Fig 2: Determination of the N-glycosylation sites on the sIL-6R in vitro and in vivo via MS.(A) HEK293 cells were transiently transfected with an expression plasmid encoding IL-6R, and cells were lysed 48 h later. Lysates were treated with either PNGase F or Deglyco-Mix or were left untreated. Proteins were resolved on a 10% SDS gel and analyzed by western blot. One representative experiment out of two performed is shown. (B) HEK293 cells were transiently transfected with an expression plasmid encoding IL-6R, and cells were stimulated after 48 h with 100 nM PMA for 2 h. The sIL-6R was precipitated from the cell supernatant and afterwards treated and analyzed as described under panel (A). (C–F) MS/MS spectra (HCD) of the different peptides of the sIL-6R that contained N-linked glycans. The four glycosylation sites (Asn-55, Asn-93, Asn-221, and Asn-245) were modified to aspartic acid residues due to PNGase F treatment in the presence of H218O-containing buffer and are shown in green. The use of Asn-350 as an N-glycosylation site was already shown in Fig 2C and 2D. Peptides were identified via database searching. (G) Schematic overview of the different domains of the IL-6R and the localization of the five N-glycosylation sites (green) and one O-glycosylation site (orange). D, domain; ICD, intracellular domain; S, stalk region; SP, signal peptide; TM, transmembrane region.

Fig 3: N- and O-linked glycosylation are dispensable for intracellular transport and signaling of the IL-6R.(A–G) Equal numbers of Ba/F3-gp130 cells were incubated for 48 h with increasing amounts (0–100 ng/ml) of either IL-6 or Hyper-IL-6. The stably transduced IL-6R variant is indicated above the diagram. One representative experiment out of three performed is shown (mean ± SD, biological triplicates). (H) Equal numbers of Ba/F3-gp130-hIL-6R and Ba/F3-gp130-hIL-6R-4N cells were labeled with an anti-IL-6R antibody and incubated at 37°C for the indicated time periods. The remaining cell-surface expression of the IL-6R was analyzed via flow cytometry. Data shown are the mean ± SD from three independent experiments.

Fig 4: Identification of the ds-sIL-6R and a novel protease-derived sIL-6R from human serum via mass spectrometry (MS).(A) Schematic illustration of the procedure to precipitate total sIL-6R from human serum. A representative coomassie-stained sodium dodecyl sulfate (SDS) gel and a western blot of the precipitated protein are shown on the right. The SDS gels were run under nonreducing conditions (see Materials and Methods for details), and the region of the SDS gel excised for MS is indicated with a red box. The precipitated sIL-6R is detected with an antibody that specifically recognizes the ectodomain of the human IL-6R (4–11). (B) Proteomics workflow including disulfide bond reduction, thiol alkylation, enzymatic N-deglycosylation, proteolysis in presence of 50% H218O, LC-MS/MS analysis, and data interpretation. (C) MS/MS spectrum (higher-energy collisional dissociation [HCD]) of the C-terminal peptide of the ds-sIL-6R identified via database searching. The N-glycan site Asn-350, which is modified to an Asp-350 because of PNGase F treatment, is shown in green. (D) MS/MS spectrum (electron-transfer dissociation [ETD]) of the C-terminal peptide of the protease-derived sIL-6R identified via database searching. The N-glycan site Asn-350, which is modified to an Asp-350 because of PNGase F treatment, is shown in green. One of the identified O-glycan structures on Thr-352 is shown.

Fig 5: ADAM17 and ADAM10 cleave the IL-6R between Pro-355 and Val-356 in vitro.(A) A schematic illustration, a representative coomassie-stained SDS gel, and a western blot of the precipitated protein after PMA-induced ADAM17-mediated sIL-6R generation are shown. The SDS gels were run under reducing conditions (see Materials and Methods for details), and the region of the SDS gel excised for MS is indicated with a red box. The precipitated sIL-6R is detected with an antibody that specifically recognizes the ectodomain of the human IL-6R (4–11). (B) MS/MS spectra (HCD) of the C-terminal peptide of the sIL-6R identified by manual spectra interpretation. One of the identified O-glycan structures on Thr-352 is shown. (C) A schematic illustration, a representative coomassie-stained SDS gel, and a western blot of the precipitated protein after ionomycin-induced ADAM10-mediated sIL-6R generation are shown. The SDS gels and the western blot were performed as described under panel (A). (D) MS/MS spectra (HCD) of the C-terminal peptide of the sIL-6R identified by manual spectra interpretation. One of the identified O-glycan structures on Thr-352 is shown.