Complex N-linked glycans on Asn-89 of Kaposi sarcoma herpes virus-encoded interleukin-6 mediate optimal function by affecting cytokine protein conformation

J Biol Chem. 2009 Oct 23;284(43):29269-82. doi: 10.1074/jbc.M109.039115. Epub 2009 Aug 18.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibodies, Viral / pharmacology
  • Cell Line, Tumor
  • Cytokine Receptor gp130 / genetics
  • Cytokine Receptor gp130 / metabolism*
  • Glucosidases / genetics
  • Glucosidases / metabolism
  • Glycosylation
  • Herpesvirus 8, Human / genetics
  • Herpesvirus 8, Human / metabolism*
  • Humans
  • Interleukin-6 / genetics
  • Interleukin-6 / metabolism*
  • Mutation
  • Polysaccharides / genetics
  • Polysaccharides / metabolism*
  • Protein Binding / drug effects
  • Protein Binding / genetics
  • Protein Folding*
  • Protein Structure, Tertiary / drug effects
  • Receptors, Interleukin-6 / genetics
  • Receptors, Interleukin-6 / metabolism*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • Antibodies, Viral
  • IL6 protein, human
  • IL6R protein, human
  • IL6ST protein, human
  • Interleukin-6
  • Polysaccharides
  • Receptors, Interleukin-6
  • Viral Proteins
  • interleukin-6, RRV-HHV-8
  • Cytokine Receptor gp130
  • Glucosidases