Limits...
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.

Show MeSH

Related in: MedlinePlus

Location of the N-linked glycosylation sites of KSHV-encoded IL-6 on the schematic representation of the cocrystal structure of vIL-6 and gp130. Carbon backbone model of vIL-6 and soluble gp130 is shown as a tetrameric complex based on previous crystal structure work, shown in side (A) and tilted view (B; reference 11). A shows site I of vIL-6, which is not a contact site with gp130, but corresponds to a site where hIL-6 interacts with IL6-Rα. B shows one vIL-6 site II (black dotted circle) in contact with D2D3 sites of gp130 molecule (green), and vIL-6 site III (red dotted circle) in contact with D1 site of a second gp130 molecule (blue). N78 (yellow) and N89 (red) are highlighted as space-filling residues and are shown to occupy site I, but not sites II or III. The figure was prepared using the RasMol program (v2.7.1).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211829&req=5

fig12: Location of the N-linked glycosylation sites of KSHV-encoded IL-6 on the schematic representation of the cocrystal structure of vIL-6 and gp130. Carbon backbone model of vIL-6 and soluble gp130 is shown as a tetrameric complex based on previous crystal structure work, shown in side (A) and tilted view (B; reference 11). A shows site I of vIL-6, which is not a contact site with gp130, but corresponds to a site where hIL-6 interacts with IL6-Rα. B shows one vIL-6 site II (black dotted circle) in contact with D2D3 sites of gp130 molecule (green), and vIL-6 site III (red dotted circle) in contact with D1 site of a second gp130 molecule (blue). N78 (yellow) and N89 (red) are highlighted as space-filling residues and are shown to occupy site I, but not sites II or III. The figure was prepared using the RasMol program (v2.7.1).

Mentions: The crystal structure of vIL-6 in a complex with the gp130 receptor has been solved (11) and may help to distinguish these alternatives. To achieve the 2.4 Å X-ray cocrystal structure resolution, it was necessary to use nonglycosylated forms of the vIL-6 and gp130 proteins produced in insect cells in the presence of the inhibitor TM. Therefore, N-linked glycosylation is not absolutely required for vIL-6 and gp130 interaction. The complex assumes a tetrameric arrangement comprising two vIL-6 proteins and two human gp130 receptors. Each vIL-6 contacts two different gp130 molecules and each gp130 interacts with two vIL-6 molecules, all through structurally distinct binding surfaces (Fig. 12; reference 11). Residues in vIL-6 designated site II interact with domains 2 and 3 (D2D3) of gp130 comprising a cytokine-binding homology region. A second vIL-6 region designated site III interacts with the NH2-terminal D1 Ig-like activation domain of a second gp130 molecule. These interactions are required for assembly of a higher order activation complex important for signal transduction (28, 29). It is important to note that the two N-linked glycosylation sites of vIL-6 are located close to each other, but are outside of sites II and III important for cytokine–gp130 receptor interaction (Fig. 12).


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)

Location of the N-linked glycosylation sites of KSHV-encoded IL-6 on the schematic representation of the cocrystal structure of vIL-6 and gp130. Carbon backbone model of vIL-6 and soluble gp130 is shown as a tetrameric complex based on previous crystal structure work, shown in side (A) and tilted view (B; reference 11). A shows site I of vIL-6, which is not a contact site with gp130, but corresponds to a site where hIL-6 interacts with IL6-Rα. B shows one vIL-6 site II (black dotted circle) in contact with D2D3 sites of gp130 molecule (green), and vIL-6 site III (red dotted circle) in contact with D1 site of a second gp130 molecule (blue). N78 (yellow) and N89 (red) are highlighted as space-filling residues and are shown to occupy site I, but not sites II or III. The figure was prepared using the RasMol program (v2.7.1).
© Copyright Policy
Related In: Results  -  Collection

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

fig12: Location of the N-linked glycosylation sites of KSHV-encoded IL-6 on the schematic representation of the cocrystal structure of vIL-6 and gp130. Carbon backbone model of vIL-6 and soluble gp130 is shown as a tetrameric complex based on previous crystal structure work, shown in side (A) and tilted view (B; reference 11). A shows site I of vIL-6, which is not a contact site with gp130, but corresponds to a site where hIL-6 interacts with IL6-Rα. B shows one vIL-6 site II (black dotted circle) in contact with D2D3 sites of gp130 molecule (green), and vIL-6 site III (red dotted circle) in contact with D1 site of a second gp130 molecule (blue). N78 (yellow) and N89 (red) are highlighted as space-filling residues and are shown to occupy site I, but not sites II or III. The figure was prepared using the RasMol program (v2.7.1).
Mentions: The crystal structure of vIL-6 in a complex with the gp130 receptor has been solved (11) and may help to distinguish these alternatives. To achieve the 2.4 Å X-ray cocrystal structure resolution, it was necessary to use nonglycosylated forms of the vIL-6 and gp130 proteins produced in insect cells in the presence of the inhibitor TM. Therefore, N-linked glycosylation is not absolutely required for vIL-6 and gp130 interaction. The complex assumes a tetrameric arrangement comprising two vIL-6 proteins and two human gp130 receptors. Each vIL-6 contacts two different gp130 molecules and each gp130 interacts with two vIL-6 molecules, all through structurally distinct binding surfaces (Fig. 12; reference 11). Residues in vIL-6 designated site II interact with domains 2 and 3 (D2D3) of gp130 comprising a cytokine-binding homology region. A second vIL-6 region designated site III interacts with the NH2-terminal D1 Ig-like activation domain of a second gp130 molecule. These interactions are required for assembly of a higher order activation complex important for signal transduction (28, 29). It is important to note that the two N-linked glycosylation sites of vIL-6 are located close to each other, but are outside of sites II and III important for cytokine–gp130 receptor interaction (Fig. 12).

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