Proteolytic Origin of the Soluble Human IL-6R In Vivo and a Decisive Role of N-Glycosylation

PLoS Biol. 2017 Jan 6;15(1):e2000080. doi: 10.1371/journal.pbio.2000080. eCollection 2017 Jan.

Abstract

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADAM10 Protein / metabolism
  • ADAM17 Protein / metabolism
  • Alternative Splicing / genetics
  • Amino Acid Sequence
  • Amyloid Precursor Protein Secretases / metabolism
  • Cell Line
  • Cell Membrane / metabolism
  • Glycosylation
  • Humans
  • Intracellular Space / metabolism
  • Mass Spectrometry
  • Membrane Proteins / metabolism
  • Mutation / genetics
  • Polysaccharides / metabolism
  • Proline / metabolism
  • Protein Domains
  • Protein Transport
  • Proteolysis*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Receptors, Interleukin-6 / blood
  • Receptors, Interleukin-6 / chemistry
  • Receptors, Interleukin-6 / genetics
  • Receptors, Interleukin-6 / metabolism*
  • Signal Transduction
  • Solubility
  • Valine / metabolism

Substances

  • Membrane Proteins
  • Polysaccharides
  • RNA, Messenger
  • Receptors, Interleukin-6
  • Proline
  • Amyloid Precursor Protein Secretases
  • ADAM10 Protein
  • ADAM10 protein, human
  • ADAM17 Protein
  • ADAM17 protein, human
  • Valine

Grants and funding

Bundesministerium für Bildung und Forschung www.bmbf.de (grant number InTraSig, project B).Received by SRJ and CG. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Deutsche Forschungsgemeinschaft http://www.dfg.de/ (grant number SFB877 projects A1, A6, A9, A10 and Z2).A1 received by SRJ, A6 received by JG, A9 received by CBP, A10 received by CG, and Z2 received by AT. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Deutsche Forschungsgemeinschaft http://www.dfg.de/ (grant number Cluster of Excellence ‘Inflammation at Interfaces’).Received by SRJ, AT, and CG. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.