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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 of hIL-6. (A) Electrophoretic mobility of wild-type hIL-6 and two N-linked glycosylation site mutants of hIL-6 expressed in HKB5/B5 cells. (B) Effect of TM on electrophoretic mobility. HKB5/B5 cells were transfected with pcDNA3.1/hIL-6 in the presence of increasing concentrations of TM (lanes 1–3) or no TM (lane 4). Both immunoblots were reacted with polyclonal antibody to hIL-6.
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fig8: N-linked glycosylation of hIL-6. (A) Electrophoretic mobility of wild-type hIL-6 and two N-linked glycosylation site mutants of hIL-6 expressed in HKB5/B5 cells. (B) Effect of TM on electrophoretic mobility. HKB5/B5 cells were transfected with pcDNA3.1/hIL-6 in the presence of increasing concentrations of TM (lanes 1–3) or no TM (lane 4). Both immunoblots were reacted with polyclonal antibody to hIL-6.

Mentions: Human cellular IL-6 is known to be N-linked and O-linked glycosylated, but the functional importance of the N-linked glycosylation sites is not known. There are two N-linked glycosylation consensus sites at N73 and N172. Single (N73K and N172K) and double (N73K/N172K) mutants of hIL-6 proteins were generated by site-directed mutagenesis. The wild-type hIL-6 and mutant proteins produced in HKB5/B5 cells were analyzed on Western blots (Fig. 8). Unlike vIL-6 in which the large 28-kD form expressed in HKB5/B5 cells was predominant (Fig. 2 B), the predominant form of secreted hIL-6 was the broad 21-kD form. The electrophoretic mobility of N172K mutant was identical to wild-type. Mutants N73K and N73K/N172K expressed only the 21-kD form of the protein. Furthermore, expression of hIL-6 in the presence of TM (Fig. 8 B) eliminated the 27-kD form. These experiments indicated that hIL-6 contained a single functional N-linked glycosylation site at N73.


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 of hIL-6. (A) Electrophoretic mobility of wild-type hIL-6 and two N-linked glycosylation site mutants of hIL-6 expressed in HKB5/B5 cells. (B) Effect of TM on electrophoretic mobility. HKB5/B5 cells were transfected with pcDNA3.1/hIL-6 in the presence of increasing concentrations of TM (lanes 1–3) or no TM (lane 4). Both immunoblots were reacted with polyclonal antibody to hIL-6.
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Related In: Results  -  Collection

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

fig8: N-linked glycosylation of hIL-6. (A) Electrophoretic mobility of wild-type hIL-6 and two N-linked glycosylation site mutants of hIL-6 expressed in HKB5/B5 cells. (B) Effect of TM on electrophoretic mobility. HKB5/B5 cells were transfected with pcDNA3.1/hIL-6 in the presence of increasing concentrations of TM (lanes 1–3) or no TM (lane 4). Both immunoblots were reacted with polyclonal antibody to hIL-6.
Mentions: Human cellular IL-6 is known to be N-linked and O-linked glycosylated, but the functional importance of the N-linked glycosylation sites is not known. There are two N-linked glycosylation consensus sites at N73 and N172. Single (N73K and N172K) and double (N73K/N172K) mutants of hIL-6 proteins were generated by site-directed mutagenesis. The wild-type hIL-6 and mutant proteins produced in HKB5/B5 cells were analyzed on Western blots (Fig. 8). Unlike vIL-6 in which the large 28-kD form expressed in HKB5/B5 cells was predominant (Fig. 2 B), the predominant form of secreted hIL-6 was the broad 21-kD form. The electrophoretic mobility of N172K mutant was identical to wild-type. Mutants N73K and N73K/N172K expressed only the 21-kD form of the protein. Furthermore, expression of hIL-6 in the presence of TM (Fig. 8 B) eliminated the 27-kD form. These experiments indicated that hIL-6 contained a single functional N-linked glycosylation site at N73.

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