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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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Comparison of binding to gp130 by wild-type and N-linked glycosylation site mutants of vIL-6. (A) Comparison of binding to sgp130 by vIL-6 expressed in HKB5/B5 cells and in E. coli. Soluble gp130 was coated on plastic plates. Serial dilutions starting at 20 ng/ml purified E. coli–derived wild-type vIL-6 from three different KSHV strains and HKB5/B5-derived wild-type vIL-6 were assessed for binding to gp130 by ELISA. (B) Comparison of binding to sgp130 by wild-type vIL-6 and N-linked glycosylation site mutants. Supernatants from transfected HKB5/B5 cells containing equal amounts of vIL-6 or its mutants were assessed for their capacity to bind to sgp130 by an ELISA. (C) Assay for the amount of immunoreactive vIL-6 added to sgp130 in the experiment shown in Fig. 5 B. Plates were coated with purified polyclonal rabbit anti–vIL-6 IgG. In experiments shown in A, B, and C, the amount of vIL-6 bound was detected with biotinylated polyclonal rabbit antibody to KSHV IL-6.
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fig5: Comparison of binding to gp130 by wild-type and N-linked glycosylation site mutants of vIL-6. (A) Comparison of binding to sgp130 by vIL-6 expressed in HKB5/B5 cells and in E. coli. Soluble gp130 was coated on plastic plates. Serial dilutions starting at 20 ng/ml purified E. coli–derived wild-type vIL-6 from three different KSHV strains and HKB5/B5-derived wild-type vIL-6 were assessed for binding to gp130 by ELISA. (B) Comparison of binding to sgp130 by wild-type vIL-6 and N-linked glycosylation site mutants. Supernatants from transfected HKB5/B5 cells containing equal amounts of vIL-6 or its mutants were assessed for their capacity to bind to sgp130 by an ELISA. (C) Assay for the amount of immunoreactive vIL-6 added to sgp130 in the experiment shown in Fig. 5 B. Plates were coated with purified polyclonal rabbit anti–vIL-6 IgG. In experiments shown in A, B, and C, the amount of vIL-6 bound was detected with biotinylated polyclonal rabbit antibody to KSHV IL-6.

Mentions: The vIL-6 protein has two potential N-linked glycosylation consensus sites, as represented by Asn-X-Ser/Thr, where X can be any amino acid. These are located at N78 and N89. The three forms of vIL-6 observed in HKB5/B5 cells (Fig. 1 A, lane 2) could be explained by forms of the protein with both sites, one site, or no sites glycosylated. To explore this possibility, site-directed mutants, N78K and N89K, and the double mutant N78K/N89K, were generated. These mutations retain a functional amine group but cannot be glycosylated. The electrophoretic mobility of the mutants was compared with wild-type vIL-6 in cell extracts and supernatants of transfected HKB5/B5 cells (Fig. 2). Wild-type vIL-6 migrated more slowly, at 28 kD, than either single N-linked glycosylation site mutant, which migrated at ∼25 kD. The double mutant that lacked both N-linked glycosylation sites migrated fastest at ∼22 kD. These findings were most consistent with N-linked glycosylation of both residues in the wild-type protein. All the mutant vIL-6 proteins were produced in amounts approximately equal to wild-type and were secreted in equal amounts (see Fig. 5 C).


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)

Comparison of binding to gp130 by wild-type and N-linked glycosylation site mutants of vIL-6. (A) Comparison of binding to sgp130 by vIL-6 expressed in HKB5/B5 cells and in E. coli. Soluble gp130 was coated on plastic plates. Serial dilutions starting at 20 ng/ml purified E. coli–derived wild-type vIL-6 from three different KSHV strains and HKB5/B5-derived wild-type vIL-6 were assessed for binding to gp130 by ELISA. (B) Comparison of binding to sgp130 by wild-type vIL-6 and N-linked glycosylation site mutants. Supernatants from transfected HKB5/B5 cells containing equal amounts of vIL-6 or its mutants were assessed for their capacity to bind to sgp130 by an ELISA. (C) Assay for the amount of immunoreactive vIL-6 added to sgp130 in the experiment shown in Fig. 5 B. Plates were coated with purified polyclonal rabbit anti–vIL-6 IgG. In experiments shown in A, B, and C, the amount of vIL-6 bound was detected with biotinylated polyclonal rabbit antibody to KSHV IL-6.
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Related In: Results  -  Collection

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fig5: Comparison of binding to gp130 by wild-type and N-linked glycosylation site mutants of vIL-6. (A) Comparison of binding to sgp130 by vIL-6 expressed in HKB5/B5 cells and in E. coli. Soluble gp130 was coated on plastic plates. Serial dilutions starting at 20 ng/ml purified E. coli–derived wild-type vIL-6 from three different KSHV strains and HKB5/B5-derived wild-type vIL-6 were assessed for binding to gp130 by ELISA. (B) Comparison of binding to sgp130 by wild-type vIL-6 and N-linked glycosylation site mutants. Supernatants from transfected HKB5/B5 cells containing equal amounts of vIL-6 or its mutants were assessed for their capacity to bind to sgp130 by an ELISA. (C) Assay for the amount of immunoreactive vIL-6 added to sgp130 in the experiment shown in Fig. 5 B. Plates were coated with purified polyclonal rabbit anti–vIL-6 IgG. In experiments shown in A, B, and C, the amount of vIL-6 bound was detected with biotinylated polyclonal rabbit antibody to KSHV IL-6.
Mentions: The vIL-6 protein has two potential N-linked glycosylation consensus sites, as represented by Asn-X-Ser/Thr, where X can be any amino acid. These are located at N78 and N89. The three forms of vIL-6 observed in HKB5/B5 cells (Fig. 1 A, lane 2) could be explained by forms of the protein with both sites, one site, or no sites glycosylated. To explore this possibility, site-directed mutants, N78K and N89K, and the double mutant N78K/N89K, were generated. These mutations retain a functional amine group but cannot be glycosylated. The electrophoretic mobility of the mutants was compared with wild-type vIL-6 in cell extracts and supernatants of transfected HKB5/B5 cells (Fig. 2). Wild-type vIL-6 migrated more slowly, at 28 kD, than either single N-linked glycosylation site mutant, which migrated at ∼25 kD. The double mutant that lacked both N-linked glycosylation sites migrated fastest at ∼22 kD. These findings were most consistent with N-linked glycosylation of both residues in the wild-type protein. All the mutant vIL-6 proteins were produced in amounts approximately equal to wild-type and were secreted in equal amounts (see Fig. 5 C).

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