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.

Two N-Linked Glycans Differentially Control Maturation, Trafficking and Proteolysis, but not Activity of the IL-11 Receptor

“…Despite this, the crystal structure of IL-11 suggested structural differences from IL-6 [34], and we have recently shown that proteases differ in their ability to cleave IL-11R and IL-6R [19]. A functional role for N-glycans in IL-11R biology has not been reported so far, but N-glycosylation is dispensable for the biological activity of the IL-6R, although it regulates ADAM17-mediated proteolysis [25]. …”

“…Interestingly, we could recently show that an N-glycan within the D1 domain of the IL-6R acts as an exosite that regulates IL-6R proteolysis [25]. Therefore, we were curious to analyze how N-linked glycosylation influences IL-11R proteolysis and transiently expressed IL-11R wt, N127Q, N194Q and the double mutant N127Q/194Q in HEK293 cells.…”

“…in [21, 39, 40]), rather less attention has been paid to the IL-11R. We have recently shown that the IL-11R has a similar modular organization like the IL-6R [16], and while glycosylation of the IL-6R is described in much detail [25, 41, 42], functional roles for glycans in IL-11 biology have not been analyzed so far. While the IL-6R contains five N-glycosylation sites, two located in the D1 and D3 domains each and one within the stalk region, the amino acid sequence of the IL-11R contains only the two sequons at N127 and N194 within its D2 domain that could be potentially used for N-glycosylation [35].…”

“…It is currently unclear why the number and the distribution of the N-glycans is different between the IL-6R and the IL-11R, which are always considered as rather similar in function and structure. Strikingly, while the D2 domain of the IL-6R is not glycosylated [25], both N-glycans of the entire IL-11R ectodomain are located at D2.…”

“…The small proportion that was transported to the cell surface was biologically active, however, letting the authors conclude that N-glycosylation of gp130 was important for the stability, but not for its signaling capacity [24]. Furthermore, we have recently shown that N-glycosylation of the IL-6 receptor (IL-6R) is dispensable for its biological function, but rather influences limited proteolysis of the IL-6R by the metalloprotease ADAM17 [25]. In contrast, the receptors for C-X-C motif chemokine 12 (CXCL12), granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF) or interleukin-5 require proper glycosylation for their biological functions [26-29].…”