title: Complex N-linked glycans on Asn-89 of Kaposi sarcoma herpes virus-encoded interleukin-6 mediate optimal function by affecting cytokine protein conformation.
abstract: Kaposi sarcoma-associated herpesvirus-encoded interleukin-6 (vIL-6) and its human cellular homologue (huIL-6) share similar biological functions. Our previous work showed that N-linked glycosylation was required for optimal function of vIL6 but not huIL-6 (1). Here we describe heterogeneity in the composition of the glycans of the two N-linked sites of vIL-6. The Asn-89 site of vIL-6, found to be required for optimal cytokine function, is composed of complex glycans. The Asn-78 site is composed of high mannose glycans, which are dispensable for cytokine function. N-Linked glycosylation at the Asn-89 site was required for intracellular production of functional vIL-6, but endoglycosidase-mediated removal of N-linked glycans from secreted vIL-6 did not impair protein function. With the use of a conformation-specific antibody and tryptic digestion assays, we showed that glycosylation at the Asn-89 site of vIL-6 affected protein conformation. Human IL-6, but not vIL-6, requires IL-6Ralpha for binding to gp130. We tested the hypothesis that the Asn-89 complex glycan of vIL-6 alone was sufficient to confer binding to gp130 independently of IL-6Ralpha. Two mutants of huIL-6, made to contain additional complex N-linked glycans in the region that interacts with IL-6Ralpha, did not confer binding to gp130 independently of IL-6Ralpha. Our findings support the conclusion that complex glycans on Asn-89 of vIL-6 specifically promote a protein conformation that allows the viral cytokine to bind gp130 independently of IL-6Ralpha.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Dela Cruz
authors.forename: Srinivas R
authors.initials: SR
authors.lastname: Viswanathan
authors.forename: Ayman S
authors.initials: AS
authors.lastname: El-Guindy
authors.forename: Duane
authors.initials: D
authors.lastname: Shedd
authors.forename: George
authors.initials: G
authors.lastname: Miller
mesh_terms: D000914:Antibodies, Viral / Q000494:pharmacology; D045744:Cell Line, Tumor; D050822:Cytokine Receptor gp130 / Q000235:genetics / Q000378:metabolism*; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D019947:Receptors, Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000914:Antibodies, Viral; C508600:IL6 protein, human; C508603:IL6R protein, human; C494199:IL6ST protein, human; D015850:Interleukin-6; D011134:Polysaccharides; D019947:Receptors, Interleukin-6; D014764:Viral Proteins; C120140:interleukin-6, RRV-HHV-8; D050822:Cytokine Receptor gp130; D005959:Glucosidases
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