Purification and Characterization of the Soluble Interleukin‐6 Receptor from Human Plasma and Identification of An Isoform Generated through Alternative Splicing

Abstract

The soluble human interleukin-6 receptor (shIL6R) was purified from human plasma. In a single immunoaffinity purification step a 140000-fold enrichment with a yield of 95% was achieved. A subsequent IL-6 affinity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the soluble receptor was clearly demonstrated by its ability to induce the synthesis of the acute-phase protein a, -antichymotrypsin in HepG2 cells stably transfected with IL-6. Upon gel filtration, the native shIL6R showed an apparent molecular mass of 93 kDa. Analysis by SDSPAGE revealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglycosylation with peptide N-glycosidase F led to a shift in molecular mass from 65 kDa to 45 kDa. It has previously been shown that the shIL6R can be generated by shedding the membrane-bound form or by expression of an alternatively spliced mRNA. Here we show that the shIL6R isolated from human plasma is recognized by an affinity-purified peptide antibody raised against an amino acid sequence unique for the alternatively spliced isoform. Thus, the shIL6R isoform generated through alternative splicing which has been previously detected in supernatants of cultured cell lines is also an in vivo product circulating in human plasma.

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

“…It has been proposed that blood-borne sIL-6R in conjunction with a soluble form of the second IL-6 receptor subunit gp130 (sgp130) acts as a buffer to prevent potentially overshooting IL-6 from acting systemically (33). Whereas in humans a substantial fraction of serum sIL-6R can be attributed to alternative splicing of the IL-6R mRNA, the bulk of circulating sIL-6R appears to be generated by mechanisms distinct from differential mRNA splicing (13). A SNP within the ADAM17 cleavage site leads to increased serum sIL-6R levels in SNP carriers suggesting a decisive role of ectodomain shedding in serum sIL-6R generation (14,15).…”

“…However, whereas serum sIL-6R levels attributed to alternative splicing vary considerably between studies, the bulk of serum sIL-6R (Ͼ65%) originates from processes other than differential mRNA splicing (12,13). A recently identified SNP in the human IL-6R gene located within the ADAM17 cleavage site, which leads to increased sIL-6R protein amounts in the circulation, strongly suggests involvement of a metalloprotease of the ADAM family in serum sIL-6R generation (14 -16).…”

Soluble receptors in human disease

“…These two mechanisms are not mutually exclusive. The soluble interleukin (IL)-4 and IL-6 receptors appear to be generated both by alternative splicing and proteolysis [42,43,[51][52][53][54][55]111]. The mechanism for generating murine soluble IL-4 receptors appears to be differentially regulated: treatment with IL-4 leads to formation of soluble IL-4 receptor by alternative mRNA splicing, whereas engagement of the T cell receptor appears to lead to proteolysis [112].…”

“…1D). In this model, the soluble receptor binds ligand in the splicing IL-6R␣ [51][52][53][54][55][56] splicing, cleavage Ag gp130 [57][58][59] splicing I CNTFR [60,61] GPI Ag LIFR [62][63][64][65][66] splicing I Leptin R [67][68][69][70] splicing I IL-11R [71,72] splicing Ag, I IL-12 p40 [73][74][75][76] splicing Ag, I Stem cell factor R (c-kit) [77][78][79] combined I Interferon R [80][81] splicing Lipopolysaccharide R (CD14) [82][83][84][85] GPI I…”

A Fusion Protein of the gp130 and Interleukin-6Rα Ligand-binding Domains Acts as a Potent Interleukin-6 Inhibitor

“…Most interestingly, the antagonizing activity of sgp130 is substantially increased by the presence of sIL-6R␣ (12). Both sIL-6R (13,14) and sgp130 (11,12) are found in high concentrations in human blood (about 50 and 300 ng/ml, respectively). This pair of soluble receptors might act as a natural IL-6 inhibitor to limit systemic IL-6 responses (12).…”