Function of DADl Protein in Programd Cell Death

我们从仓鼠BHK21细胞系中分离到一系列温度敏感型生长突变体。在这些ts突变体中，我们发现了在限制性温度下进入细胞死亡的突变体。在其中一个tsBN7中，染色体DNA以典型的依赖于蛋白质合成的凋亡性细胞死亡的方式碎裂。我们克隆了一个与tsBN7突变互补的人类基因，命名为DAD1，编码一个12.5 kDa的蛋白，该蛋白在进化过程中非常保守。作为合作，我们克隆了秀丽隐杆线虫的dadl同源基因(ce-dadl)。Ce-DAD1与脊椎动物DAD1在氨基酸水平上有60%以上的同源性，并且与tsBN7突变有很好的互补。通过过表达，人DAD1抑制了优雅线虫的程序性细胞死亡。最近，人们发现DAD1是参与N连接糖基化的酵母OST2的同源物。基于这一发现，我们发现在tsBN7细胞中，P450的糖基化是温度敏感的。据报道，N连接糖基化发生在进入高尔基体之前，DAD1似乎位于内质网。我们不知道N连接糖基化缺陷是如何导致细胞凋亡的。为了阐明这一问题，我们通过基因打靶的方法从小鼠染色体上删除了DAD1基因。

We have isolated a series of temperature sensitive growth mutant from the hamster BHK21 cell line. Among those ts mutants, we found the mutants which enter apoptotic cell death at the restrictive temperature. In one of them tsBN7, chromosomal DNA was fragmented with a typical manner like apoptptic cell death depending on protein synthesis. We have cloned a human gene complementing tsBN7 mutation and designated as DAD1 encoding a protein of 12.5 kDa which is well conserved through evolution. As a co-work, we have cloned a dadl homologue of Caenorhabditis elegans (Ce-dadl). Ce-dadl is more than 60% identical to vertebrate DAD1 in amino acid level and well complemented tsBN7 mutation. By overexpression, human DAD1 suppressed programd cell death in C.elegance.Recently, DAD1 was found to be a homologue of yeast OST2 involved in N linked glycosylation. Based on this finding, we found that a glycosylation of p450 is temperature sensitive in tsBN7 cells. As reported that N linked glycosylation occurs prior to entering Golgi apparatus, DAD1 seems to be located in endoplasmic reticulum. We don't know how a defect of N linked glycosylation causes apoptotic cell death. In order to clarify this issue, we are deleting DAD1 gene from mouse chromosome by gene targeting.