Muresan Z, Paul D L, Goodenough D A
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Biol Cell. 2000 Feb;11(2):627-34. doi: 10.1091/mbc.11.2.627.
Occludin and claudin are the major integral membrane components of the mammalian tight junction. Although more than 11 distinct claudins have been identified, only 1 occludin transcript has been reported thus far. Therefore, we searched by reverse transcription-PCR for occludin-related sequences in Madin-Darby canine kidney (MDCK) mRNA and identified a transcript encoding an alternatively spliced form of occludin, designated occludin 1B. The occludin 1B transcript contained a 193-base pair insertion encoding a longer form of occludin with a unique N-terminal sequence of 56 amino acids. Analysis of the MDCK occludin gene revealed an exon containing the 193-base pair sequence between the exons encoding the original N terminus and the distal sequence, suggesting that occludin and occludin 1B arise from alternative splicing of one transcript. To assess the expression and distribution of occludin 1B, an antibody was raised against its unique N-terminal domain. Immunolabeling of occludin 1B in MDCK cells revealed a distribution indistinguishable from that of occludin. Furthermore, occludin 1B staining at cell-to-cell contacts was also found in cultured T84 human colon carcinoma cells and in frozen sections of mouse intestine. Immunoblots of various mouse tissues revealed broad coexpression of occludin 1B with occludin. The wide epithelial distribution and the conservation across species suggests a potentially important role for occludin 1B in the structure and function of the tight junction.
闭合蛋白和紧密连接蛋白是哺乳动物紧密连接的主要整合膜成分。尽管已鉴定出11种以上不同的紧密连接蛋白,但迄今为止仅报道了1种闭合蛋白转录本。因此,我们通过逆转录-聚合酶链反应在犬肾Madin-Darby(MDCK)mRNA中搜索闭合蛋白相关序列,并鉴定出一种编码闭合蛋白可变剪接形式的转录本,命名为闭合蛋白1B。闭合蛋白1B转录本包含一个193个碱基对的插入片段,编码一种更长形式的闭合蛋白,其独特的N端序列有56个氨基酸。对MDCK闭合蛋白基因的分析显示,在编码原始N端的外显子和远端序列之间有一个包含193个碱基对序列的外显子,这表明闭合蛋白和闭合蛋白1B来自一个转录本的可变剪接。为了评估闭合蛋白1B的表达和分布,制备了一种针对其独特N端结构域的抗体。MDCK细胞中闭合蛋白1B的免疫标记显示其分布与闭合蛋白无法区分。此外,在培养的T84人结肠癌细胞和小鼠肠道冰冻切片的细胞间接触处也发现了闭合蛋白1B染色。各种小鼠组织的免疫印迹显示闭合蛋白1B与闭合蛋白广泛共表达。其广泛的上皮分布和跨物种保守性表明闭合蛋白1B在紧密连接的结构和功能中可能具有重要作用。