Joint Study on Post-Translational Protein Tyrosine Sulfation

蛋白质的硫酸化是一种翻译后修饰，通过共价连接硫酸酯。在已经提出的一些可能的职能作用中，最近我们在牛肝中发现了一个位于高尔基体上的175 kDa的膜结合酪氨酸-O-硫酸盐结合蛋白，这有力地支持了酪氨酸硫酸化参与细胞内蛋白的分选和转运。使用TyrS-Affi-gel 10亲和层析、羟基磷灰石层析和电洗脱的组合从牛肝高度纯化TyrS受体。经还原条件下SDS-PAGE测定，纯化的受体的表观分子量为175 kDa。我们使用P3-X63-Ag 8-U1细胞作为融合伴侣细胞建立了与纯化的受体反应的小鼠单克隆抗体。获得两株稳定分泌抗TyrS受体单克隆抗体的杂交瘤细胞株（R3-1、R2-4）。 关于我们 在溶解含有TyrS受体的膜结合蛋白后，我们分析了与TyrS的结合。将胰蛋白酶加入悬浮液中，并消化混合物。结果表明，65 k、63 k和59 k Da的三个片段具有与TyrS结合的能力，TyrS受体呈双蛋白带迁移，表观分子量约为10000。为了获得有关这些蛋白和三个片段的结构形式的信息，我们研究了V8肽图。这些TyrS受体的部分V8蛋白酶图谱相似，显示同源性。TyrS是一种糖蛋白，具有N-糖链。因此，TyrS受体的糖链被糖苷酶不同程度地修剪，表现出反应性的变化或丧失与TyrS结合的能力。由此得出结论，TyrS受体是一种分子量为175 K的糖蛋白。该蛋白的N连接的糖残基在识别TyrS中非常重要，并且在受体活性中具有功能贡献。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