Kido M, Obata S, Tanihara H, Rochelle J M, Seldin M F, Taketani S, Suzuki S T
Doheny Eye Institute, University of Southern California School of Medicine, Los Angeles 90033, USA.
Genomics. 1998 Mar 1;48(2):186-94. doi: 10.1006/geno.1997.5152.
Cloning of rat cadherin-8 cDNA demonstrated two types of cDNAs. The overall structure of the protein defined by one type of the cDNA is essentially the same as that of classic cadherins, whereas the protein defined by the other type of cDNA ends near the N-terminus of the fifth repeat of the extracellular domain (EC5) and contains a short unique sequence at the C-terminus. The same truncated type of cDNA was also obtained from a human cDNA library. In Northern blot analysis of rat brain mRNA, a probe for EC5 detected multiple bands of about 3.5-4.3 knt, whereas a probe for the alternative form hybridized with a band of about 3.5 knt. Western blot experiments showed that an antibody against the extracellular domain of rat cadherin-8 stained a band of about 95 kDa and a faint band of about 130 kDa in rat brain extract. These results suggest that cadherin-8 is expressed in two forms, a complete form and a truncated form without a transmembrane domain or cytoplasmic domain, in brain. The complete form of cadherin-8 expressed in L cells was about 130 kDa in molecular mass and was located at the cell periphery, mainly at the cell-cell contact sites. However, we failed to express the truncated form in L cells. The transfectants of the complete form showed weak cell adhesion activity. The complete form of cadherin-8 was sensitive to trypsin digestion, and Ca2+ did not protect cadherin-8 from digestion, in contrast to the classic cadherins. The complete form of cadherin-8 coprecipitated with beta-catenin, but did not immunoprecipitate well with alpha-catenin or gamma-catenin. Cadherin-8, as well as cadherin-11, was mapped to a specific region of chromosome 8 that also includes cadherins-1, -3, and -5.
大鼠钙黏蛋白-8 cDNA 的克隆显示出两种类型的 cDNA。由一种类型的 cDNA 所定义的蛋白质的整体结构与经典钙黏蛋白基本相同，而由另一种类型的 cDNA 所定义的蛋白质在细胞外结构域（EC5）的第五个重复序列的 N 端附近终止，并在 C 端含有一个短的独特序列。同样的截短型 cDNA 也从人 cDNA 文库中获得。在大鼠脑 mRNA 的 Northern 印迹分析中，针对 EC5 的探针检测到多条约 3.5 - 4.3 kb 的条带，而针对另一种形式的探针与一条约 3.5 kb 的条带杂交。蛋白质印迹实验表明，抗大鼠钙黏蛋白-8 细胞外结构域的抗体在大鼠脑提取物中染出一条约 95 kDa 的条带和一条约 130 kDa 的 faint 条带。这些结果表明，钙黏蛋白-8 在脑中以两种形式表达，一种是完整形式，另一种是没有跨膜结构域或细胞质结构域的截短形式。在 L 细胞中表达的完整形式的钙黏蛋白-8 分子量约为 130 kDa，位于细胞周边，主要在细胞 - 细胞接触位点。然而，我们未能在 L 细胞中表达截短形式。完整形式的转染子显示出较弱的细胞黏附活性。与经典钙黏蛋白相反，完整形式的钙黏蛋白-8 对胰蛋白酶消化敏感，并且 Ca2+ 不能保护钙黏蛋白-8 不被消化。完整形式的钙黏蛋白-8 与β - 连环蛋白共沉淀，但与α - 连环蛋白或γ - 连环蛋白的免疫沉淀效果不佳。钙黏蛋白-8 以及钙黏蛋白-11 被定位到染色体 8 的一个特定区域，该区域还包括钙黏蛋白-1、-3 和 -5。
