title: Proteolytic Origin of the Soluble Human IL-6R In Vivo and a Decisive Role of N-Glycosylation.
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.
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Riethmueller
authors.affiliation: Systematic Proteomics & Bioanalytics, Institute for Experimental Medicine, Kiel University, Kiel, Germany.
authors.forename: Prasath
authors.initials: P
authors.lastname: Somasundaram
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Johanna C
authors.initials: JC
authors.lastname: Ehlers
authors.affiliation: Systematic Proteomics & Bioanalytics, Institute for Experimental Medicine, Kiel University, Kiel, Germany.
authors.forename: Chien-Wen
authors.initials: CW
authors.lastname: Hung
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Charlotte M
authors.initials: CM
authors.lastname: Flynn
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Lokau
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Agthe
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Düsterhöft
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Yijue
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Grötzinger
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Inken
authors.initials: I
authors.lastname: Lorenzen
authors.affiliation: Systematic Proteomics & Bioanalytics, Institute for Experimental Medicine, Kiel University, Kiel, Germany.
authors.forename: Tomas
authors.initials: T
authors.lastname: Koudelka
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Pickhinke
authors.affiliation: Institute of Biochemistry, Unit for Degradomics of the Protease Web, Kiel University, Kiel, Germany.
authors.forename: Rielana
authors.initials: R
authors.lastname: Wichert
authors.affiliation: Institute of Biochemistry, Unit for Degradomics of the Protease Web, Kiel University, Kiel, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Becker-Pauly
authors.affiliation: Bioorganic Chemistry, Gebaeude NWI, University of Bayreuth, Bayreuth, Germany.
authors.forename: Marisa
authors.initials: M
authors.lastname: Rädisch
authors.affiliation: Bioorganic Chemistry, Gebaeude NWI, University of Bayreuth, Bayreuth, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Albrecht
authors.affiliation: Bioorganic Chemistry, Gebaeude NWI, University of Bayreuth, Bayreuth, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Hessefort
authors.affiliation: Bioorganic Chemistry, Gebaeude NWI, University of Bayreuth, Bayreuth, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Stahnke
authors.affiliation: Bioorganic Chemistry, Gebaeude NWI, University of Bayreuth, Bayreuth, Germany.
authors.forename: Carlo
authors.initials: C
authors.lastname: Unverzagt
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Rose-John
authors.affiliation: Systematic Proteomics & Bioanalytics, Institute for Experimental Medicine, Kiel University, Kiel, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
authors.affiliation: Institute of Biochemistry, Kiel University, Kiel, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Garbers
mesh_terms: D000072197:ADAM10 Protein / Q000378:metabolism; D000072198:ADAM17 Protein / Q000378:metabolism; D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D000072417:Protein Domains; D021381:Protein Transport; D059748:Proteolysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019947:Receptors, Interleukin-6 / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D012995:Solubility; D014633:Valine / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011134:Polysaccharides; D012333:RNA, Messenger; D019947:Receptors, Interleukin-6; D011392:Proline; D053829:Amyloid Precursor Protein Secretases; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; D014633:Valine