Abstract
Soluble Interleukin-6 receptor (sIL-6R) mediated trans-signaling is an important pro-inflammatory stimulus associated with pathological conditions, such as arthritis, neurodegeneration and inflammatory bowel disease. The sIL-6R is generated proteolytically from its membrane bound form and A Disintegrin And Metalloprotease (ADAM) 10 and 17 were shown to perform ectodomain shedding of the receptor in vitro and in vivo. However, under certain conditions not all sIL-6R could be assigned to ADAM10/17 activity. Here, we demonstrate that the IL-6R is a shedding substrate of soluble meprin α and membrane bound meprin β, resulting in bioactive sIL-6R that is capable of inducing IL-6 trans-signaling. We determined cleavage within the N-terminal part of the IL-6R stalk region, distinct from the cleavage site reported for ADAM10/17. Interestingly, meprin β can be shed from the cell surface by ADAM10/17 and the observation that soluble meprin β is not capable of shedding the IL-6R suggests a regulatory mechanism towards trans-signaling. Additionally, we observed a significant negative correlation of meprin β expression and IL-6R levels on human granulocytes, providing evidence for in vivo function of this proteolytic interaction.
Highlights
ADAM10/17 can cleave the membrane-bound interleukin-6 receptor (IL-6R)[20,21,22,23,24], which plays a critical role in the initiation and propagation of inflammation[25,26,27]
It has been demonstrated recently that negatively charged amino acid residues at the P1’ position are disliked by ADAM1747, but highly favoured by the metalloproteases meprin αand meprin β(Fig. 1B,C)[50], which were further analyzed in our study
The use of two different mass spectrometric technologies (LC-ESI MS and MALDI-MS) in parallel enabled identification of cleavage sites within the peptide (Fig. 1D,E). These results clearly showed that the peptide was cleaved, predominantly between amino acid residues glutamine and aspartate, after incubation with recombinant soluble meprin αor meprin β(Fig. 1F), with slightly faster kinetics of peptide digestion for meprin β(data not shown)
Summary
ADAM10/17 can cleave the membrane-bound interleukin-6 receptor (IL-6R)[20,21,22,23,24], which plays a critical role in the initiation and propagation of inflammation[25,26,27]. IL-1837–39 as well as IL-640, which are released in response to tissue injury and inflammation This is further supported by distinct immunological phenotypes observed in meprin αand meprin βdeficient mice[41,42,43]. Meprin αknock-out animals showed more severe inflammation and intestinal injury in a DSS (dextran sodium sulfate)-induced colitis model compared to wild-type animals[44], which is comparable to that observed in studies with significantly reduced expression of ADAM17 in mice[45]. Recombinant ADAM17 cleaved an IL-6R peptide comprising a part of the stalk region, situated two residues N-terminal of QD between Pro[355] and Val35648. This cleavage site was further substantiated by molecular modelling[49]. Ectodomain shedding of the IL-6R by meprins results in the generation of biologically active soluble IL-6R capable of inducing IL-6 trans-signaling
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