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 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 Cys(469)), and one (Cys(397)) is modified by S-cysteinylation. Of the 11 potential N-glycosylation 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(224) and Asn(368). The disulfide bonds, Cys(112)-Cys(122) and Cys(150)-Cys(160), 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 (Cys(6)-Cys(32) and Cys(26)-Cys(81)) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys(436)-Cys(444). We have created a model predicting 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.
Moritz
R L
RL
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Tumor Biology Branch) and the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia.
Hall
N E
NE
Connolly
L M
LM
Simpson
R J
RJ
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry* / Q000302:isolation & purification; D050822:Cytokine Receptor gp130; D004220:Disulfides / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping
D015703:Antigens, CD; D004220:Disulfides; C494199:IL6ST protein, human; D008562:Membrane Glycoproteins; D050822:Cytokine Receptor gp130