Determination of the Disulfide Structure and N-Glycosylation Sites of the Extracellular Domain of the Human Signal Transducer gp130

Abstract

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 NH 2-terminal Ig-like domain. Domains 2 and 3 constitute the cytokine-binding region defined by a set of four conserved cysteines and a WSXWS motif, respectively. Here we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman degradation and mass spectrometry. Of the 13 cysteines present, 10 form disulfide bonds, two are present as free cysteines (Cys 279 and Cys469), and one (Cys397) is modified by S-cysteinylation. Of the 11 potential N-glycosylation sites, Asn 21, Asn61, Asn109, Asn135, Asn 205, Asn357, Asn361, Asn531, and Asn542 are glycosylated but not Asn224 and Asn 368. The disulfide bonds, Cys112-Cys122 and Cys150-Cys160, are consistent with known cytokine-binding region motifs. Unlike granulocyte colony-stimulating factor receptor, the connectivities of the four cysteines in the NH2-terminal domain of gp130 (Cys6-Cys32 and Cys26-Cys81) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys436-Cys444. We have created a model predicting that this loop maintains Cys469 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.