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

Steffen Riethmueller; Prasath Somasundaram; Johanna C Ehlers; Chien-Wen Hung; Charlotte M Flynn; Juliane Lokau; Maria Agthe; Stefan Düsterhöft; Yijue Zhu; Joachim Grötzinger; Inken Lorenzen; Tomas Koudelka; Kosuke Yamamoto; Ute Pickhinke; Rielana Wichert; Christoph Becker-Pauly; Marisa Rädisch; Alexander Albrecht; Markus Hessefort; Dominik Stahnke; Carlo Unverzagt; Stefan Rose-John; Andreas Tholey; Christoph Garbers

doi:10.1371/journal.pbio.2000080

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.

Authors

Steffen Riethmueller¹, Prasath Somasundaram², Johanna C Ehlers¹, Chien-Wen Hung², Charlotte M Flynn¹, Juliane Lokau¹, Maria Agthe¹, Stefan Düsterhöft¹, Yijue Zhu¹, Joachim Grötzinger¹, Inken Lorenzen¹, Tomas Koudelka², Kosuke Yamamoto¹, Ute Pickhinke¹, Rielana Wichert³, Christoph Becker-Pauly³, Marisa Rädisch⁴, Alexander Albrecht⁴, Markus Hessefort⁴, Dominik Stahnke⁴, Carlo Unverzagt⁴, Stefan Rose-John¹, Andreas Tholey², Christoph Garbers¹

Affiliations

¹ Institute of Biochemistry, Kiel University, Kiel, Germany.
² Systematic Proteomics & Bioanalytics, Institute for Experimental Medicine, Kiel University, Kiel, Germany.
³ Institute of Biochemistry, Unit for Degradomics of the Protease Web, Kiel University, Kiel, Germany.
⁴ Bioorganic Chemistry, Gebaeude NWI, University of Bayreuth, Bayreuth, Germany.

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.