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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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N-linked glycosylation is not required for hIL-6 receptor binding, STAT signaling or cytokine-dependent B9.11 cell proliferation. (A) Calibration of quantities of hIL-6 and N-linked glycosylation site mutants secreted from transfected HKB5/B5 cells. ELISA plates were coated with polyclonal goat antibody to hIL-6, and hIL-6 was quantitated with monoclonal mouse antibody to hIL-6 conjugated to HRP. All the hIL-6 supernatants contained 100 ng/ml hIL-6 protein. (B) Binding to sgp130 in the presence or absence of soluble IL-6Rα. ELISA plates were coated with sgp130. Culture supernatants mixed with soluble IL-6Rα, or untreated, were assessed for binding to sgp130 by ELISA. (C) Supernatants from HKB5/B5 cells transfected with wild-type hIL-6 or N-linked glycosylation site mutants were assessed for their capacity to activate a STAT1-responsive reporter, pGAS3/Luc. (D) B9.11 cell proliferation assay in response to wild-type and N-linked glycosylation site mutants of hIL-6.
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fig9: N-linked glycosylation is not required for hIL-6 receptor binding, STAT signaling or cytokine-dependent B9.11 cell proliferation. (A) Calibration of quantities of hIL-6 and N-linked glycosylation site mutants secreted from transfected HKB5/B5 cells. ELISA plates were coated with polyclonal goat antibody to hIL-6, and hIL-6 was quantitated with monoclonal mouse antibody to hIL-6 conjugated to HRP. All the hIL-6 supernatants contained 100 ng/ml hIL-6 protein. (B) Binding to sgp130 in the presence or absence of soluble IL-6Rα. ELISA plates were coated with sgp130. Culture supernatants mixed with soluble IL-6Rα, or untreated, were assessed for binding to sgp130 by ELISA. (C) Supernatants from HKB5/B5 cells transfected with wild-type hIL-6 or N-linked glycosylation site mutants were assessed for their capacity to activate a STAT1-responsive reporter, pGAS3/Luc. (D) B9.11 cell proliferation assay in response to wild-type and N-linked glycosylation site mutants of hIL-6.

Mentions: N-linked glycosylation did not affect the secretion of cellular IL-6 (Fig. 9 A). Wild-type hIL-6 and the N-linked glycosylation site mutants bound to sgp130 to a similar extent, only in the presence of soluble IL6-Rα (Fig. 9 B). N-linked glycosylation site mutagenesis did not alter binding to sgp130. Furthermore, unlike vIL-6 in which the N89K mutant cytokine was impaired at stimulating cell proliferation, the N73K glycosylation site mutation in hIL-6 did not impair JAK/STAT signaling (Fig. 9 C), B9.11, or DS-1 cell proliferation (Fig. 9 D and unpublished data).


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)

N-linked glycosylation is not required for hIL-6 receptor binding, STAT signaling or cytokine-dependent B9.11 cell proliferation. (A) Calibration of quantities of hIL-6 and N-linked glycosylation site mutants secreted from transfected HKB5/B5 cells. ELISA plates were coated with polyclonal goat antibody to hIL-6, and hIL-6 was quantitated with monoclonal mouse antibody to hIL-6 conjugated to HRP. All the hIL-6 supernatants contained 100 ng/ml hIL-6 protein. (B) Binding to sgp130 in the presence or absence of soluble IL-6Rα. ELISA plates were coated with sgp130. Culture supernatants mixed with soluble IL-6Rα, or untreated, were assessed for binding to sgp130 by ELISA. (C) Supernatants from HKB5/B5 cells transfected with wild-type hIL-6 or N-linked glycosylation site mutants were assessed for their capacity to activate a STAT1-responsive reporter, pGAS3/Luc. (D) B9.11 cell proliferation assay in response to wild-type and N-linked glycosylation site mutants of hIL-6.
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Related In: Results  -  Collection

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fig9: N-linked glycosylation is not required for hIL-6 receptor binding, STAT signaling or cytokine-dependent B9.11 cell proliferation. (A) Calibration of quantities of hIL-6 and N-linked glycosylation site mutants secreted from transfected HKB5/B5 cells. ELISA plates were coated with polyclonal goat antibody to hIL-6, and hIL-6 was quantitated with monoclonal mouse antibody to hIL-6 conjugated to HRP. All the hIL-6 supernatants contained 100 ng/ml hIL-6 protein. (B) Binding to sgp130 in the presence or absence of soluble IL-6Rα. ELISA plates were coated with sgp130. Culture supernatants mixed with soluble IL-6Rα, or untreated, were assessed for binding to sgp130 by ELISA. (C) Supernatants from HKB5/B5 cells transfected with wild-type hIL-6 or N-linked glycosylation site mutants were assessed for their capacity to activate a STAT1-responsive reporter, pGAS3/Luc. (D) B9.11 cell proliferation assay in response to wild-type and N-linked glycosylation site mutants of hIL-6.
Mentions: N-linked glycosylation did not affect the secretion of cellular IL-6 (Fig. 9 A). Wild-type hIL-6 and the N-linked glycosylation site mutants bound to sgp130 to a similar extent, only in the presence of soluble IL6-Rα (Fig. 9 B). N-linked glycosylation site mutagenesis did not alter binding to sgp130. Furthermore, unlike vIL-6 in which the N89K mutant cytokine was impaired at stimulating cell proliferation, the N73K glycosylation site mutation in hIL-6 did not impair JAK/STAT signaling (Fig. 9 C), B9.11, or DS-1 cell proliferation (Fig. 9 D and unpublished data).

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