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Elucidation of Functional Implication of Post-translational Tyrosine Sulfation

翻译后酪氨酸硫酸化的功能意义的阐明

基本信息

批准号：
06660117
负责人：
SUIKO Masahito
金额：
$ 1.34万
依托单位：
Miyazaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

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.

通过采用亲和凝胶馏分技术，我们已经检测到175 kDa酪氨酸-O-硫酸（TyrS）结合蛋白的胆酸钠提取物的微粒体膜组分的牛肝脏和胰腺，以及犬肝脏和胰腺。蛋白质印迹分析揭示了牛肝脏TyrS结合蛋白在体内和体外与酪氨酸硫酸化蛋白的复合物中的存在，表明前者的推定作用是后者的受体。使用过滤生长的Madin-Darby犬肾（MDCK）细胞作为模型，证明了合成的酪氨酸硫酸化蛋白主要分泌到顶端培养基中。结果进一步表明，MDCK细胞产生的两种主要分泌蛋白，纤连蛋白（FN）和80 kDa糖蛋白（gp 80）的不同硫酸化形式的生产和差异极化分泌，其酪氨酸硫酸化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mannojirimycin处理过滤生长的MDCK细胞，似乎能增强酪氨酸硫酸化FN和gp 80的顶端分泌。类似的175 kDa膜结合的“TyrS受体。“与犬肝TyrS受体抗血清交叉反应。显示存在于MDCK细胞中。脉冲追踪实验表明其存在于新合成的FN和gp 80的复合物中。酪氨酸硫酸化蛋白生物合成转运过程中作为顶端靶向信号的TyrS残基的假设模型。由TyrS受体介导。在MDCK细胞中。