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

Abstract

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 α-subunit and sequentially forms a high affinity complex of IL-2·IL-2 receptor α-, β-, and γ-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 vitrotranscription and translation method. We found that wild-type35S-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-2Rα, -β, -γ)2, and the hetero-octamer stimulates IL-2-dependent T-cell proliferation by intensifying cellular signaling. 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 α-subunit and sequentially forms a high affinity complex of IL-2·IL-2 receptor α-, β-, and γ-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 vitrotranscription and translation method. We found that wild-type35S-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-2Rα, -β, -γ)2, and the hetero-octamer stimulates IL-2-dependent T-cell proliferation by intensifying cellular signaling. interleukin-2 phosphate-buffered saline mannose IL-2 receptor Man5GlcNAc2 Man6GlcNAc2 Man7GlcNAc2 Man8GlcNAc2 Man9GlcNAc2 Man3GlcNAc2 endo-β-N-acetylglucosaminidase H recombinant human polymerase chain reaction polyacrylamide gel electrophoresis mannose-binding proteins. Interleukin-2 (IL-2)1has been widely studied as a mediator of cellular signaling in the immune system. The receptor for IL-2 (IL-2R) consists of α-, β-, and γ-subunits (IL-2Rα, -β, and -γ), and the intracellular portions of the β- and γ-subunits are associated with a variety of cytoplasmic proteins including tyrosine kinases Jak1 and Jak3 (1Nelson B.H. Willerford D.M. Adv. Immunol. 1998; 70: 1-81Crossref PubMed Google Scholar, 2Asao H. Tanaka N. Ishii N. Higuchi M. Takeshita T. Nakamura M. Shirasawa T. Sugamura K. FEBS Lett. 1994; 351: 201-206Crossref PubMed Scopus (55) Google Scholar). The phosphorylated cytoplasmic domain of IL-2Rβ especially plays a critical role in attracting downstream signaling molecules including the transcription factors STAT3 and STAT5 into the activated receptor complex (1Nelson B.H. Willerford D.M. Adv. Immunol. 1998; 70: 1-81Crossref PubMed Google Scholar, 3Gaffen S.L. Lai S.Y. Ha M. Liu X. Hennighausen L. Greene W.C. Goldsmith M.A. J. Biol. Chem. 1996; 271: 21381-21390Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). IL-2Rα, -β, and -γ bind to distinct sites of IL-2, and these associations appear to occur in a stepwise manner (4Zurawski S.M. Imler J.L. Zurawski G. EMBO J. 1990; 9: 3899-3905Crossref PubMed Scopus (58) Google Scholar, 5Imler J.L. Miyajima A. Zurawski G. EMBO J. 1992; 11: 2047-2053Crossref PubMed Scopus (23) Google Scholar, 6Voss S.D. Leary T.P. Sondel P.M. Robb R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2428-2432Crossref PubMed Scopus (73) Google Scholar, 7Moreau J.L. Bossus M. de Groote D. Francois C. Jacques Y. Tartar A. Theze J. Mol. Immunol. 1995; 32: 1047-1056Crossref PubMed Scopus (10) Google Scholar, 8Sauve 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). When IL-2Rα, -β, or -γ was independently expressed, IL-2Rα bound IL-2 with low affinity (K d ∼10 nm); IL-2Rβ bound IL-2 with very low affinity (K d ∼100 nm), and IL-2Rγ had no measurable affinity for IL-2, whereas co-expressed IL-2Rα, -β, and -γ bound IL-2 with a very high affinity constant (K d ∼pm) (9Arima 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, 10Takeshita T. Ohtani K. Asao H. Kumaki S. Nakamura M. Sugamura K. J. Immunol. 1992; 148: 2154-2158PubMed Google Scholar, 11Anderson 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.V. Shapiro D.N. Morris S.W. Park L.S. Cosman D. J. Biol. Chem. 1995; 270: 29862-29869Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). We reported previously that IL-2 binds IL-2Rα through Man5GlcNAc2 and a specific peptide sequence in IL-2Rα on the surface of CTLL-2 cells and that this dual recognition triggers formation of the high affinity complex of IL-2·IL-2Rα, -β, -γ, leading to downstream signaling (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar).IL-2Rα binds Lys-35, Arg-38, Phe-42, and Lys-43 residues of IL-2 (8Sauve 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), 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) (13Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar). 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.DISCUSSIONWe reported in a previous paper that the carbohydrate recognition of IL-2 is essential for expression of its physiological function (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In this study, we prepared several mutant forms of IL-2 based on the homology with three mannose-binding proteins (13Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar), 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 anin 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 vitrotranscription 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 to and IL-2-dependent cell the of of with M5 or M6 of the to the cells was As reported since IL-2 both a high mannose-type glycan and a specific peptide in IL-2Rα (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), the in the between and the high mannose-type glycan to IL-2Rα on the cell surface of IL-2 and As as IL-2 binds to both Man5GlcNAc2 and a specific peptide in IL-2Rα, the bound complex might be and not be by the the is not for in vitro of IL-2, IL-2 bind in a specific portion including to or sites of IL-2Rα determine the carbohydrate recognition site of IL-2, we prepared several IL-2 muteins on the of the reported previously (13Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar). Since IL-2 is a cytokine Immunol. 1990; 11: Full Text PDF PubMed Scopus Google Scholar), a to its physiological activity. of the muteins could not the dimeric form which exclusively has the cell proliferation activity. although the muteins at Asn-26 could the dimeric form, carbohydrate activities were affected and that of was whereas that of was Accordingly, Asn-26 was to be involved in the carbohydrate recognition site. When a of IL-2 to IL-2Rα, -β, and -γ was on the of the in the P. 1994; Full Text Full Text PDF Scopus Google Scholar), which was by both and the of Asn-26 and the of the residues involved in each of the receptor were found to be but not to be This also the that IL-2 both the IL-2 receptor and high mannose-type and that to these is for expression of IL-2-induced cellular When the high affinity complex of IL-2Rα, -β, and -γ was the IL-2 carbohydrate recognition site including Asn-26, intracellular might be IL-2Rα of CTLL-2 cells has three and A. S. S. J. N. H. H. T. T. T. PubMed Scopus Google Scholar), and human IL-2Rα has two and J.M. S. J. Robb R.J. M. PubMed Scopus Google Scholar), high mannose-type glycan also Y. and K. Yamashita, in The which carbohydrate at in IL-2Rα to the of IL-2 be in the showed in a previous paper (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google that the formation of complex triggers the formation of a high affinity complex that consists of IL-2, IL-2Rα, -β, -γ, and tyrosine The cell proliferation activity and carbohydrate recognition activity of IL-2 could be in the dimeric form, which was on Superose 12 column Since IL-2 and IL-2Rα were by a of by G. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar), was that as as the hetero-octamer of (IL-2·IL-2Rα, -β, the tyrosine kinases to the cytoplasmic domains of IL-2Rβ and -γ are and cellular signaling might be of the for and been M. 1992; PubMed Scopus Google and S.W. C. Liu H. J. J. S. K. D.J. J. 1998; PubMed Scopus Google were to form a complex with based on M. N. T. Y. K. PubMed Scopus Google Scholar), A. M. P. D.M. 1991; Full Text PDF Scopus Google Scholar), and K. H. T. T. T. K. M. EMBO J. PubMed Scopus Google form a complex with and receptor and form a complex A. J.M. L. J. R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). be by or IL-2 and IL-2Rα, -β, and -γ form a hetero-octamer complex as in this Interleukin-2 (IL-2)1has been widely studied as a mediator of cellular signaling in the immune system. The receptor for IL-2 (IL-2R) consists of α-, β-, and γ-subunits (IL-2Rα, -β, and -γ), and the intracellular portions of the β- and γ-subunits are associated with a variety of cytoplasmic proteins including tyrosine kinases Jak1 and Jak3 (1Nelson B.H. Willerford D.M. Adv. Immunol. 1998; 70: 1-81Crossref PubMed Google Scholar, 2Asao H. Tanaka N. Ishii N. Higuchi M. Takeshita T. Nakamura M. Shirasawa T. Sugamura K. FEBS Lett. 1994; 351: 201-206Crossref PubMed Scopus (55) Google Scholar). The phosphorylated cytoplasmic domain of IL-2Rβ especially plays a critical role in attracting downstream signaling molecules including the transcription factors STAT3 and STAT5 into the activated receptor complex (1Nelson B.H. Willerford D.M. Adv. Immunol. 1998; 70: 1-81Crossref PubMed Google Scholar, 3Gaffen S.L. Lai S.Y. Ha M. Liu X. Hennighausen L. Greene W.C. Goldsmith M.A. J. Biol. Chem. 1996; 271: 21381-21390Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). IL-2Rα, -β, and -γ bind to distinct sites of IL-2, and these associations appear to occur in a stepwise manner (4Zurawski S.M. Imler J.L. Zurawski G. EMBO J. 1990; 9: 3899-3905Crossref PubMed Scopus (58) Google Scholar, 5Imler J.L. Miyajima A. Zurawski G. EMBO J. 1992; 11: 2047-2053Crossref PubMed Scopus (23) Google Scholar, 6Voss S.D. Leary T.P. Sondel P.M. Robb R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2428-2432Crossref PubMed Scopus (73) Google Scholar, 7Moreau J.L. Bossus M. de Groote D. Francois C. Jacques Y. Tartar A. Theze J. Mol. Immunol. 1995; 32: 1047-1056Crossref PubMed Scopus (10) Google Scholar, 8Sauve 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). When IL-2Rα, -β, or -γ was independently expressed, IL-2Rα bound IL-2 with low affinity (K d ∼10 nm); IL-2Rβ bound IL-2 with very low affinity (K d ∼100 nm), and IL-2Rγ had no measurable affinity for IL-2, whereas co-expressed IL-2Rα, -β, and -γ bound IL-2 with a very high affinity constant (K d ∼pm) (9Arima 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, 10Takeshita T. Ohtani K. Asao H. Kumaki S. Nakamura M. Sugamura K. J. Immunol. 1992; 148: 2154-2158PubMed Google Scholar, 11Anderson 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.V. Shapiro D.N. Morris S.W. Park L.S. Cosman D. J. Biol. Chem. 1995; 270: 29862-29869Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). We reported previously that IL-2 binds IL-2Rα through Man5GlcNAc2 and a specific peptide sequence in IL-2Rα on the surface of CTLL-2 cells and that this dual recognition triggers formation of the high affinity complex of IL-2·IL-2Rα, -β, -γ, leading to downstream signaling (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). IL-2Rα binds Lys-35, Arg-38, Phe-42, and Lys-43 residues of IL-2 (8Sauve 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), 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) (13Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar). 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 reported in a previous paper that the carbohydrate recognition of IL-2 is essential for expression of its physiological function (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In this study, we prepared several mutant forms of IL-2 based on the homology with three mannose-binding proteins (13Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar), 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 anin 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 vitrotranscription 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 to and IL-2-dependent cell the of of with M5 or M6 of the to the cells was As reported since IL-2 both a high mannose-type glycan and a specific peptide in IL-2Rα (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), the in the between and the high mannose-type glycan to IL-2Rα on the cell surface of IL-2 and As as IL-2 binds to both Man5GlcNAc2 and a specific peptide in IL-2Rα, the bound complex might be and not be by the the is not for in vitro of IL-2, IL-2 bind in a specific portion including to or sites of IL-2Rα determine the carbohydrate recognition site of IL-2, we prepared several IL-2 muteins on the of the reported previously (13Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar). Since IL-2 is a cytokine Immunol. 1990; 11: Full Text PDF PubMed Scopus Google Scholar), a to its physiological activity. of the muteins could not the dimeric form which exclusively has the cell proliferation activity. although the muteins at Asn-26 could the dimeric form, carbohydrate activities were affected and that of was whereas that of was Accordingly, Asn-26 was to be involved in the carbohydrate recognition site. When a of IL-2 to IL-2Rα, -β, and -γ was on the of the in the P. 1994; Full Text Full Text PDF Scopus Google Scholar), which was by both and the of Asn-26 and the of the residues involved in each of the receptor were found to be but not to be This also the that IL-2 both the IL-2 receptor and high mannose-type and that to these is for expression of IL-2-induced cellular When the high affinity complex of IL-2Rα, -β, and -γ was the IL-2 carbohydrate recognition site including Asn-26, intracellular might be IL-2Rα of CTLL-2 cells has three and A. S. S. J. N. H. H. T. T. T. PubMed Scopus Google Scholar), and human IL-2Rα has two and J.M. S. J. Robb R.J. M. PubMed Scopus Google Scholar), high mannose-type glycan also Y. and K. Yamashita, in The which carbohydrate at in IL-2Rα to the of IL-2 be in the showed in a previous paper (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google that the formation of complex triggers the formation of a high affinity complex that consists of IL-2, IL-2Rα, -β, -γ, and tyrosine The cell proliferation activity and carbohydrate recognition activity of IL-2 could be in the dimeric form, which was on Superose 12 column Since IL-2 and IL-2Rα were by a of by G. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar), was that as as the hetero-octamer of (IL-2·IL-2Rα, -β, the tyrosine kinases to the cytoplasmic domains of IL-2Rβ and -γ are and cellular signaling might be of the for and been M. 1992; PubMed Scopus Google and S.W. C. Liu H. J. J. S. K. D.J. J. 1998; PubMed Scopus Google were to form a complex with based on M. N. T. Y. K. PubMed Scopus Google Scholar), A. M. P. D.M. 1991; Full Text PDF Scopus Google Scholar), and K. H. T. T. T. K. M. EMBO J. PubMed Scopus Google form a complex with and receptor and form a complex A. J.M. L. J. R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). be by or IL-2 and IL-2Rα, -β, and -γ form a hetero-octamer complex as in this We reported in a previous paper that the carbohydrate recognition of IL-2 is essential for expression of its physiological function (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In this study, we prepared several mutant forms of IL-2 based on the homology with three mannose-binding proteins (13Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar), 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 anin 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 vitrotranscription 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 To investigate the inhibitory effects of M5 and M6 on the to and IL-2-dependent cell the of of with M5 or M6 of the to the cells was As reported since IL-2 both a high mannose-type glycan and a specific peptide in IL-2Rα (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), the in the between and the high mannose-type glycan to IL-2Rα on the cell surface of IL-2 and As as IL-2 binds to both Man5GlcNAc2 and a specific peptide in IL-2Rα, the bound complex might be and not be by the the is not for in vitro of IL-2, IL-2 bind in a specific portion including to or sites of IL-2Rα glycans. To determine the carbohydrate recognition site of IL-2, we prepared several IL-2 muteins on the of the reported previously (13Sherblom A.P. Sathyamoorthy N. Decker J.M. Muchmore A.V. J. Immunol. 1989; 143: 939-944PubMed Google Scholar). Since IL-2 is a cytokine Immunol. 1990; 11: Full Text PDF PubMed Scopus Google Scholar), a to its physiological activity. of the muteins could not the dimeric form which exclusively has the cell proliferation activity. although the muteins at Asn-26 could the dimeric form, carbohydrate activities were affected and that of was whereas that of was Accordingly, Asn-26 was to be involved in the carbohydrate recognition site. When a of IL-2 to IL-2Rα, -β, and -γ was on the of the in the P. 1994; Full Text Full Text PDF Scopus Google Scholar), which was by both and the of Asn-26 and the of the residues involved in each of the receptor were found to be but not to be This also the that IL-2 both the IL-2 receptor and high mannose-type and that to these is for expression of IL-2-induced cellular When the high affinity complex of IL-2Rα, -β, and -γ was the IL-2 carbohydrate recognition site including Asn-26, intracellular might be IL-2Rα of CTLL-2 cells has three and A. S. S. J. N. H. H. T. T. T. PubMed Scopus Google Scholar), and human IL-2Rα has two and J.M. S. J. Robb R.J. M. PubMed Scopus Google Scholar), high mannose-type glycan also Y. and K. Yamashita, in The which carbohydrate at in IL-2Rα to the of IL-2 be in the We showed in a previous paper (12Fukushima K. Yamashita K. J. Biol. Chem. 2001; 276: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (22) Google that the formation of complex triggers the formation of a high affinity complex that consists of IL-2, IL-2Rα, -β, -γ, and tyrosine The cell proliferation activity and carbohydrate recognition activity of IL-2 could be in the dimeric form, which was on Superose 12 column Since IL-2 and IL-2Rα were by a of by G. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar), was that as as the hetero-octamer of (IL-2·IL-2Rα, -β, the tyrosine kinases to the cytoplasmic domains of IL-2Rβ and -γ are and cellular signaling might be of the for and been M. 1992; PubMed Scopus Google and S.W. C. Liu H. J. J. S. K. D.J. J. 1998; PubMed Scopus Google were to form a complex with based on M. N. T. Y. K. PubMed Scopus Google Scholar), A. M. P. D.M. 1991; Full Text PDF Scopus Google Scholar), and K. H. T. T. T. K. M. EMBO J. PubMed Scopus Google form a complex with and receptor and form a complex A. J.M. L. J. R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). be by or IL-2 and IL-2Rα, -β, and -γ form a hetero-octamer complex as in this