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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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Effect of expression of vIL-6 in the presence of TM on binding to sgp130 and on B9.11 cell proliferation. (A and B) Electrophoretic mobility of vIL-6 expressed in HKB5/B5 cells in the absence or presence of 1.0 μg/ml TM. Immunoblots of culture supernatants (A) or cell extracts (B) were reacted with polyclonal rabbit antibody to vIL-6. The preparation shown in A was used for experiments illustrated in panels C–E. (C) ELISA assay for the amount of vIL-6 present in supernatants of HKB5/B5 cells transfected with pcDNA3.1 or pcDNA3.1/vIL-6, treated or untreated with TM. (D) ELISA assay for binding of vIL-6 to sgp130. In one sample, vIL-6 expressed in the absence of TM was spiked with TM before assaying its binding to sgp130. (E) B9.11 assay of vIL-6 expressed in the absence of TM (solid bars), the presence of TM (shaded bars), or vIL-6 expressed in the presence of TM to which untreated vIL-6 was added (hatched bars).
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fig6: Effect of expression of vIL-6 in the presence of TM on binding to sgp130 and on B9.11 cell proliferation. (A and B) Electrophoretic mobility of vIL-6 expressed in HKB5/B5 cells in the absence or presence of 1.0 μg/ml TM. Immunoblots of culture supernatants (A) or cell extracts (B) were reacted with polyclonal rabbit antibody to vIL-6. The preparation shown in A was used for experiments illustrated in panels C–E. (C) ELISA assay for the amount of vIL-6 present in supernatants of HKB5/B5 cells transfected with pcDNA3.1 or pcDNA3.1/vIL-6, treated or untreated with TM. (D) ELISA assay for binding of vIL-6 to sgp130. In one sample, vIL-6 expressed in the absence of TM was spiked with TM before assaying its binding to sgp130. (E) B9.11 assay of vIL-6 expressed in the absence of TM (solid bars), the presence of TM (shaded bars), or vIL-6 expressed in the presence of TM to which untreated vIL-6 was added (hatched bars).

Mentions: To establish whether the impaired binding of the N89K and N78K/N89K mutants was due to alterations of glycosylation, we compared binding to sgp130 of wild-type vIL-6 expressed in the presence or absence of TM (Fig. 6). The effect of TM on the secreted vIL-6 preparation used for the experiments was to change it from a fully glycosylated to an unglycosylated form (Fig. 6, A and B). TM slightly reduced the amount of immunoreactive vIL-6 that was secreted, however, equal amounts of vIL-6 were used for the binding studies (Fig. 6 C). vIL-6 expressed in the presence of TM bound to sgp130 approximately two- to threefold less well than wild-type vIL-6 (Fig. 6 D). The extent of impairment in binding of vIL-6 expressed in the presence of TM (Fig. 6 D) was similar to the impairment in binding by the N89K and N78K/N89K mutants (Fig. 5 B). TM added in the binding assay to wild-type vIL-6 expressed in the absence of the inhibitor did not affect binding of vIL-6 to gp130 (Fig. 6 D).


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)

Effect of expression of vIL-6 in the presence of TM on binding to sgp130 and on B9.11 cell proliferation. (A and B) Electrophoretic mobility of vIL-6 expressed in HKB5/B5 cells in the absence or presence of 1.0 μg/ml TM. Immunoblots of culture supernatants (A) or cell extracts (B) were reacted with polyclonal rabbit antibody to vIL-6. The preparation shown in A was used for experiments illustrated in panels C–E. (C) ELISA assay for the amount of vIL-6 present in supernatants of HKB5/B5 cells transfected with pcDNA3.1 or pcDNA3.1/vIL-6, treated or untreated with TM. (D) ELISA assay for binding of vIL-6 to sgp130. In one sample, vIL-6 expressed in the absence of TM was spiked with TM before assaying its binding to sgp130. (E) B9.11 assay of vIL-6 expressed in the absence of TM (solid bars), the presence of TM (shaded bars), or vIL-6 expressed in the presence of TM to which untreated vIL-6 was added (hatched bars).
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Related In: Results  -  Collection

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fig6: Effect of expression of vIL-6 in the presence of TM on binding to sgp130 and on B9.11 cell proliferation. (A and B) Electrophoretic mobility of vIL-6 expressed in HKB5/B5 cells in the absence or presence of 1.0 μg/ml TM. Immunoblots of culture supernatants (A) or cell extracts (B) were reacted with polyclonal rabbit antibody to vIL-6. The preparation shown in A was used for experiments illustrated in panels C–E. (C) ELISA assay for the amount of vIL-6 present in supernatants of HKB5/B5 cells transfected with pcDNA3.1 or pcDNA3.1/vIL-6, treated or untreated with TM. (D) ELISA assay for binding of vIL-6 to sgp130. In one sample, vIL-6 expressed in the absence of TM was spiked with TM before assaying its binding to sgp130. (E) B9.11 assay of vIL-6 expressed in the absence of TM (solid bars), the presence of TM (shaded bars), or vIL-6 expressed in the presence of TM to which untreated vIL-6 was added (hatched bars).
Mentions: To establish whether the impaired binding of the N89K and N78K/N89K mutants was due to alterations of glycosylation, we compared binding to sgp130 of wild-type vIL-6 expressed in the presence or absence of TM (Fig. 6). The effect of TM on the secreted vIL-6 preparation used for the experiments was to change it from a fully glycosylated to an unglycosylated form (Fig. 6, A and B). TM slightly reduced the amount of immunoreactive vIL-6 that was secreted, however, equal amounts of vIL-6 were used for the binding studies (Fig. 6 C). vIL-6 expressed in the presence of TM bound to sgp130 approximately two- to threefold less well than wild-type vIL-6 (Fig. 6 D). The extent of impairment in binding of vIL-6 expressed in the presence of TM (Fig. 6 D) was similar to the impairment in binding by the N89K and N78K/N89K mutants (Fig. 5 B). TM added in the binding assay to wild-type vIL-6 expressed in the absence of the inhibitor did not affect binding of vIL-6 to gp130 (Fig. 6 D).

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