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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 TM on electrophoretic mobility of vIL-6. HKB5/B5 cells were transfected with wild-type or N-linked glycosylation site mutants of vIL-6. The cells were untreated (lanes 2, 5, 8, 12, and 15) or received 0.2 (lanes 3, 6, 9,13, and 16) or 1.0 μg/ml (lanes 4, 7, 11, 14, and 17) TM. Lanes 1 and 10, HH-B2 cell extracts; lanes 2–4, vIL-6; lanes 5–7, N78K/N89K; lanes 8, 9, and 11, pcDNA3; lanes 12–14, N78K; lanes 15–17, N89K.
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fig3: Effect of TM on electrophoretic mobility of vIL-6. HKB5/B5 cells were transfected with wild-type or N-linked glycosylation site mutants of vIL-6. The cells were untreated (lanes 2, 5, 8, 12, and 15) or received 0.2 (lanes 3, 6, 9,13, and 16) or 1.0 μg/ml (lanes 4, 7, 11, 14, and 17) TM. Lanes 1 and 10, HH-B2 cell extracts; lanes 2–4, vIL-6; lanes 5–7, N78K/N89K; lanes 8, 9, and 11, pcDNA3; lanes 12–14, N78K; lanes 15–17, N89K.

Mentions: To confirm that the differences in electrophoretic mobility of wild-type vIL-6 and mutant forms of vIL-6 were the result of N-linked glycosylation, the proteins were expressed in HKB5/B5 cells treated with TM (Fig. 3). When wild-type vIL-6 was produced in the presence of TM, the mobility of the vIL-6 polypeptide shifted from a predominantly 28-kD form (Fig. 3, lane 2) to a 22-kD form (Fig. 3, lanes 3 and 4), which comigrated with the N78K/N89K double mutant (Fig. 3, lane 5). TM treatment of cells expressing the single glycosylation site mutants, N78K and N89K, resulted in a change in electrophoretic mobility from a predominant 25-kD form to the 22-kD form. TM treatment did not affect the electrophoretic mobility of vIL-6 expressed by the mutant N78K/N89K (Fig. 3, lanes 5–7). These results showed that in wild-type vIL-6, both N78 and N89 were N-linked glycosylated. The predominant 28-kD protein represents the doubly N-linked glycosylated form of vIL-6, the 25-kD protein represents the two singly N-linked glycosylated species, and the 22-kD protein represents a non-N–linked glycosylated form of vIL-6 protein.


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 TM on electrophoretic mobility of vIL-6. HKB5/B5 cells were transfected with wild-type or N-linked glycosylation site mutants of vIL-6. The cells were untreated (lanes 2, 5, 8, 12, and 15) or received 0.2 (lanes 3, 6, 9,13, and 16) or 1.0 μg/ml (lanes 4, 7, 11, 14, and 17) TM. Lanes 1 and 10, HH-B2 cell extracts; lanes 2–4, vIL-6; lanes 5–7, N78K/N89K; lanes 8, 9, and 11, pcDNA3; lanes 12–14, N78K; lanes 15–17, N89K.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Effect of TM on electrophoretic mobility of vIL-6. HKB5/B5 cells were transfected with wild-type or N-linked glycosylation site mutants of vIL-6. The cells were untreated (lanes 2, 5, 8, 12, and 15) or received 0.2 (lanes 3, 6, 9,13, and 16) or 1.0 μg/ml (lanes 4, 7, 11, 14, and 17) TM. Lanes 1 and 10, HH-B2 cell extracts; lanes 2–4, vIL-6; lanes 5–7, N78K/N89K; lanes 8, 9, and 11, pcDNA3; lanes 12–14, N78K; lanes 15–17, N89K.
Mentions: To confirm that the differences in electrophoretic mobility of wild-type vIL-6 and mutant forms of vIL-6 were the result of N-linked glycosylation, the proteins were expressed in HKB5/B5 cells treated with TM (Fig. 3). When wild-type vIL-6 was produced in the presence of TM, the mobility of the vIL-6 polypeptide shifted from a predominantly 28-kD form (Fig. 3, lane 2) to a 22-kD form (Fig. 3, lanes 3 and 4), which comigrated with the N78K/N89K double mutant (Fig. 3, lane 5). TM treatment of cells expressing the single glycosylation site mutants, N78K and N89K, resulted in a change in electrophoretic mobility from a predominant 25-kD form to the 22-kD form. TM treatment did not affect the electrophoretic mobility of vIL-6 expressed by the mutant N78K/N89K (Fig. 3, lanes 5–7). These results showed that in wild-type vIL-6, both N78 and N89 were N-linked glycosylated. The predominant 28-kD protein represents the doubly N-linked glycosylated form of vIL-6, the 25-kD protein represents the two singly N-linked glycosylated species, and the 22-kD protein represents a non-N–linked glycosylated form of vIL-6 protein.

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