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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

N89 of KSHV IL-6 is required for optimal function in a B9.11 cell cytokine–dependent proliferation assay. Two-fold serially diluted supernatants, containing 25 ng/ml vIL-6 protein as determined by vIL-6–specific ELISA, derived from HKB5/B5 cells transfected with plasmids encoding wild-type vIL-6, or mutants N78K, N89K, and N78K/N89K were assessed for their capacity to stimulate proliferation of B9.11 cells. Cells were pulsed with tritiated thymidine from 48 to 64 h after addition of the supernatants, and radioactivity incorporation (cpm) was determined.
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fig4: N89 of KSHV IL-6 is required for optimal function in a B9.11 cell cytokine–dependent proliferation assay. Two-fold serially diluted supernatants, containing 25 ng/ml vIL-6 protein as determined by vIL-6–specific ELISA, derived from HKB5/B5 cells transfected with plasmids encoding wild-type vIL-6, or mutants N78K, N89K, and N78K/N89K were assessed for their capacity to stimulate proliferation of B9.11 cells. Cells were pulsed with tritiated thymidine from 48 to 64 h after addition of the supernatants, and radioactivity incorporation (cpm) was determined.

Mentions: To determine whether specific N-linked glycosylation of vIL-6 was required for its biological function and could explain the nonoptimal function of nonglycosylated, bacterially produced vIL-6, supernatants from HKB5/B5 cells transfected with different vIL-6 N-linked glycosylation mutant constructs containing equal amounts of immunoreactive vIL-6 were assessed for their capacity to stimulate B9.11 cell growth. By comparison to wild-type vIL-6 and the N78K mutant, the N89K and N78K/N89K double mutants of vIL-6 were 16-fold impaired in their ability to stimulate proliferation of B9.11 cells (Fig. 4). This result suggested that N-linked glycosylation at site N89 was important for vIL-6 function, whereas N78 site was not essential. Another interpretation of this result was that the N89K mutation created a form of vIL-6 that inhibited B9.11 cell proliferation. Experiments were performed in which N89K vIL-6 protein was combined with wild-type vIL-6 to determine whether N89K acted as a dominant negative mutant. An additive effect on B9.11 cell proliferation was observed when N89K vIL-6 supernatants were mixed with wild-type vIL-6 (unpublished data). Similarly, HKB5/B5 cells were cotransfected with N89K vIL-6 and wild-type vIL-6 plasmids to determine if N89K mutant vIL-6 could block wild-type vIL-6 intracellularly. Results of B9.11 cell proliferation assays using supernatants from the cotransfected cells showed no inhibition of wild-type vIL-6 activity by the mutant N89K form (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)

N89 of KSHV IL-6 is required for optimal function in a B9.11 cell cytokine–dependent proliferation assay. Two-fold serially diluted supernatants, containing 25 ng/ml vIL-6 protein as determined by vIL-6–specific ELISA, derived from HKB5/B5 cells transfected with plasmids encoding wild-type vIL-6, or mutants N78K, N89K, and N78K/N89K were assessed for their capacity to stimulate proliferation of B9.11 cells. Cells were pulsed with tritiated thymidine from 48 to 64 h after addition of the supernatants, and radioactivity incorporation (cpm) was determined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211829&req=5

fig4: N89 of KSHV IL-6 is required for optimal function in a B9.11 cell cytokine–dependent proliferation assay. Two-fold serially diluted supernatants, containing 25 ng/ml vIL-6 protein as determined by vIL-6–specific ELISA, derived from HKB5/B5 cells transfected with plasmids encoding wild-type vIL-6, or mutants N78K, N89K, and N78K/N89K were assessed for their capacity to stimulate proliferation of B9.11 cells. Cells were pulsed with tritiated thymidine from 48 to 64 h after addition of the supernatants, and radioactivity incorporation (cpm) was determined.
Mentions: To determine whether specific N-linked glycosylation of vIL-6 was required for its biological function and could explain the nonoptimal function of nonglycosylated, bacterially produced vIL-6, supernatants from HKB5/B5 cells transfected with different vIL-6 N-linked glycosylation mutant constructs containing equal amounts of immunoreactive vIL-6 were assessed for their capacity to stimulate B9.11 cell growth. By comparison to wild-type vIL-6 and the N78K mutant, the N89K and N78K/N89K double mutants of vIL-6 were 16-fold impaired in their ability to stimulate proliferation of B9.11 cells (Fig. 4). This result suggested that N-linked glycosylation at site N89 was important for vIL-6 function, whereas N78 site was not essential. Another interpretation of this result was that the N89K mutation created a form of vIL-6 that inhibited B9.11 cell proliferation. Experiments were performed in which N89K vIL-6 protein was combined with wild-type vIL-6 to determine whether N89K acted as a dominant negative mutant. An additive effect on B9.11 cell proliferation was observed when N89K vIL-6 supernatants were mixed with wild-type vIL-6 (unpublished data). Similarly, HKB5/B5 cells were cotransfected with N89K vIL-6 and wild-type vIL-6 plasmids to determine if N89K mutant vIL-6 could block wild-type vIL-6 intracellularly. Results of B9.11 cell proliferation assays using supernatants from the cotransfected cells showed no inhibition of wild-type vIL-6 activity by the mutant N89K form (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