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 consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular 'soluble' part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig- 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 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys¹⁰²-Cys¹¹³ and Cys¹⁴⁶-Cys¹⁵⁷ are consistent with known cytokine-binding domain motifs, and Cys²⁸-Cys⁷⁷ with known Ig superfamily domains. An unusual cysteine connectivity between Cys⁶-Cys¹⁷⁴, which links the Ig-like and NH₂-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys¹⁹² and Cys²⁵⁸) were detected as cysteinyl- cysteines, although a small proportion of Cys²⁵⁸ was reactive with the alkylating agent 4-vinylpyridine. Of the four potential N-glycosylation sites, carbohydrate moieties were identified on Asn³⁶, Asn⁷⁴, and Asn²⁰², but not on Asn²²⁶.