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PE anti-human CD130 (gp130) Antibody
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Please wait while we load your content Thus, our results confirm previous studies that found that a fraction of the sIL-6R in humans is generated via alternative splicing but that the majority must originate from a different mechanism. 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. Cell viability was assessed after 48 h. E Determination of the total sIL-6R levels in the serum of eight healthy donors.
In order to identify other human sIL-6R forms that exist in vivo, we performed an isotopic labeling strategy that is based on proteolysis in the presence of H 2 18 O [ 32 ]. We isolated human serum, depleted the endogenous antibodies, and precipitated all sIL-6R forms by the sepharose-coupled 4—11 antibody, which binds to the N-terminal D1 domain Fig 2A. We separated the precipitated proteins via SDS-PAGE under nonreducing conditions, confirmed sIL-6R presence in the precipitate via western blot, and excised the corresponding region of the coomassie-stained gel Fig 2A.
Using an anti-IL-6R antibody, we detected several proteins of different molecular weights, which most likely are different sIL-6R isoforms and complexes of these with other serum proteins that are not dissociated under nonreducing conditions Fig 2A. This endoglycosidase hydrolyzes the bond formed between the carbohydrate and the Asn side chain, resulting in the formation of an Asp residue at the former glycosylation site. By using this method, all proteolytically generated neo -C-termini contain an 18 O-isotope incorporated in the carboxyl group, while the original canonical or truncated protein C-terminus remains unmodified Fig 2B.
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 precipitated sIL-6R is detected with an antibody that specifically recognizes the ectodomain of the human IL-6R 4— One of the identified O-glycan structures on Thr is shown. With this MS-based strategy, we were for the first time able to identify an sIL-6R variant in human serum that does not originate from alternative splicing but appears to be generated by proteolysis.
Having shown that a protease-derived sIL-6R exists in vivo, we sought to identify the protease that could be responsible for its generation. The strongest band detected in the western blot corresponded to a cleavage product of the IL-6R, whereas the lower band at around 55 kDa most likely was the heavy chain of the antibody used for precipitation Fig 3A.
Indeed, MS analysis revealed a C-terminal peptide that ended with Pro Fig 3B , indicating exactly the same cleavage site that was used in vivo Fig 2D. We could further confirm the N-glycan site on Asn and the O-glycan site at Thr within the same sequon, which was again decorated with the same pattern of glycan structures found in vivo Fig 3B and S2A—S2E Fig. The SDS gels and the western blot were performed as described under panel A. Similar to ADAMmediated shedding Fig 3A , we detected cleaved IL-6R and a band of lower molecular weight, which most likely corresponded to the heavy chain of the antibody used for precipitation Fig 3C.
Surprisingly, we identified the same cleavage site between Pro and Val Fig 3D and the same pattern of O-glycans at Thr S3A—S3D Fig , indicating that both proteases share the same cleavage site. In general, we exchanged Pro and Val into amino-acid residues that have been shown to be disfavored by ADAM17 [ 23 ]. 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. One out of three experiments with similar outcomes is shown. 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. The experiments were performed as described in C to E. Mutations are colored in either green or blue, the amino-acid residues of the wild-type cleavage site are shown in red, and the AspAla single nucleotide polymorphism SNP is colored in orange.
The stably transduced IL-6R variant is indicated above the diagram. Thus, mutation of Val is also sufficient to prevent the enhanced shedding of the AspAla mutant. Finally, we analyzed whether these mutations influenced the biological activity of the IL-6R. As an internal control, we incubated all cell lines with Hyper-IL-6, which is a fusion protein of IL-6 and the sIL-6R and thus activates proliferation via gp homodimerization independent of membrane-bound IL-6R.
Although N-linked glycosylation of the IL-6R has been reported [ 13 , 14 ], no functional role has been addressed so far. While determining the cleavage site, we had already successfully detected an N-glycosylation site at Asn and an O-glycosylation site at Thr, both in vitro and in vivo Figs 2D and 3B. A HEK cells were transiently transfected with an expression plasmid encoding IL-6R, and cells were lysed 48 h later. One representative experiment out of two performed is shown. The sIL-6R was precipitated from the cell supernatant and afterwards treated and analyzed as described under panel A.
The four glycosylation sites Asn, Asn, Asn, and Asn were modified to aspartic acid residues due to PNGase F treatment in the presence of H 2 18 O-containing buffer and are shown in green. 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.
Flow cytometry analysis revealed that all IL-6R mutants were transported to and expressed at the cell surface, albeit the amount slightly decreased when more N-glycans were deleted S6A Fig. The remaining cell-surface expression of the IL-6R was analyzed via flow cytometry. Finally, we analyzed protein stability and turnover at the cell surface of the wild-type IL-6R compared to the 4N mutant.
Collectively, our data show that glycosylation of the IL-6R is not needed for transport to and expression at the cell surface, is dispensable for ILmediated signal transduction, and does not alter the half-life of the IL-6R at the cell surface.
Gene group | HUGO Gene Nomenclature Committee
Having excluded that glycosylation is important for the signaling capacity of the IL-6R, we sought to analyze a possible role for the glycans in terms of IL-6R proteolysis. Interestingly, addition of a larger biantennary N-glycan blocked cleavage of the 5 QD peptide but did not alter processing of the 6 QA peptide Fig 7C. Thus, in line with a recent report [ 25 ], we can detect an inhibitory influence of the glycosylation on the capacity of ADAM17 to cleave the IL-6R peptide, but overall, the influence appears to be rather small. Cleavage was determined as described in panel A.
The IL-6R variant is indicated above the respective diagram. Finally, we analyzed proteolysis of the 5N variant, which lacks all N-glycans. Surprisingly, we detected significantly reduced PMA-induced shedding 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. One experiment out of three with similar outcomes is shown. The control staining is shown in gray. One experiment out of two with similar outcomes is shown. Soluble cytokine receptors play pivotal roles in health and disease. As long as the ligand-binding domains are retained within the soluble protein, they have similar affinities towards their ligands as the membrane-tethered counterparts.
Most of them have antagonistic properties, because they compete with the membrane-bound receptors for the same cytokine, and cytokines bound to soluble receptors are no longer able to bind to their cognate target cells in order to activate them. A rare example of an agonistic soluble receptor is the sIL-6R. Surprisingly, despite its importance in terms of disease and therapy, little is known about the mechanisms that lead to sIL-6R generation in vivo.
In the present study, we confirm the presence of the ds-sIL-6R in human serum and detect for the first time a second form of the sIL-6R with a novel C-terminus generated by proteolytic cleavage. Interestingly, the cleavage event occurs between Pro and Val within the stalk region of the IL-6R. We cannot absolutely exclude that exoprotease activity led to trimming of the C-terminal peptide in vivo or during sample preparation, which might confound the identification of the cleavage site via LC-MS. However, our mutational analysis of the novel cleavage site, which suggests that substitution of Val by glycine or glutamic acid is sufficient to render the IL-6R resistant towards ADAM-mediated proteolysis, further corroborates this finding.
The major genetic determinant of human sIL-6R serum levels is a single nucleotide polymorphism rs , which leads to the exchange of Asp to an alanine residue [ 38 ]. Homozygous carriers have strongly increased sIL-6R serum levels, which reduces their risk of suffering from coronary heart disease [ 43 , 44 ]. Although differential splicing of the IL6R mRNA is increased in these individuals [ 45 ], the majority of the sIL-6R is nevertheless generated by an alternative mechanism.
Because we have now determined the cleavage site between Pro and Val, this explanation for the observed effects of the AspAla mutation has to be revised. However, the cleavage site profiling by Tucher et al. This is in contrast to other cytokine receptors like epidermal growth factor receptor EGFR and granulocyte macrophage colony-stimulating factor receptor GM-CSFR [ 10 , 11 ], in which N-linked glycosylation is essential for ligand binding. However, the transport of the unglycosylated IL-6R mutant to the cell surface was only marginally affected, and the protein turnover compared to the wild-type IL-6R was not altered at all.
N- and O-linked glycosylation are common post-translational modifications that have been described for a variety of ADAM substrates besides the IL-6R, e. Substrates with and without glycans have also been used to generate novel ADAMspecific inhibitors [ 48 ]. Here, we show that the IL-6R is indeed O-glycosylated on Thr in vivo and confirm that this glycan reduces cleavage of an IL-6R peptide in conjunction with an N-linked glycan on Asn In contrast, an N-linked glycan on Asn, located in the D1 domain of the IL-6R far away from the cleavage site, was surprisingly identified as a protease-regulatory exosite, whose deletion caused increased shedding of the IL-6R.
Importantly, this glycan on Asn has no role in ILmediated signaling. Consequently, Hyper-IL-6 does not contain this domain [ 49 ], and a membrane-bound IL-6R variant lacking D1 is biologically active [ 50 ]. Thus, our data suggest that the glycan on Asn has a unique role in the regulation of proteolysis but is dispensable for signaling of IL Furthermore, an IL-6R mutant without any N-glycans reached the cell surface and was able to mediate ILdependent signaling, but its shedding was severely impaired.
It is currently unclear why this IL-6R variant, which contains the cleavage site and is able to coprecipitate with the protease via CANDIS, is nevertheless largely resistant towards proteolysis. A possible explanation is cooperativity between the individual glycosylation site, and simultaneous loss of all five N-glycans results in structural changes within the IL-6R ectodomain that disturb cleavage by the protease but do not perturb ILdependent signaling.
In summary, we identify a soluble form of the IL-6R in human serum that is generated by a protease in vivo and map the cleavage site by mass spectrometry. Furthermore, we map the occupancy of all N- and O-glycosylation sites of the sIL-6R and find that glycosylation is dispensable for trafficking, stabilization, and signaling of the IL-6R but is an important regulatory mechanism in terms of proteolysis. The peroxidase conjugated secondary antibodies were obtained from Pierce Thermo Scientific, Perbio, Bonn, Germany , and the APC-conjugated anti-mouse monoclonal secondary antibodies for flow cytometry experiments were obtained from Dianova Hamburg, Germany.
G and puromycin were from Carl Roth Karlsruhe, Germany. Hyper-IL-6 was produced as described previously [ 54 , 55 ].
IL-6 was expressed, purified, and refolded as described previously [ 56 ]. Forty-eight h later, expressing cells were selected with 0. The proteins were further purified via size exclusion chromatography on a Superdex column GE Healthcare. Construction of the different expression plasmids encoding IL-6R variants was performed using standard molecular biology techniques with restriction-enzyme-based cloning. Mutations within the protease cleavage site of the IL-6R were performed similarly.
Cells stably expressing IL-6R were selected with puromycin 1.
Values were measured in triplicates per experiment, and relative light units RLU obtained after 60 min were normalized by subtraction of control values obtained after 0 min. Peripheral blood from healthy volunteers was collected by venipuncture and serum isolated via centrifugation. Database of Orthologous Groups More OrthoDB i. Database for complete collections of gene phylogenies More PhylomeDB i.
TreeFam database of animal gene trees More TreeFam i. Integrated resource of protein families, domains and functional sites More InterPro i. Pfam protein domain database More Pfam i. PIRSF i. Protein Motif fingerprint database; a protein domain database More SMART i. Superfamily database of structural and functional annotation More Interleukin-6 Interleukin Corresponds to variant dbSNP:rs Ensembl.
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CCDS i. Protein sequence database of the Protein Information Resource More PIR i. RefSeq i. Ensembl eukaryotic genome annotation project More Ensembl i. GeneID i. KEGG i. Wikipedia Interleukin-6 entry. Atlas of Genetics and Cytogenetics in Oncology and Haematology. PDBj i Links Updated. DrugCentral More DrugCentral i.
ABCD curated depository of sequenced antibodies More ABCD i. DNASU i.
Comparative Toxicogenomics Database More CTD i. GeneCards: human genes, protein and diseases More GeneCards i. GenAtlas: human gene database More GenAtlas i. ChiTaRS i. IL6 human. The Gene Wiki collection of pages on human genes and proteins More GeneWiki i. GenomeRNAi i. Pharos More Pharos i. Protein Ontology More PRO i. ProtoNet; Automatic hierarchical classification of proteins More ProtoNet i.
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