Elucidation of Functional Implication of Post-translational Tyrosine Sulfation

采用亲和凝胶组分技术，我们在牛肝脏、胰腺以及犬肝脏和胰腺的微粒体膜组分的胆酸钠提取物中检测到一个175 kDa的酪氨酸- o -硫酸盐（TyrS）结合蛋白。Western blot分析显示，牛肝脏tyrs结合蛋白在体内和体外均与酪氨酸-硫酸化蛋白复合物中存在，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，证实其合成的酪氨酸磺酸蛋白主要分泌到根尖培养基中。结果进一步表明，MDCK细胞产生的两种主要的分泌蛋白，纤维连接蛋白（FN）和80 kDa糖蛋白（gp 80）的不同硫酸盐形式的产生和差异极化分泌，其中酪氨酸-硫酸盐形式主要从顶端表面分泌。用糖基化抑制剂，苦马豆素和1-脱氧甘露诺吉霉素处理滤过培养的MDCK细胞，似乎可以增强酪氨酸磺酸化FN和gp 80的顶端分泌。一个类似的175 kDa的膜结合的“TyrS受体”与犬肝TyrS受体的抗血清交叉反应。存在于MDCK细胞中。脉冲追踪实验显示其存在于新合成的FN和gp 80配合物中。在酪氨酸硫酸盐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

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.