A FUNCTIONAL ROLE OF N-GLYCANS AS SORTING SIGNALS FOR GLYCOPROTEINS IN EPITHELIAL CELLS

细胞极化是生物功能中组织形成的一个重要特征。为了阐明n链糖链是否与细胞极化有关，我们以Madin-Darby犬肾（MDCK）细胞在膜过滤器上极化为模型。首先，分析了凝集素a对[^<35>S]蛋氨酸标记细胞顶端和基底外侧分泌的蛋白的亲和力。这些结果表明，根尖蛋白和基底外侧蛋白对Con A和AAL柱的结合有很大差异，说明根尖侧分泌的糖蛋白与基底外侧分泌的糖蛋白具有不同的糖链组成。其次，分析了膜结合糖蛋白的糖链结构。用[^3H]甘露糖标记极化的MDCK细胞后，对每侧的膜结合糖蛋白进行生物素化，用营养剂X-100裂解，通过亲和素柱纯化。将[^3H]甘露糖标记的低聚糖通过肼水解从这些糖蛋白中释放出来后，用纸电泳、唾液酸酶切和甲醇水解对其进行分析。结果表明，中性糖链：唾液化糖链：两侧硫酸化糖链的比例约为20:3:1。在Bio-Gel P-4柱层析和几种凝集素柱层析上，中性糖链主要为高甘露糖型，酸性糖链主要为复杂型，包括n -乙酰乳胺重复结构。根尖膜糖蛋白比基底膜糖蛋白更集中。另一方面，由于转运囊泡蛋白VIP 36的n端编码结构域与豆科植物凝集素具有同源性，因此对其进行了分析。利用RT-PCR方法从MDCK细胞总RNA中获得编码VIP 36细胞外结构域的cDNA，并与pGEX中编码谷胱甘氨酸- s -转移酶（gluthatione-S-transferase， GST）的cDNA下游相连。质粒在大肠杆菌中转化，表达融合蛋白GST / Vip36，用谷胱甘肽Sepharose 4B纯化。纯化后的蛋白用于检测与[^<35>S]蛋氨酸标记的顶端分泌蛋白的结合。结果表明，融合蛋白在pH 5.5条件下与多种分泌蛋白结合，在pH 7.4条件下分离。VIP 36与主要存在于顶端膜蛋白中的集中糖链的关系目前正在研究中。少

Cell polarization is an important character on tissue formation of biological functions. To clarify whether N-linked sugar chains are related to cell polarization, Madin-Darby canine kidney (MDCK) cells polarized on membrane filter were used as the model. First, the lectin a affinity against proteins secreted from apical and basolateral sides of [^<35>S]methionine labeled cells were analyzed. These results showed that the binding against Con A and AAL columns was great different between apical and basolateral proteins, indicationg that secreted glycoproteins from apical side have distinct sugar chain compositions from those of basolateral side. Second, the sugar chain structures of membrane-bound glycoproteins from each side were analyzed. After labeling polarized MDCK cells with [^3H]mannose, membrane-bound glycoproteins of each side were biotinylated, lysed with trition X-100 and purified through avidin column.After [^3H] mannose-labeled oligosaccharides were released from these glyc … More oproteins by hydrazinolysis, they were analyzed with paper electrophoresis, sialidase digestion and methanolysis. These results showed that the ratio of neutral sugar chains : sialylated sugar chains : sulfated sugar chains of both sides were about 20 : 3 : 1. On Bio-Gel P-4 column chromatography and severallectin column chromatographies, the neutral sugar chains were mainly high mannose type and acidic sugar chains were complex type including N-acetyllactosamine repeating structures. It was also elucidated that apical membrane glycoproteins were more fucosylated than basolateral ones. On the other hand, transport vesicle protein VIP 36 was analyzed because the N-terminal encoded domain shows homology to leguminous plant lectins. The cDNA encoded extracelldomain of VIP 36 was prepared from total RNA of MDCK cells by the method of RT-PCR,and linked to the downtream of cDNA encoded gluthatione-S-transferase (GST) in pGEX.The plasmids were transformed in E.coli, and the fusion proteins (GST / Vip36) were expressed and purified with gluthatione Sepharose 4B.The purified proteins were used for examination of binding to [^<35>S]methionine labeled apical secreted proteins. It was found that the fusion proteins bind several secreted proteins under the condition of pH 5.5 and they dissociate under pH 7.4. The relationship of VIP 36 and fucosylated sugar chains found in mainly apical membrane proteins is now investigated. Less