Determination of the disulfide structure and N-glycosylation sites of the extracellular domain of the human signal transducer gp130

gp130 is the common signal transducing receptor subunit for the interleukin -6-type family of cytokines, Its extracellular region (sgp130) is predicted to consist of five fibronectin type III-like domains and an NH2-terminal I g-like domain. Domains 2 and 3 constitute the cytokine-binding region defin ed by a set of four conserved cysteines and a WSXWS motif, respectively. He re we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman d egradation and mass spectrometry. Of the 13 cysteines present, 10 form disu lfide bonds, two are present as free cysteines (Cys(279) and Cys(469)), and one (Cys(397)) is modified by S-cysteinylation. Of the 11 potential N-glyc osylation sites, Asn(21), Asn(61), Asn(109), Asn(135), Asn(205), Asn(357), Asn(361), Asn(531), and Asn(542) are glycosylated but not Asn and Asn(368). The disulfide bonds, Cys(112)-Cys(122) and Cys(150)-Cys(160), are consiste nt with known cytokine-binding region motifs. Unlike granulocyte colony-sti mulating factor receptor, the connectivities of the four cysteines in the N H2-terminal domain of gp130 (Cys(6)-Cys(32) and Cys(26)-Cys(81)) are consis tent with known superfamily of Ig-like domains. An eight-residue loop in do main 5 is tethered by Cys(436)- Cys(444). We have created a model predictin g that this loop maintains Cys(469) in a reduced form, available for ligand -induced intramolecular disulfide bond formation. Furthermore, we postulate that domain 5 may play a role in the disulfide-linked homodimerization and activation process of gp130.