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

• Published in: PLoS biology 2017-01, Vol.15 (1), p.e2000080-e2000080
• Main Authors: Riethmueller, Steffen, Somasundaram, Prasath, Ehlers, Johanna C, Hung, Chien-Wen, Flynn, Charlotte M, Lokau, Juliane, Agthe, Maria, Düsterhöft, Stefan, Zhu, Yijue, Grötzinger, Joachim, Lorenzen, Inken, Koudelka, Tomas, Yamamoto, Kosuke, Pickhinke, Ute, Wichert, Rielana, Becker-Pauly, Christoph, Rädisch, Marisa, Albrecht, Alexander, Hessefort, Markus, Stahnke, Dominik, Unverzagt, Carlo, Rose-John, Stefan, Tholey, Andreas, Garbers, Christoph
• Format: Article
• Language: English
• Subjects: ADAM10 Protein - metabolism; ADAM17 Protein - metabolism; Alternative Splicing - genetics; Amino Acid Sequence; Amyloid Precursor Protein Secretases - metabolism; Biology and Life Sciences; Cell Line; Cell Membrane - metabolism; Colleges & universities; Cytokines; Editing; Experiments; Funding; Glycosylation; Humans; Interleukins; Intracellular Space - metabolism; Mass Spectrometry; Medical research; Medicine; Medicine and Health Sciences; Membrane Proteins - metabolism; Mutation - genetics; Observations; Physiological aspects; Plasmids; Polysaccharides - metabolism; Proline - metabolism; Protein Domains; Protein Transport; Proteolysis; Proteomics; Receptors, Interleukin-6 - blood; Receptors, Interleukin-6 - chemistry; Receptors, Interleukin-6 - genetics; Receptors, Interleukin-6 - metabolism; Research and Analysis Methods; RNA, Messenger - genetics; RNA, Messenger - metabolism; Roles; Signal Transduction; Solubility; Supervision; Valine - metabolism; Writing
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.2000080

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.