Collier E, Gorden P
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Diabetes. 1991 Feb;40(2):197-203. doi: 10.2337/diab.40.2.197.
The insulin receptor, an integral membrane glycoprotein, is synthesized as a single-chain precursor that is cleaved to produce two mature subunits, both of which contain N-linked oligosaccharide chains and covalently linked fatty acids. We report that the beta-subunit also contains O-linked oligosaccharides. The proreceptor, alpha-subunit, and beta-subunit were labeled with [3H]mannose and [3H]galactose in the presence or absence of an inhibitor of O-linked glycosylation. Tryptic peptides from each component were separated by reverse-phase high-performance liquid chromatography. N- and O-linked oligosaccharide chains were identified on these peptides by specific enzymatic digestions. The proreceptor and alpha-subunit contained only N-linked oligosaccharides, whereas the beta-subunit contained both N- and O-linked oligosaccharides. The O-linked oligosaccharide chains were attached to a single tryptic fraction of the beta-subunit, which also contained N-linked chains. This fraction was further localized to the NH2-terminal tryptic peptide of the beta-subunit by specific immunoprecipitation with an anti-peptide antibody with specificity for this region. Binding of insulin and autophosphorylation of the beta-subunit were not dependent on O-linked glycosylation, because cells grown in the presence of the inhibitor exhibited a normal dose response to insulin. Therefore, the insulin receptor contains O-linked oligosaccharides on the NH2-terminal tryptic peptide of the beta-subunit, and these O-linked oligosaccharides are not necessary to the binding or autophosphorylation function of the receptor.
胰岛素受体是一种整合膜糖蛋白，最初作为单链前体合成，随后被切割产生两个成熟亚基，这两个亚基均含有N - 连接寡糖链和共价连接的脂肪酸。我们报告β亚基还含有O - 连接寡糖。在存在或不存在O - 连接糖基化抑制剂的情况下，用[³H]甘露糖和[³H]半乳糖标记前受体、α亚基和β亚基。通过反相高效液相色谱法分离各组分的胰蛋白酶肽段。通过特异性酶切鉴定这些肽段上的N - 和O - 连接寡糖链。前受体和α亚基仅含有N - 连接寡糖，而β亚基同时含有N - 和O - 连接寡糖。O - 连接寡糖链连接到β亚基的一个单一胰蛋白酶片段上，该片段也含有N - 连接链。通过使用针对该区域的抗肽抗体进行特异性免疫沉淀，该片段进一步定位到β亚基的NH₂末端胰蛋白酶肽段。胰岛素结合和β亚基的自磷酸化不依赖于O - 连接糖基化，因为在抑制剂存在下生长的细胞对胰岛素表现出正常的剂量反应。因此，胰岛素受体在β亚基的NH₂末端胰蛋白酶肽段上含有O - 连接寡糖，并且这些O - 连接寡糖对于受体的结合或自磷酸化功能不是必需的。
