Red display of women in the unattractive and attractive experimenter condition.

<p>Red display of women in the unattractive and attractive experimenter condition.</p

ADAM17 and ADAM10 cleave the IL-6R between Pro-355 and Val-356 in vitro.

<p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000080#sec013" target="_blank">Materials and Methods</a> 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.</p

Mutation of Val-356 is sufficient to block proteolysis of the IL-6R and the Asp358Ala variant.

<p>(A) Amino-acid residues from Asp-340 to Ala-370 of the human IL-6R. The identified cleavage site between Pro-355 (P1) and Val-356 (P1′) is indicated. (B) Overview of the six different IL-6R cleavage site mutants. Mutations are colored in either green or blue; the amino-acid residues of the wild-type cleavage site are shown in red. (C) HEK293 cells were transiently transfected with expression plasmids encoding the wild-type IL-6R (PV) or the double mutants (IE, DG) as indicated. Cells were either treated with 100 nM PMA for 2 h or DMSO as vehicle control. sIL-6R was precipitated from the supernatant with concanavalin A-covered sepharose beads, and the cells were lysed. Both were analyzed via western blot, and β-actin served as the loading control. One out of three experiments with similar outcomes is shown. (D, E) The experiment was performed as described under (C), but sIL-6R generation was analyzed via ELISA. In (D), the amount of sIL-6R generated after PMA stimulation of the wild-type IL-6R (PV) was set to 100%, and all other values were calculated accordingly. In (E), the amount of sIL-6R without stimulation was considered as constitutive shedding and set to 1, and the increase of sIL-6R was calculated. Data shown are the mean ± SD from at least three independent experiments (*<i>p</i> < 0.05, ns = no significant difference). (F–H) HEK293 cells were transiently transfected with expression plasmids encoding the wild-type IL-6R (PV) or the single mutants (DV, IV, PE, PG) as indicated. The experiments were performed as described in (C) to (E). (I) Overview of the four different IL-6R cleavage site mutants of the IL-6R Asp358Ala variant. Mutations are colored in either green or blue, the amino-acid residues of the wild-type cleavage site are shown in red, and the Asp358Ala single nucleotide polymorphism (SNP) is colored in orange. (J) ADAM17-mediated proteolysis of the IL-6R variants depicted in (I) were analyzed as described in (D). (K–M) 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).</p

The Asn-55 N-glycan in domain D1 is a protease-regulatory exosite.

<p>(A–K) PMA-mediated ectodomain shedding of IL-6R variants lacking single or multiple N-glycans. Experiments were performed with stably transduced Ba/F3-gp130 cell lines. The IL-6R variant is indicated above the respective diagram. Data shown are the mean ± SD from three independent experiments, which were compared to wild-type IL-6R (*<i>p</i> < 0.05, compared to DMSO-treated Ba/F3-gp130-IL-6R cells; ^§<i>p</i> < 0.05, compared to PMA-stimulated Ba/F3-gp130-IL-6R cells).</p

Identification of the ds-sIL-6R and a novel protease-derived sIL-6R from human serum via mass spectrometry (MS).

<p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000080#sec013" target="_blank">Materials and Methods</a> 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.</p

The majority of sIL-6R in human serum is not generated by alternative mRNA splicing.

<p>(A) Multiple sequence alignment of amino acids Met-310 to Ala-370 of the full-length human IL-6R with the sIL-6R (Met-310 to Gln-357) and amino acids Met-310 to Leu-365 of the differentially spliced sIL-6R (ds-sIL-6R). The ten unique C-terminal amino-acid residues of the sIL-6R, which represent the epitope against which the antibody ds6R was raised, are colored in green. (B, C) Proliferation assay of Ba/F3-gp130 cells in response to 10 ng/ml IL-6 and increasing amounts (0–100 ng/ml) of either (B) recombinant sIL-6R or (C) recombinant ds-sIL-6R. Cell viability was assessed after 48 h. Data shown are the mean ± the standard deviation (SD) of one representative experiment with three biological replicates. (D) Schematic representation of the sandwich ELISA that recognizes all forms of the sIL-6R (left) and proof-of-principle experiment with recombinant proteins (mean ± SD, <i>n</i> = 3). (E) Determination of the total sIL-6R levels in the serum of eight healthy donors. Values for the mean ± the standard error of the mean (SEM) are indicated. (F) Schematic representation of the sandwich ELISA that recognizes only the ds-sIL-6R (left) and proof-of-principle experiment with recombinant proteins (mean ± SD, <i>n</i> = 3). (G) Determination of the ds-sIL-6R levels in the serum of the same eight healthy donors as in (E). The values for the mean ± SEM are indicated.</p

An IL-6R devoid of all N-glycans is defective in ADAM17-mediated proteolysis.

<p>(A) PMA-mediated ectodomain shedding of IL-6R-5N with a stably transduced Ba/F3-gp130 cell line. Data shown are the mean ± SD from three independent experiments compared to wild-type IL-6R. (B) HEK293 cells were transiently transfected with a cDNA encoding wild-type IL-6R. Cells were pretreated with the ADAM10-specific inhibitor GI or the combined ADAM10/ADAM17-specific inhibitor GW for 30 min and then stimulated with 100 nM PMA for 2 h as indicated. The amount of sIL-6R in the cell supernatant was determined by ELISA. One out of three experiments with similar outcomes is shown (mean ± SD, <i>n</i> = 3). (C) The experiment was performed as described under (B), but sIL-6R was precipitated from cell supernatant and analyzed by western blot. Furthermore, the cells were lysed, and IL-6R expression in the lysates was also determined by western blot. β-actin served as loading control. One experiment out of three with similar outcomes is shown. (D, E) The experiment was performed as described in (C) and (D), but HEK293 cells were transiently transfected with a cDNA encoding IL-6R-5N. nd, not detected. (F) Cell-surface expression of the IL-6R-5N variant on transiently transfected HEK293 cells analyzed via flow cytometry (black histogram). The control staining is shown in gray. (G) HEK293 cells were transiently transfected with expression plasmids encoding wild-type IL-6R or IL-6R-5N in combination with either an expression plasmid encoding mCherry or MPD17-CANDIS. After 48 h, cells were lysed and the MPD17-CANDIS precipitated via its protein C (PC) tag. The expression of all proteins (input) and the interaction of the IL-6R variants with MPD17-CANDIS were analyzed via western blot. One experiment out of two with similar outcomes is shown.</p

N- and O-linked glycosylation are dispensable for intracellular transport and signaling of the IL-6R.

<p>(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.</p