Disulfide bond structure and N-glycosylation sites of the extracellular domain of the human interleukin-6 receptor

The high affinity interleukin-6 (IL-6) receptor is a hexameric complex cons isting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high a ffinity converter and signaling molecule, gp130, The extracellular "soluble " part of the IL-GR (sIL-6R) consists of three domains: an amino-terminal I g-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserv ed cysteine residues and a WSXWS sequence motif. Here, we have determined t he disulfide structure of the human sIL-6R by peptide mapping in the absenc e and presence of reducing agent. Mass spectrometric analysis of these pept ides revealed four disulfide bonds and two free cysteines. The disulfides C ys(102)-Cys(113) and Cys(146)-Cys(157),, consistent with known cytokine-bin ding domain motifs, and Cys(28)-Cys(77) With known Ig superfamily domains. An unusual cysteine connectivity between Cys(6)-Cys(174), which links the I g-like and NH2-rminal FN III domains causing them to fold back onto each ot her, has not previously been observed among cytokine receptors, The two fre e cysteines (Cys(192) and Cys(258)) were detected as cysteinyl-cysteines, a lthough a small proportion of Cys(258) was reactive with the alkylating age nt 4-vinylpyridine. Of the four potential N-glycosylation sites, carbohydra te moieties were identified on Asn(36), Asn(74), and Asn(202), but not on A sn(226).