Determination of the disulfide structure and N-glycosylation sites of the extracellular domain of the human signal transducer gp130
Determination of the disulfide structure and N-glycosylation sites of the extracellular domain of the human signal transducer gp130
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DOI：
10.1074/jbc.m009979200
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发表时间：
2001-03-16
影响因子：
4.8
通讯作者：
Simpson, RJ
中科院分区：
文献类型：
--
作者：
Moritz, RL;Hall, NE;Simpson, RJ
gp 130是白细胞介素-6型细胞因子家族共同的信号转导受体亚单位，其胞外区（sgp 130）由5个纤连蛋白III型样结构域和一个氨基端Ig样结构域组成。结构域2和3分别构成由一组四个保守的半胱氨酸和WSXWS基序限定的精氨酸结合区。在这里，我们确定的二硫键结构的人sgp 130肽图谱，在还原剂的存在和不存在下，结合埃德曼降解和质谱。在存在的13个半胱氨酸中，10个形成二硫键，两个作为游离半胱氨酸（Cys（279）和Cys（469））存在，一个（Cys（397））通过S-半胱氨酸化修饰。在11个潜在的N-糖基化位点中，Asn（21）、Asn（61）、Asn（109）、Asn（135）、Asn（205）、Asn（357）、Asn（361）、Asn（531）和Asn（542）被糖基化，但Asn和Asn（368）未被糖基化。二硫键Cys（112）-Cys（122）和Cys（150）-Cys（160）与已知的精氨酸结合区基序一致。与粒细胞集落刺激因子受体不同，gp 130的NH 2端结构域中的四个半胱氨酸（Cys（6）-Cys（32）和Cys（26）-Cys（81））的连接性与已知的Ig样结构域超家族一致。结构域5中的八残基环由Cys（436）- Cys（444）栓系。我们已经建立了一个模型，预测这个环保持Cys（469）在一个还原的形式，可用于配体诱导的分子内二硫键形成。此外，我们推测，域5可能在二硫键连接的同源二聚化和活化过程中发挥作用的gp 130。
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 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.