Elucidation of Functional Implication of Post-translational Tyrosine Sulfation

利用亲和凝胶部分技术，我们在牛肝和胰腺以及犬肝和胰腺的微体膜部分的胆酸钠提取物中检测到175 kDa酪氨酸-O-硫酸盐(TyrS)结合蛋白。Western印迹分析表明，在体内和体外，牛肝TyrS结合蛋白均存在于酪氨酸硫化蛋白的复合体中，提示前者可能是后者的受体。以过滤培养的Madin-Darby犬肾(MDCK)细胞为模型，证明合成的酪氨酸硫化蛋白主要分泌到根尖上清液中。结果进一步表明，MDCK细胞产生的两种主要分泌蛋白，纤维连接蛋白(FN)和80 kDa糖蛋白(GP 80)的不同硫酸形式的产生和分化极化分泌，其酪氨酸硫酸形式主要从顶端表面分泌。用糖基化抑制剂苦马豆素和1-脱氧甘露糖苷处理MDCK细胞，似乎能促进酪氨酸硫化的FN和GP-80的顶端分泌。一个类似的175 kDa的膜结合的“TyrS拦截器”。对犬肝脏TyrS受体抗血清的交叉反应。被证明存在于MDCK细胞中。脉冲追逐实验表明，它存在于新合成的FN和GP-80的络合物中。在酪氨酸硫化蛋白的生物合成运输过程中，TyrS残基作为顶端靶向信号的假设模型。由TyrS受体介导。在MDCK细胞中的表达。

By employing the affinity gel fraction technique, we have detected a 175 kDa tyrosine-O-sulfate (TyrS)-binding protein in sodium choleate extracts of the microsomal membrane fractions of bovine liver and pancreas, as well as canine liver and pancreas. Western blot analysis revealed the presence of the bovine liver TyrS-binding protein in complexes with tyrosine-sulfated proteins both in vivo and in vitro, suggesting the putative role of the former being the receptor for the latter. Using filter-grown Madin-Darby canine kidney (MDCK) cells as a model, it was demonstrated that the tyrosine-sulfated proteins synthesized were predominantly secreted into the apical medium. The results further indicate the production and differential polarized secretion of different sulfated forms of the two major secretory proteins produced by MDCK cells, fibronectin (FN) and an 80 kDa glycoprotein (gp 80), with their tyrosine-sulfated forms being predominantly secreted from the apical surface. Treatment of filtergrown MDCK cells with glycosylation inhibitors, swainsonine and 1-deoxymannojirimycin, appeared to enhance the apical secretion of tyrosine-sulfated FN and gp 80. A similar 175 kDa membrane-bound "TyrS trceptor." cross-reactive toward antiserum against the canine liver TyrS receptor. was shown to be present in MDCK cells. Pulse-chase experiments revealed its presence in complexes with newly synthesized FN and gp 80. A hypothetical model for TyrS residues serving as an apical targeting signal during the biosynthetic transport of tyrosine-sulfated proteins. as mediated by the TyrS receptor. in MDCK cells is proposed.