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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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Comparison of vIL-6 expressed in E. coli and in eukaryotic cells. (A) Comparison of electrophoretic mobility of vIL-6 from various sources. An immunoblot reacted with polyclonal rabbit antibody to vIL-6. Lane 1, extract of HKB5 cells transfected with pcDNA3.1; lane 2, extract of HKB5/B5 cells transfected with pcDNA3.1/vIL-6; lane 3, purified vIL-6 expressed in E. coli; lane 4, extracts of HHB2 cells, a KSHV+ PEL cell line; lane 5, extract of HH514-16 (clone16), an EBV+ KSHV− Burkitt lymphoma cell line. (B) Comparison of biological activity of vIL-6–expressed mammalian cells or E. coli in a B9.11 cell proliferation assay. B9.11 cells were incubated with supernatants of HKB5/B5 cells transfected with pcDNA3.1 or pcDNA3.1/vIL-6 (BC-1). B9.11 cells were also incubated with vIL-6 from three KSHV strains cloned in pET22, expressed in E. coli, and purified on Ni+1 columns. The concentration of vIL-6 expressed in eukaryotic cells was 25 ng/ml. The concentration of vIL-6 expressed in E. coli varied from 17 to 147 μg/ml.
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fig1: Comparison of vIL-6 expressed in E. coli and in eukaryotic cells. (A) Comparison of electrophoretic mobility of vIL-6 from various sources. An immunoblot reacted with polyclonal rabbit antibody to vIL-6. Lane 1, extract of HKB5 cells transfected with pcDNA3.1; lane 2, extract of HKB5/B5 cells transfected with pcDNA3.1/vIL-6; lane 3, purified vIL-6 expressed in E. coli; lane 4, extracts of HHB2 cells, a KSHV+ PEL cell line; lane 5, extract of HH514-16 (clone16), an EBV+ KSHV− Burkitt lymphoma cell line. (B) Comparison of biological activity of vIL-6–expressed mammalian cells or E. coli in a B9.11 cell proliferation assay. B9.11 cells were incubated with supernatants of HKB5/B5 cells transfected with pcDNA3.1 or pcDNA3.1/vIL-6 (BC-1). B9.11 cells were also incubated with vIL-6 from three KSHV strains cloned in pET22, expressed in E. coli, and purified on Ni+1 columns. The concentration of vIL-6 expressed in eukaryotic cells was 25 ng/ml. The concentration of vIL-6 expressed in E. coli varied from 17 to 147 μg/ml.

Mentions: To study the function of KSHV IL-6, the protein was prepared from both bacterial and mammalian sources. vIL-6 was cloned with a six-residue histidine tag into the pET-22 vector and purified from E. coli using a nickel column. vIL-6 was also cloned into the mammalian vector pcDNA3.1 and expressed in human HKB5/B5 cells. Western blot analysis using polyclonal rabbit antiserum to vIL-6 revealed differences in electrophoretic mobility of vIL-6 depending on the source of the protein. There were three major immunoreactive protein products present in extracts of HKB5/B5 cells transfected with vIL-6 (Fig. 1 A, lane 2). The largest and most abundant of these products, ∼28 kD, comigrated with vIL-6 expressed during the KSHV lytic cycle in HHB2 cells, a KSHV-infected PEL cell line (Fig. 1 A, lane 4). An EBV-infected Burkitt lymphoma cell line contained no immunoreactive vIL-6 (Fig. 1 A, lane 5). E. coli–produced His-tagged vIL-6 protein (Fig. 1 A, lane 3) showed one predominant polypeptide, ∼21 kD, which was slightly larger in molecular weight than the smallest form of vIL-6 present in human cells (Fig. 1 A, lane 2). We postulated that the electrophoretic mobility of the E. coli–expressed vIL-6 could be accounted for by an unmodified form of the protein containing additional residues from the histidine tags.


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)

Comparison of vIL-6 expressed in E. coli and in eukaryotic cells. (A) Comparison of electrophoretic mobility of vIL-6 from various sources. An immunoblot reacted with polyclonal rabbit antibody to vIL-6. Lane 1, extract of HKB5 cells transfected with pcDNA3.1; lane 2, extract of HKB5/B5 cells transfected with pcDNA3.1/vIL-6; lane 3, purified vIL-6 expressed in E. coli; lane 4, extracts of HHB2 cells, a KSHV+ PEL cell line; lane 5, extract of HH514-16 (clone16), an EBV+ KSHV− Burkitt lymphoma cell line. (B) Comparison of biological activity of vIL-6–expressed mammalian cells or E. coli in a B9.11 cell proliferation assay. B9.11 cells were incubated with supernatants of HKB5/B5 cells transfected with pcDNA3.1 or pcDNA3.1/vIL-6 (BC-1). B9.11 cells were also incubated with vIL-6 from three KSHV strains cloned in pET22, expressed in E. coli, and purified on Ni+1 columns. The concentration of vIL-6 expressed in eukaryotic cells was 25 ng/ml. The concentration of vIL-6 expressed in E. coli varied from 17 to 147 μg/ml.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211829&req=5

fig1: Comparison of vIL-6 expressed in E. coli and in eukaryotic cells. (A) Comparison of electrophoretic mobility of vIL-6 from various sources. An immunoblot reacted with polyclonal rabbit antibody to vIL-6. Lane 1, extract of HKB5 cells transfected with pcDNA3.1; lane 2, extract of HKB5/B5 cells transfected with pcDNA3.1/vIL-6; lane 3, purified vIL-6 expressed in E. coli; lane 4, extracts of HHB2 cells, a KSHV+ PEL cell line; lane 5, extract of HH514-16 (clone16), an EBV+ KSHV− Burkitt lymphoma cell line. (B) Comparison of biological activity of vIL-6–expressed mammalian cells or E. coli in a B9.11 cell proliferation assay. B9.11 cells were incubated with supernatants of HKB5/B5 cells transfected with pcDNA3.1 or pcDNA3.1/vIL-6 (BC-1). B9.11 cells were also incubated with vIL-6 from three KSHV strains cloned in pET22, expressed in E. coli, and purified on Ni+1 columns. The concentration of vIL-6 expressed in eukaryotic cells was 25 ng/ml. The concentration of vIL-6 expressed in E. coli varied from 17 to 147 μg/ml.
Mentions: To study the function of KSHV IL-6, the protein was prepared from both bacterial and mammalian sources. vIL-6 was cloned with a six-residue histidine tag into the pET-22 vector and purified from E. coli using a nickel column. vIL-6 was also cloned into the mammalian vector pcDNA3.1 and expressed in human HKB5/B5 cells. Western blot analysis using polyclonal rabbit antiserum to vIL-6 revealed differences in electrophoretic mobility of vIL-6 depending on the source of the protein. There were three major immunoreactive protein products present in extracts of HKB5/B5 cells transfected with vIL-6 (Fig. 1 A, lane 2). The largest and most abundant of these products, ∼28 kD, comigrated with vIL-6 expressed during the KSHV lytic cycle in HHB2 cells, a KSHV-infected PEL cell line (Fig. 1 A, lane 4). An EBV-infected Burkitt lymphoma cell line contained no immunoreactive vIL-6 (Fig. 1 A, lane 5). E. coli–produced His-tagged vIL-6 protein (Fig. 1 A, lane 3) showed one predominant polypeptide, ∼21 kD, which was slightly larger in molecular weight than the smallest form of vIL-6 present in human cells (Fig. 1 A, lane 2). We postulated that the electrophoretic mobility of the E. coli–expressed vIL-6 could be accounted for by an unmodified form of the protein containing additional residues from the histidine tags.

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