TOPOLOGICAL DISTRIBUTION OF 2 CONNEXIN32 ANTIGENIC SITES IN INTACT AND SPLIT RODENT HEPATOCYTE GAP-JUNCTIONS
TOPOLOGICAL DISTRIBUTION OF 2 CONNEXIN32 ANTIGENIC SITES IN INTACT AND SPLIT RODENT HEPATOCYTE GAP-JUNCTIONS
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DOI：
10.1083/jcb.107.5.1817
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发表时间：
1988-11-01
影响因子：
7.8
通讯作者：
JESAITIS, L
中科院分区：
文献类型：
--
作者：
GOODENOUGH, DA;PAUL, DL;JESAITIS, L
在大鼠和小鼠肝细胞缝隙连接中，缝隙连接蛋白32是一种主要的细胞间隙连接多肽。用合成的寡肽抗血清研究了缝隙连接蛋白32的膜拓扑结构。根据已发表的亲水性图谱和确定的蛋白酶敏感切割位点，我们合成了与连接蛋白32的两个亲水结构域相对应的寡肽，一个结构域面向细胞质，另一个结构域被预测为定向于细胞外。将抗血清提纯为寡肽的锁孔帽状血蓝蛋白结合物，并用间接免疫细胞化学方法在分离的缝隙连接上定位其抗原的分布。这些结果直接证明了包含在连接蛋白32序列的98-124个氨基酸中的抗原的细胞质定向。在完整的、孤立的缝隙连接中，细胞外空间太小，不能与抗体分子结合，因此有必要通过实验分离连接膜，以使用尿素/碱程序暴露其细胞外表面。虽然在一些分离的连接膜的细胞外表面可以看到包含在氨基酸164-189中的抗原，但在分离连接膜所需的变性条件之后，观察和分裂过程中的可变性留下了关于观察的生物学相关性的含糊不清。然而，使用一种不同的方法，抗原可以通过高渗二糖连接裂解程序在完整的肝脏中暴露。在细胞表面暴露抗原的时间在30 S时达到高峰，2-4分钟后消失。综上所述，这些数据表明了包含在氨基酸164-189中的抗原的细胞外定位，这可能参与缝隙连接内的细胞间相互作用。
The membrane topology of connexin32, a principal polypeptide of gap junctions in diverse cell types, has been studied in rat and mouse hepatocyte gap junctions using site-specific antisera raised against synthetic oligopeptides corresponding to amino acid sequences deduced from cDNA clones. Based on published hydropathicity maps and identified protease-sensitive cleavage sites, oligopeptides were synthesized corresponding to two hydrophilic domains of connexin32, one predicted to face the cytoplasm, the other predicted to be directed extracellularly. Antisera were raised to keyhole limpet hemocyanin conjugates of the oligopeptides and used to map the distribution of their antigens using indirect immunocytochemistry on isolated gap junctions. The results directly demonstrated the cytoplasmic orientation of an antigen contained within amino acids 98-124 of the connexin32 sequence. The extracellular space in intact, isolated gap junctions is too small to permit binding of antibody molecules, necessitating the experimental separation of the junctional membranes to expose their extracellular surfaces using a urea/alkali procedure. While an antigen contained within amino acids 164-189 was visualized on the extracellular surfaces of some of the separated junctional membranes, variability in the observations and in the splitting procedure left ambiguities concerning the biological relevance of the observations after the denaturing conditions necessary to separate the junctional membranes. Using a different approach, however, the antigen could be exposed in intact liver using a hypertonic disaccharide junction-splitting procedure. The period of time of antigen exposure at the cell surface appears to peak at 30 s and disappear by 2-4 min. Taken together, these data demonstrate the extracellular orientation of an antigen contained within amino acids 164-189, which may be involved in cell-cell interaction within the gap junction.