazolium compound and an electron coupling reagent, phenazine ethosulfate, in Dulbecco's phosphate-buffered saline, pH 6.0.

Oligosaccharides-- Man₅GlcNAc₂Asn (M5) and Man₆GlcNAc₂Asn (M6) 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). Man₅GlcNAc₂ and Man₆GlcNAc₂ were prepared by hydrazinolysis of Man₅GlcNAc₂Asn and Man₆GlcNAc₂Asn followed by re-N-acetylation (16). Man₅GlcNAc and Man₆GlcNAc were obtained from Man_5-6GlcNAc₂Asn by Endo H digestion (15). Man₇GlcNAc₂, Man₈GlcNAc₂, Man₉GlcNAc₂, and Man₃GlcNAc₂ were prepared from 3 g of porcine thyroglobulin by hydrazinolysis followed by re-N-acetylation, and Man_7-9GlcNAc₂ was isolated by Bio-Gel P-4 (under 400 mesh, 2.0 × 100 cm) column chromatography. Each oligosaccharide was converted to aspartyloligosaccharide by treatment with Arthrobacter protophormiae endo--N-acetylglucosaminidase (Endo A) as described by Kuge et al. (17). Man_5-6GlcNAcGlcNAc_OH and Man₅GlcNAc_OH were obtained from Man_5-6GlcNAc₂ and Man₅GlcNAc by reduction using NaBH₄, respectively. The structures of these different oligosaccharyl-asparagines and oligosaccharides were determined by a combination of methylation analysis (18), -mannosidase digestion, partial acetolysis (15), and matrix-assisted laser desorption ionization/time of flight mass spectrometry (Shimadzu Corp., Kyoto, Japan).

Preparation of Mutant ³⁵S-rhIL-2-- The mutant forms of cDNAs encoding ³⁵S-rhIL-2, E15D, L19D, L21D, N26D, N26Q, G27A, N30D, and E67Q were generated by a two-step PCR method, as described by Mikaelian and Sergeant (19). The following primers were used: 5'-TAATACGACTCACTATAGGG (common I); 5'-GCTAGTTATTGCTCAGCGG (common II); 5'-CTGACTCAGTACTTCCTCCAG (common III); 5'-CTGCAATTGGACCATCTGCTG (for E15D); 5'-CATCTGCTGGACGATCTGCAG (for L19D); 5'-CTGCTGGATGACCAGATGATT (for L21D); 5'-ATGATTCTGGACGGCATCAAT (for N26D); 5'-ATGATTCTGCAAGGCATCAAT (for N26Q); 5'-ATTCTGAATGCCATCAATAAC (for G27A); 5'-GGCATCAATCAATACAAGAAC (for N30D); and 5'-AAGCCGCTGGACGAAGTACTT (for E67Q). Primers specific to each mutant contained the desired mutation. Common I and common III primers were used as a pair in one reaction; and common II and mutant-specific primer were used in a separate reaction in the first round of PCR. In the second round of PCR, the resulting two individual fragments were purified and mixed and then amplified using common I and common II primers. The fragments obtained were then digested with NdeI and HindIII and ligated into pET3a. The primary structure of all PCR-derived inserts was verified by DNA sequencing. After DNA sequencing of each mutant, [³⁵S]methionine-labeled mutant IL-2 was prepared in the same way as wild-type IL-2.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Forms of rhIL-2-- In order to investigate the carbohydrate-binding site in IL-2, we prepared wild-type and several mutant forms of ³⁵S-rhIL-2 by an in vitro transcription and translation method. In this paper, rhIL-2 indicates IL-2 produced in E. coli, and ³⁵S-rhIL-2 indicates ³⁵S-IL-2 synthesized in vitro. First of all, we studied whether ³⁵S-rhIL-2 can be used to measure the carbohydrate binding activity and CTLL-2 cell proliferation activity in comparison with rhIL-2. The molecular forms of rhIL-2 having cell proliferating activity were separated by Superose 12 column chromatography. The elution patterns of rhIL-2 (4 unit/ng) showed several molecular forms (Fig. 1A). Since the ratio of each molecular form was different in each sample, we showed one typical pattern. However, the CTLL-2 proliferation activities in the respective fractions of rhIL-2 were detected at the elution position corresponding to 30 kDa (Fig. 1B). On the other hand, when ³⁵S-rhIL-2 was immediately size-fractionated by Superose 12 column chromatography after the excess [³⁵S]methionine had been removed on a PD-10 column; ~80% of ³⁵S-rhIL-2 was reproducibly eluted at the elution volume corresponding to 30 kDa, and the remaining 20% was eluted at the elution volume corresponding to 60 kDa (Fig. 1C). However, the ³⁵S-rhIL-2 corresponding to 60 kDa increased with time (data not shown). Only the fraction corresponding to 30 kDa showed cell proliferation activity (Fig. 1D), and the specific activity was 50 units/ng (see "Experimental Procedures"). Since the molecular mass of ³⁵S-rhIL-2 is calculated as 15,030 Da on the basis of the amino acid sequence (20), ³⁵S-rhIL-2 spontaneously forms a dimeric structure in the PBS buffer. These results showed that this in vitro translated method is useful for studying the relationship between carbohydrate recognition activity and the cell proliferation activity of IL-2 muteins.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Size fractionation of rhIL-2 produced in E. coli and 35S-rhIL-2 synthesized by in vitro transcription and translation, and the cell proliferation activity of each fractionate. rhIL-2 produced in E. coli (A and B) and 35S-rhIL-2 synthesized by in vitro transcription and translation (C and D) as described under "Experimental Procedures" were subjected to gel filtration using Superose 12 columns. The amount of rhIL-2 produced in E. coli was measured by protein assay (Bradford method) (A), and the cell proliferation activity of each fractionate was determined using Cell Titer 96® Aqueous one solution reagent (see "Experimental Procedures") (B). C, radioactivity of 35S-rhIL-2; D, the cell proliferation activity of 35S-rhIL-2. Arrows indicate the elution positions of blue dextran 2000 (Vo, void volume), bovine serum albumin (67 kDa) (1), ovalbumin (43 kDa) (2), chymotrypsinogen (25 kDa) (3), ribonuclease A (13.7 kDa) (4), and mannose (180 Da) (5).

Conditions for the Solid-phase Binding Assay of ³⁵S-rhIL-2-- We established a direct analytical method to investigate the precise carbohydrate binding specificity of IL-2, because the inhibitory concentrations of carbohydrates determined by solid-phase enzyme-linked immunosorbent assay method was dependent on the activities of the antibodies used (data not shown). ³⁵S-rhIL-2 was translated in vitro in the presence of [³⁵S]methionine and then was separated from excess [³⁵S]methionine by PD-10 column chromatography (see "Experimental Procedures"). After the preparation, ³⁵S-rhIL-2 was immediately used for the direct solid-phase binding assay. To select the glycoprotein to be immobilized on the plates, a preliminary experiment was performed using ovalbumin (15), porcine thyroglobulin (21), and bovine ribonuclease B (22) containing high mannose-type glycans. It was found that ribonuclease B bound well to ³⁵S-rhIL-2 and had the lowest level of nonspecific binding activity among these three glycoproteins. Accordingly, ribonuclease B was immobilized on the plates. In subsequent experiments, 20 µg/ml ribonuclease B in 100 µl of PBS was immobilized in each well by incubation overnight, and the remaining sites were blocked with PBS containing 3% human serum albumin. For a blocking reagent, bovine serum albumin was not appropriate because of strong nonspecific binding. Thereafter, 5 × 10⁴ to 4 × 10⁵ dpm/ml ³⁵S-rhIL-2 was added to each well and allowed to stand for 2 h at 37 °C. The plates were then washed with 0.05% Tween 20 in PBS, and the radioactivity of bound ³⁵S-rhIL-2 was measured after being released by 1% SDS. As shown in Fig. 2A (), the binding of ³⁵S-rhIL-2 to ribonuclease B-coated plates was concentration-dependent up to 4 × 10⁵ dpm/ml (30 pM). In contrast, ³⁵S-rhIL-2 did not bind to ribonuclease A-coated plates, although ribonuclease A has the same amino acid sequence as ribonuclease B without any N-glycan (Fig. 2A, ). The same results were obtained using Endo H-treated ribonuclease B-coated plates (Fig. 2A, ). These results suggested that the binding of ³⁵S-rhIL-2 to immobilized ribonuclease B occurs via Man₅GlcNAc₂ or Man₆GlcNAc₂, which are linked to ribonuclease B (22).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Binding of 35S-rhIL-2 to ribonuclease B-coated plates (A) and effects of high mannose-type glycans on binding (B). Plates were coated with ribonuclease B, ribonuclease B treated with Endo H, or ribonuclease A as described under "Experimental Procedures," and the amount of bound 35S-rhIL-2 was measured using a liquid scintillation counter after being released with 1% SDS. A, 35S-rhIL-2 concentration dependence. Plates coated with 20 µg/ml ribonuclease B (), ribonuclease A (), or ribonuclease B treated with Endo H () were incubated for 2 h with various concentrations of 35S-rhIL-2 (up to 4 × 105dpm/ml) in PBS. B, inhibition curves obtained for the binding of 35S-rhIL-2 to ribonuclease B-coated plates in the presence of various high mannose-type glycans, Man5GlcNAc2Asn (), Man6GlcNAc2Asn (), Man9GlcNAc2Asn (), and Man3GlcNAc2 (). The inhibition curves obtained using Man7-8GlcNAc2Asn were the same as those obtained using Man3GlcNAc2 (data not shown). Results are means of three experiments (standard deviations were less than 5%).

Inhibitory Effects of Various High Mannose-type Glycans on the Carbohydrate Binding Activity of ³⁵S-rhIL-2-- It had been already reported that IL-2 interacts with Man₅GlcNAc₂ and Man₆GlcNAc₂Asn (13). However, it has remained unclear how the sugar chain structures of the reducing terminal side affect the carbohydrate-binding ability. We examined the inhibitory effects of various high mannose-type glycans (see Table I) on the binding of ³⁵S-rhIL-2 to ribonuclease B-coated plates. As shown in Fig. 2B, M5 and M6 showed inhibitory effects, whereas M7, M8, M9, and M3 did not. As summarized in Table I, in the case of M5 or M6, the 50% inhibition concentration was estimated to be 0.2 µM, whereas in the case of M7, M8, M9, or M3, it was greater than 1 mM. Since Man₅GlcNAc₂Asn, Man₅GlcNAc₂, and Man₅GlcNAc showed the same inhibitory effects, with respect to the structural requirement of the reducing terminal end, neither asparagine nor N, N'-diacetylchitobiose structure was required, but Man₅GlcNAc_OH did not show any inhibitory effect. These results indicate that two non-substituted -mannosyl residues linked to the 16 side of the tri-mannosyl core and reducing terminal 4-O-(-mannosyl)-N-acetylglucosaminyl pyranoside are required for the inhibitory activity.

Effects of Point Mutations of Asn-26 on the Carbohydrate Recognition Activity of IL-2-- We tried to produce mutant forms of in vitro translated ³⁵S-rhIL-2 with different levels of carbohydrate binding activities to directly determine whether the carbohydrate recognition activity of IL-2 is co-related to IL-2-induced cell proliferation activity. It has been reported by Sherblom et al. (13) that the amino-terminal portion of IL-2 exhibits a limited degree of sequence homology with human mannose-binding protein, MBP(H) (23), and two rat liver mannose-binding proteins, MBP(A) and MBP(C) (24). Glu-15, Asn-26, Gly-27, Asn-30, Cys-58, and Glu-67 are the amino acids conserved among MBP(H), MBP(A), MBP(C), and IL-2. His-16 and Leu-19 are common between MBP(H) and IL-2, whereas Leu-21 is conserved among MBP(C), MBP(H), and IL-2 (24). Moreover, it is confirmed that Asp-20 is the binding site for IL-2R; Gln-126 is the binding site for IL-2R; and Lys-35, Arg-38, Phe-42, and Lys-43 are the binding sites for IL-2R (8). On the basis of these data, we introduced point mutations at Glu-15, Leu-19, Leu-21, Asn-26, Gly-27, Asn-30, Cys-58, and Glu-67 into IL-2 cDNA by the PCR method (see "Experimental Procedures"), and we synthesized ³⁵S-rhIL-2 muteins in vitro in the presence of [³⁵S]methionine. When the respective products were developed on SDS-PAGE and visualized by autoradiography, all of them showed single bands corresponding to 16 kDa (data not shown), and the wild type of ³⁵S-rhIL-2 and eight mutant forms of ³⁵S-rhIL-2 were equally labeled with five [³⁵S]methionine residues per molecule. Among these eight mutant forms, three ³⁵S-labeled mutants showed similar yields of dimeric forms as the wild type on Superose 12 column chromatography, as summarized in Fig. 3 and Table II. IL-2 analogues E15D, L19D, L21D, G27A, and N30D which mostly could not construct dimeric forms had no carbohydrate recognition activities and cell proliferation activities up to 50 pM (Fig. 4,, , , , ). On the other hand, mutated N26D, N26Q, and E67Q had the same dimeric forms as the wild type (Fig. 3, E, F, and I). The carbohydrate recognition activities of these IL-2 analogues with conserved dimeric forms were assayed by a ribonuclease B-coated plate method. Although mutated E67Q showed the same binding activity as the wild type (Fig. 4A, ), mutated N26Q showed higher carbohydrate-binding ability (Fig. 4A, ) compared with wild-type IL-2 (Fig. 4A, ), whereas mutated N26D showed lower carbohydrate binding activity (Fig. 4A, ). The carbohydrate binding specificities of N26D and N26Q seemed not to be altered in comparison with that of wild-type IL-2, because the binding activities of N26D and N26Q activity on the ribonuclease B-coated plates were inhibited by M5 or M6, whereas M7, M8, or M9 did not show inhibitory effect as summarized in Table III. The 50% inhibition concentration with M5 or M6 was 0.2 µM, whereas those of M7, M8, or M9 were greater than 1 mM. Since the side chain of Asn-26 is directed toward the outside of the -helix structure as calculated on the basis of x-ray crystallography of IL-2 (25) (Fig. 5A), even if Asn-26 is replaced by Asp or Gln as a result of a point mutation, it is thought that the three-dimensional structure of these IL-2 molecules would not be affected. These results suggested that Asn-26 is involved in the carbohydrate binding of IL-2.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Size fractionation of wild-type and mutant forms of 35S-rhIL-2. Wild-type and mutant forms of 35S-rhIL-2 synthesized by in vitro transcription and translation were analyzed using Superose 12 column chromatography. A, wild type; B, E15D; C, L19D; D, L21D; E, N26D; F, N26Q; G, G27A; H, N30D; I, E67Q. Arrows indicate the elution positions of bovine serum albumin (67 kDa) (1), ovalbumin (43 kDa) (2), chymotrypsinogen (25 kDa) (3), and ribonuclease A (13.7 kDa) (4).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Comparison between wild-type and mutant forms of IL-2 with respect to binding to ribonuclease B-coated plates (A) and inducing CTLL-2 cell proliferation (B). The binding assay and the CTLL-2 cell proliferation assay were performed as described under "Experimental Procedures." , N26Q-mutated IL-2; , wild type IL-2; , N26D-mutated IL-2; , E67Q-mutated IL-2; , E15D-mutated IL-2; , L19D-mutated IL-2; , L21D-mutated IL-2; , G27A-mutated IL-2; , N30D-mutated IL-2. Results are means of three experiments (standard deviations were less than 5%).

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Molecular models of human IL-2 and human IL-2 bound to IL-2 receptor subunits. A, a molecular model of human IL-2 taken from the Rutgers University Protein Data Base (code 1irl) shown as a turquoise ribbon structure. The side chain of Asn-26 is shown as a ball and stick structure. Lys-35, Arg-38, Phe-42, and Lys-43, which bind to the IL-2R peptide portion, are red. B, a theoretical model of IL-2 attached to its three receptor subunits, which was also taken from the Protein Data Base (code 1iln). IL-2 is shown in blue; the receptor subunits are red (), green (), and turquoise (). The Asn-26 residue is white.

Effects of Point Mutation of Asn-26 on the Induction of IL-2-dependent Cell Proliferation-- We also investigated whether the mutated forms of IL-2 showed any change in effectiveness to induce T-cell proliferation, as compared with wild-type IL-2. As shown in a previous paper (12), CTLL-2 cells, a mouse T-cell line, proliferate in a manner dependent on IL-2. Upon incubation (1 × 10⁴ cells/well) in the presence of in vitro translated ³⁵S-rhIL-2 for 48 h, the cells showed a proliferative response that was dependent on the concentration of ³⁵S-rhIL-2. The extent of cell proliferation was determined colorimetrically (see "Experimental Procedures"). The mutated E67Q had the same effect as the wild type of IL-2 on the T-cell proliferation activity (Fig. 4B, ), whereas the mutated N26Q showed higher effectiveness in inducing T-cell proliferation, as well as higher carbohydrate binding activity. The concentration at which 50% of the maximal proliferative response was observed in the case of the mutated N26Q (0.8 pM) (Fig. 4B, ) was one-tenth that of the wild-type (7 pM) (Fig. 4B, ). In contrast, the mutated N26D showed lower effectiveness in inducing T-cell proliferation, as well as lower carbohydrate binding activity. The concentration at which 50% of the maximal proliferative response was observed (21 pM) (Fig. 4B, ) was three times that of wild-type IL-2. Furthermore, N26D- or N26Q-inducible cell proliferation activity was inhibited by the preincubation with M5 (data not shown). These results suggest that Asn-26 is located within the carbohydrate recognition site of IL-2, and Asn-26 and the specific amino acids Lys-35, Arg-38, Phe-42, and Lys-43 in IL-2 as shown in Fig. 5A bind both Man₅GlcNAc₂ and a specific peptide in IL-2R, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We reported in a previous paper that the carbohydrate recognition of IL-2 is essential for expression of its physiological function (12). In this study, we prepared several mutant forms of IL-2 based on the homology with three mannose-binding proteins (13), and we compared the proliferation activities and carbohydrate recognition activities among them. We clearly demonstrated that the carbohydrate recognition site in IL-2 involves Asn-26. Before accomplishing this study, we needed to characterize the molecular forms of IL-2, which were synthesized by an in vitro translation method. As shown in Fig. 1, rhIL-2 produced in E. coli showed several molecular forms from polymer to monomer on Superose 12 column chromatography, although the dimeric form of IL-2 exclusively has the cell proliferation activity. Since rhIL-2 produced in E. coli is incorporated into inclusion bodies, the denaturing and refolding is necessary. These treatments might cause the oligomerization of IL-2. Freezing and thawing also caused oligomerization (data not shown). In contrast, ³⁵S-labeled rhIL-2 prepared by an in vitro transcription and translation method in the presence of [³⁵S]methionine reproducibly yielded the dimeric form, which has the same cell proliferation activity as the dimeric form of rhIL-2 produced in E. coli and was stable at 37 °C for several hours. Accordingly, ³⁵S-labeled rhIL-2 could be used to analyze precisely the relationship between the carbohydrate recognition activity and IL-2-induced proliferation activity of CTLL-2 cells.

To investigate the inhibitory effects of M5 and M6 on the binding to ribonuclease B-coated plates and IL-2-dependent cell proliferation, the 2 h of preincubation of ³⁵S-rhIL-2 with M5 or M6 before addition of the mixture to the cells was critical. As reported previously, since IL-2 recognizes both a high mannose-type glycan and a specific peptide in IL-2R (12), the difference in the conformation between free oligomannosides and the high mannose-type glycan linked to IL-2R on the cell surface may require preincubation of IL-2 and hapten. As soon as IL-2 binds to both Man₅GlcNAc₂ and a specific peptide in IL-2R, the tightly bound IL-2·IL-2R complex might be formed and not be replaced by the haptenic sugar. Although the N,N'-diacetyl chitobiosyl asparagine structure is not necessary for in vitro binding of IL-2, IL-2 may bind in vivo more strongly a specific glycopeptide portion including Man₅GlcNAcGlcNAc linked to Asn-33, Asn-43, or Asn-200 N-glycosylation potential sites of IL-2R than free glycans.

To determine the carbohydrate recognition site of IL-2, we prepared several IL-2 muteins on the basis of the data reported previously (13). Since IL-2 is a helical cytokine (26), it should hold a helical structure to retain its physiological activity. However, most of the muteins could not conserve the dimeric form which exclusively has the cell proliferation activity. Interestingly, although the muteins at Asn-26 could retain the dimeric form, their carbohydrate binding activities were affected and that of N26Q was increased, whereas that of N26D was decreased. Accordingly, Asn-26 was considered to be involved in the carbohydrate recognition site. When a theoretical model of IL-2 attached to IL-2R, -, and - was constructed on the basis of the structural model in the Rutgers University Protein Data base (27), which was obtained by both x-ray crystallographic and NMR studies, the position of Asn-26 and the positions of the residues involved in binding each of the receptor subunits were found to be close but not to be overlapped (Fig. 5B). This model also supports the view that IL-2 recognizes both the IL-2 receptor subunits and high mannose-type glycans and that binding to these ligands is necessary for expression of IL-2-induced cellular signal transduction. When the high affinity complex of IL-2R, -, and - was formed via the IL-2 carbohydrate recognition site including Asn-26, intracellular signal transduction might be stimulated. IL-2R of mouse CTLL-2 cells has three potential N-glycosylation sites, Asn-33, Asn-43, and Asn-200 (28), and human IL-2R has two potential N-glycosylation sites, Asn-70 and Asn-89 (29), where high mannose-type glycan also binds.² The question which carbohydrate at Asn-33, Asn-43, and/or Asn-200 in murine IL-2R contributes to the binding of IL-2 should be resolved in the near future.

We showed in a previous paper (12) that the formation of IL-2·IL-2R complex triggers the formation of a high affinity complex that consists of IL-2, IL-2R, -, -, and other tyrosine kinases. The cell proliferation activity and carbohydrate recognition activity of IL-2 could be seen mostly in the dimeric form, which was separated on Superose 12 column chromatography. Since co-crystallized IL-2 and IL-2R were constructed by a ratio of 1:1 by x-ray crystallography (30), it was considered that as soon as the hetero-octamer consisting of (IL-2·IL-2R, -, -)₂ is tightly formed, all the tyrosine kinases linked to the cytoplasmic domains of IL-2R and - are immediately phosphorylated, and cellular signaling might be effectively intensified.

Several types of the binding mechanisms for cytokines and their receptors have been reported. First, growth hormone (31) and erythropoietin (32) were considered to form a 1:2 heterotrimer complex with their receptors based on x-ray crystallographic analysis. Second, granulocyte colony-stimulating factor (33), basic fibroblast growth factor (34), and midkine (35) form a 2:2 heterotetramer complex with their receptors, and IL-6, IL-6 receptor -subunit, and gp130 form a 2:2:2 heterohexamer complex (36). It should be further confirmed by x-ray crystallography or NMR analysis whether IL-2 and IL-2R, -, and - form a 2:2:2:2 hetero-octamer complex as suggested in this study.

	FOOTNOTES

^* This work was supported in part by Grants-in-aid for Scientific Research on Priority Area 10178104 from the Ministry of Education, Science, Sports, and Culture of Japan.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.

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, June 4, 2001, DOI 10.1074/jbc.M102789200

² K. Fukushima, Y. Kanaya, and K. Yamashita, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: IL-2, interleukin-2; PBS, phosphate-buffered saline; Man, mannose; IL-2R, IL-2 receptor; M5, Man₅GlcNAc₂; M6, Man₆GlcNAc₂; M7, Man₇GlcNAc₂; M8, Man₈GlcNAc₂; M9, Man₉GlcNAc₂; M3, Man₃GlcNAc₂; Endo H, endo--N-acetylglucosaminidase H; rh, recombinant human; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MBP, mannose-binding proteins..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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