Joint Study on Post-Translational Protein Tyrosine Sulfation

蛋白质的硫酸盐化是一种硫酸盐共价结合的翻译后修饰。在已提出的许多可能的功能作用中，酪氨酸硫酸酯参与细胞内蛋白质的分类和运输得到了我们最近从牛肝中发现的175kDa膜结合酪氨酸-O-硫酸盐结合蛋白的有力支持。在这里，我们描述了酪氨酸-O-硫酸盐(TyrS)受体的结合特性。在还原条件下，经SDS-PAGE测定，纯化的受体表观分子量为175kDa。我们以P3-X63-Ag8-Ul细胞为融合配对细胞，建立了与纯化受体反应的小鼠单抗。获得两株稳定分泌抗酪氨酸受体单抗的杂交瘤细胞株(R3-1、R2-4)…进一步，我们分析了含有TyrS受体的膜结合蛋白溶解后与TyrS的结合。在悬浮液中加入胰酶，混合物被消化。将反应产物与TyrS-Affi-Gel 10共孵育，经SDS-PAGE分析，发现65k、63k和59k大小的三个片段具有与TyrS结合的能力。在SDS-PAGE上，TyrS受体以双蛋白带的形式迁移，表观分子量约为175k。这些TyrS受体的部分V8蛋白酶图谱相似，显示了同源性。TyrS是一种具有类N碳水化合物链的糖蛋白。因此，TyrS受体被糖苷酶不同程度地修剪其糖链，表现出与TyrS结合活性的改变或结合能力的丧失。结论：TyrS受体是一种分子量为175K的糖蛋白，该蛋白的N连接碳水化合物残基在识别TyrS过程中起着非常重要的作用，并在受体活性中具有功能贡献。较少

The sulfation of protein is a post-translational modification by covalent attachment of sulfate. Among a number of possible funcitonal roles that have been proposed, the involvement of tyrosine sulfation in intracellular protein sorting and transport received strong support from our recent finding of Golgi located 175 k Da membrane-bound tyrosine-O-sulfate binding protein from bovine liver.Here we described the bindingproperties fo the tyrosine-O-sulfate (TyrS) receptor.TyrS receptor was highly purified from bovine liver using a combination of TyrS-Affi-gel 10 affinity chromatography, hydroxylapatite chromatography and electroelution. The purified receptor exhibited an apparent molecular weight of 175k Da as determined by SDS-PAGE under reducing conditions. We established mouse monoclonal antibodies reactive to the purified receptor using P3-X63-Ag8-Ul cells as fusion partner cells. Two stable hybridoma clone secreting anti TyrS receptor monoclonal antibodies were obtained (R3-1, R2-4) … More .We analyzed for the binding against TyrS,after solubilization the membrane-bound protein containing TyrS receptor. Trypsin was added to the suspension and the mixture was digested. To examine the ligand binding specificity, the reaction mixtures were incubated with TyrS-Affi-Gel 10.Then we analyzed by SDS-PAGE.It was apparent that three fragments of 65k, 63k and 59k Da have the ability to bind against TyrS.TyrS receptor migrated as double protein bands with apparent molecular weights of ca. 175K upon SDS-PAGE.In order to obtain information concerning the sturctural form of these proteins and three fragments we investigated V8 peptide maps. Partial V8 protease mapping of these TyrS receptor were similar showing the homology. TyrS is a glycoprotein which has N-liked carbohydrate chain. So, TyrS receptor variously trimmed their carbohydrate chains by glycosidase showing the change of reactivity or loss of the ability to bind against TyrS.It is concluded that TyrS receptor is a glycoprotein which is having a molecular weight of 175 K.The N linked carbohydrate residue of this protein is very important in recognition of the TyrS and has a functional contribution in receptor activity. Less