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N-linked glycosylation is required for optimal function of Kaposi's sarcoma herpesvirus-encoded, but not cellular, interleukin 6.

Dela Cruz CS, Lee Y, Viswanathan SR, El-Guindy AS, Gerlach J, Nikiforow S, Shedd D, Gradoville L, Miller G - J. Exp. Med. (2004)

Bottom Line: Although hIL-6 is also N-glycosylated at N73 and multiply O-glycosylated, neither N-linked nor O-linked glycosylation is necessary for IL-6 receptor alpha-dependent binding to gp130 or signaling through JAK1-STAT1/3.As distinct from vIL-6, unglycosylated hIL-6 is as potent as glycosylated hIL-6 in stimulating B cell proliferation.These findings highlight distinct functional roles of N-linked glycosylation in viral and cellular IL-6.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus interleukin-6 (vIL-6) is a structural and functional homologue of the human cytokine IL-6 (hIL-6). hIL-6 and vIL-6 exhibit similar biological functions and both act via the gp130 receptor subunit to activate the Janus tyrosine kinase (JAK)1 and signal transducer and activator of transcription (STAT)1/3 pathway. Here we show that vIL-6 is N-linked glycosylated at N78 and N89 and demonstrate that N-linked glycosylation at site N89 of vIL-6 markedly enhances binding to gp130, signaling through the JAK1-STAT1/3 pathway and functions in a cytokine-dependent cell proliferation bioassay. Although hIL-6 is also N-glycosylated at N73 and multiply O-glycosylated, neither N-linked nor O-linked glycosylation is necessary for IL-6 receptor alpha-dependent binding to gp130 or signaling through JAK1-STAT1/3. As distinct from vIL-6, unglycosylated hIL-6 is as potent as glycosylated hIL-6 in stimulating B cell proliferation. These findings highlight distinct functional roles of N-linked glycosylation in viral and cellular IL-6.

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Related in: MedlinePlus

Unglycosylated and fully glycosylated forms of hIL-6 proteins have similar biological potency. (A) Electrophoretic mobility of glycosylation site mutants of hIL-6. Lane 1, wild-type hIL-6; lane 2, T6A hIL-6; lane 3, T166A hIL-6; lane 4, T170A/T171A hIL-6; lane 5, T166A/T170A/T171A hIL-6 (lacking all O-glycans); lane 6, T166A/T170A/T171A/N73K hIL-6 (lacking all O- and N-glycans). (B) Electrophoretic mobility of hIL-6 glycoforms expressed in HKB5/B5 cells. Lanes 1–2 and 7, wild-type hIL-6; lane 3, supernatant from pcDNA3.1-transfected cells; lane 4, hIL-6 produced in the presence of TM; lane 5, hIL-6 produced in the presence of monensin; lane 6, hIL-6 produced in the presence of both monensin and TM. (C) ELISA assay for the binding to gp130/IL-6Rα of hIL-6, hIL-6 made in the presence of monensin and TM, and hIL-6 mutant lacking both O- and N-glycans (T166A/T170A/T171A/N73K). (D) B9.11 proliferative activity of wild-type hIL-6 (•), O-glycan lacking hIL-6 mutant (T166A/T170A/T177A; ▴), or fully unglycosylated hIL-6 mutant (T166A/T170A/T171A/N73K; ×). (E) B9.11 proliferation assay using hIL-6 expressed in the absence of inhibitors (•), the presence of monensin (♦), the presence of TM (▴), or the presence of both inhibitors (□). Equal amounts of hIL-6 preparations were used in the above experiments as assessed by hIL-6–specific ELISA.
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fig10: Unglycosylated and fully glycosylated forms of hIL-6 proteins have similar biological potency. (A) Electrophoretic mobility of glycosylation site mutants of hIL-6. Lane 1, wild-type hIL-6; lane 2, T6A hIL-6; lane 3, T166A hIL-6; lane 4, T170A/T171A hIL-6; lane 5, T166A/T170A/T171A hIL-6 (lacking all O-glycans); lane 6, T166A/T170A/T171A/N73K hIL-6 (lacking all O- and N-glycans). (B) Electrophoretic mobility of hIL-6 glycoforms expressed in HKB5/B5 cells. Lanes 1–2 and 7, wild-type hIL-6; lane 3, supernatant from pcDNA3.1-transfected cells; lane 4, hIL-6 produced in the presence of TM; lane 5, hIL-6 produced in the presence of monensin; lane 6, hIL-6 produced in the presence of both monensin and TM. (C) ELISA assay for the binding to gp130/IL-6Rα of hIL-6, hIL-6 made in the presence of monensin and TM, and hIL-6 mutant lacking both O- and N-glycans (T166A/T170A/T171A/N73K). (D) B9.11 proliferative activity of wild-type hIL-6 (•), O-glycan lacking hIL-6 mutant (T166A/T170A/T177A; ▴), or fully unglycosylated hIL-6 mutant (T166A/T170A/T171A/N73K; ×). (E) B9.11 proliferation assay using hIL-6 expressed in the absence of inhibitors (•), the presence of monensin (♦), the presence of TM (▴), or the presence of both inhibitors (□). Equal amounts of hIL-6 preparations were used in the above experiments as assessed by hIL-6–specific ELISA.

Mentions: The majority of hIL-6 molecules are O-glycosylated, whereas a minority are N-glycosylated (Fig. 10, A and B). Through mutagenesis, we found that O-glycosylation of hIL-6 occurs at sites T166, T170, and/or T171. A triple mutant of hIL-6 with T to A substitutions at each of these sites showed the absence of the ∼22-kD band representing the O-glycosylated form of hIL-6 and the presence of the ∼20-kD unglycosylated form of the protein (Fig. 10, A and B). This triple mutant was maximally functionally active in stimulating the IL-6–dependent B9.11 cells as compared with the wild-type protein (Fig. 10 D). Moreover, a triple mutant of hIL-6 lacking the three O-glycosylation sites that additionally lacks the N73 glycan was found to lack all forms of glycosylation as shown in Fig. 10 A. This fully unglycosylated mutant form of hIL-6 was equally capable of binding to gp130/IL-6Rα and stimulating proliferation of the B9.11 cells as the wild-type protein (Fig. 10, C and D). We also made hIL-6 in the presence of monensin, an inhibitor of O-glycosylation, and TM to produce hIL-6 lacking both O-glycans and N-glycans (Fig. 10 B, lanes 5 and 6). hIL-6 made in the presence of these inhibitors was as active as wild-type in binding gp130/IL-6Rα and in stimulating B9.11 cell proliferation (Fig. 10, C and E). Thus, both N-linked and O-linked glycans on hIL-6 made in mammalian cells are dispensable for the functional assays we used. Moreover, the lack of importance of N-glycans in hIL-6 cannot be accounted for by the presence of O-glycans. Recombinant hIL-6 protein expressed in E. coli bacteria, which are not known to glycosylate protein, was equally active in stimulating B9.11 cell proliferation as comparable amounts of the fully glycosylated hIL-6 protein were made in mammalian cells (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20031205/DC1).


N-linked glycosylation is required for optimal function of Kaposi's sarcoma herpesvirus-encoded, but not cellular, interleukin 6.

Dela Cruz CS, Lee Y, Viswanathan SR, El-Guindy AS, Gerlach J, Nikiforow S, Shedd D, Gradoville L, Miller G - J. Exp. Med. (2004)

Unglycosylated and fully glycosylated forms of hIL-6 proteins have similar biological potency. (A) Electrophoretic mobility of glycosylation site mutants of hIL-6. Lane 1, wild-type hIL-6; lane 2, T6A hIL-6; lane 3, T166A hIL-6; lane 4, T170A/T171A hIL-6; lane 5, T166A/T170A/T171A hIL-6 (lacking all O-glycans); lane 6, T166A/T170A/T171A/N73K hIL-6 (lacking all O- and N-glycans). (B) Electrophoretic mobility of hIL-6 glycoforms expressed in HKB5/B5 cells. Lanes 1–2 and 7, wild-type hIL-6; lane 3, supernatant from pcDNA3.1-transfected cells; lane 4, hIL-6 produced in the presence of TM; lane 5, hIL-6 produced in the presence of monensin; lane 6, hIL-6 produced in the presence of both monensin and TM. (C) ELISA assay for the binding to gp130/IL-6Rα of hIL-6, hIL-6 made in the presence of monensin and TM, and hIL-6 mutant lacking both O- and N-glycans (T166A/T170A/T171A/N73K). (D) B9.11 proliferative activity of wild-type hIL-6 (•), O-glycan lacking hIL-6 mutant (T166A/T170A/T177A; ▴), or fully unglycosylated hIL-6 mutant (T166A/T170A/T171A/N73K; ×). (E) B9.11 proliferation assay using hIL-6 expressed in the absence of inhibitors (•), the presence of monensin (♦), the presence of TM (▴), or the presence of both inhibitors (□). Equal amounts of hIL-6 preparations were used in the above experiments as assessed by hIL-6–specific ELISA.
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Related In: Results  -  Collection

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fig10: Unglycosylated and fully glycosylated forms of hIL-6 proteins have similar biological potency. (A) Electrophoretic mobility of glycosylation site mutants of hIL-6. Lane 1, wild-type hIL-6; lane 2, T6A hIL-6; lane 3, T166A hIL-6; lane 4, T170A/T171A hIL-6; lane 5, T166A/T170A/T171A hIL-6 (lacking all O-glycans); lane 6, T166A/T170A/T171A/N73K hIL-6 (lacking all O- and N-glycans). (B) Electrophoretic mobility of hIL-6 glycoforms expressed in HKB5/B5 cells. Lanes 1–2 and 7, wild-type hIL-6; lane 3, supernatant from pcDNA3.1-transfected cells; lane 4, hIL-6 produced in the presence of TM; lane 5, hIL-6 produced in the presence of monensin; lane 6, hIL-6 produced in the presence of both monensin and TM. (C) ELISA assay for the binding to gp130/IL-6Rα of hIL-6, hIL-6 made in the presence of monensin and TM, and hIL-6 mutant lacking both O- and N-glycans (T166A/T170A/T171A/N73K). (D) B9.11 proliferative activity of wild-type hIL-6 (•), O-glycan lacking hIL-6 mutant (T166A/T170A/T177A; ▴), or fully unglycosylated hIL-6 mutant (T166A/T170A/T171A/N73K; ×). (E) B9.11 proliferation assay using hIL-6 expressed in the absence of inhibitors (•), the presence of monensin (♦), the presence of TM (▴), or the presence of both inhibitors (□). Equal amounts of hIL-6 preparations were used in the above experiments as assessed by hIL-6–specific ELISA.
Mentions: The majority of hIL-6 molecules are O-glycosylated, whereas a minority are N-glycosylated (Fig. 10, A and B). Through mutagenesis, we found that O-glycosylation of hIL-6 occurs at sites T166, T170, and/or T171. A triple mutant of hIL-6 with T to A substitutions at each of these sites showed the absence of the ∼22-kD band representing the O-glycosylated form of hIL-6 and the presence of the ∼20-kD unglycosylated form of the protein (Fig. 10, A and B). This triple mutant was maximally functionally active in stimulating the IL-6–dependent B9.11 cells as compared with the wild-type protein (Fig. 10 D). Moreover, a triple mutant of hIL-6 lacking the three O-glycosylation sites that additionally lacks the N73 glycan was found to lack all forms of glycosylation as shown in Fig. 10 A. This fully unglycosylated mutant form of hIL-6 was equally capable of binding to gp130/IL-6Rα and stimulating proliferation of the B9.11 cells as the wild-type protein (Fig. 10, C and D). We also made hIL-6 in the presence of monensin, an inhibitor of O-glycosylation, and TM to produce hIL-6 lacking both O-glycans and N-glycans (Fig. 10 B, lanes 5 and 6). hIL-6 made in the presence of these inhibitors was as active as wild-type in binding gp130/IL-6Rα and in stimulating B9.11 cell proliferation (Fig. 10, C and E). Thus, both N-linked and O-linked glycans on hIL-6 made in mammalian cells are dispensable for the functional assays we used. Moreover, the lack of importance of N-glycans in hIL-6 cannot be accounted for by the presence of O-glycans. Recombinant hIL-6 protein expressed in E. coli bacteria, which are not known to glycosylate protein, was equally active in stimulating B9.11 cell proliferation as comparable amounts of the fully glycosylated hIL-6 protein were made in mammalian cells (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20031205/DC1).

Bottom Line: Although hIL-6 is also N-glycosylated at N73 and multiply O-glycosylated, neither N-linked nor O-linked glycosylation is necessary for IL-6 receptor alpha-dependent binding to gp130 or signaling through JAK1-STAT1/3.As distinct from vIL-6, unglycosylated hIL-6 is as potent as glycosylated hIL-6 in stimulating B cell proliferation.These findings highlight distinct functional roles of N-linked glycosylation in viral and cellular IL-6.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus interleukin-6 (vIL-6) is a structural and functional homologue of the human cytokine IL-6 (hIL-6). hIL-6 and vIL-6 exhibit similar biological functions and both act via the gp130 receptor subunit to activate the Janus tyrosine kinase (JAK)1 and signal transducer and activator of transcription (STAT)1/3 pathway. Here we show that vIL-6 is N-linked glycosylated at N78 and N89 and demonstrate that N-linked glycosylation at site N89 of vIL-6 markedly enhances binding to gp130, signaling through the JAK1-STAT1/3 pathway and functions in a cytokine-dependent cell proliferation bioassay. Although hIL-6 is also N-glycosylated at N73 and multiply O-glycosylated, neither N-linked nor O-linked glycosylation is necessary for IL-6 receptor alpha-dependent binding to gp130 or signaling through JAK1-STAT1/3. As distinct from vIL-6, unglycosylated hIL-6 is as potent as glycosylated hIL-6 in stimulating B cell proliferation. These findings highlight distinct functional roles of N-linked glycosylation in viral and cellular IL-6.

Show MeSH
Related in: MedlinePlus