Department of Biochemistry, University of Texas Health Center at Tyler, Tyler, TX, 75710, USA.
Cytotechnology. 1997 Jan;23(1-3):143-9. doi: 10.1023/A:1007903315687.
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 filter-grown 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 receptor', 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.
利用亲和凝胶层析技术，我们在牛肝和胰腺以及犬肝和胰腺的微粒体膜部分的胆酸钠提取物中检测到一种 175kDa 的酪氨酸-O-硫酸盐（TyrS）结合蛋白。Western blot 分析表明，牛肝 TyrS 结合蛋白存在于体内和体外与酪氨酸硫酸化蛋白形成的复合物中，这表明前者可能是后者的受体。使用滤膜培养的犬肾 (MDCK) 细胞作为模型，证明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 80kDa 糖蛋白 (gp80)）的不同硫酸化形式的产生和差异极化分泌，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 swainsonine 和 1-deoxymannojirimycin 处理滤膜培养的 MDCK 细胞，似乎增强了酪氨酸硫酸化 FN 和 gp80 的顶端分泌。在 MDCK 细胞中也显示存在一种类似的 175kDa 膜结合的“TyrS 受体”，它与抗犬肝 TyrS 受体的抗血清交叉反应。脉冲追踪实验表明它存在于与新合成的 FN 和 gp80 形成的复合物中。提出了一种假设模型，即 TyrS 残基作为 MDCK 细胞中酪氨酸硫酸化蛋白生物合成转运过程中的顶端靶向信号，由 TyrS 受体介导。
