Yokoyama H, Mukae N, Sakahira H, Okawa K, Iwamatsu A, Nagata S
Department of Genetics, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2000 Apr 28;275(17):12978-86. doi: 10.1074/jbc.275.17.12978.
Caspase-activated DNase (CAD) is an enzyme that cleaves chromosomal DNA in apoptotic cells. Here, we identified a DNase in Drosophila Schneider cells that can be activated by caspase 3, and purified it as a complex of two subunits (p32 and p20). Using primers based on the amino acid sequence of the purified proteins, a cDNA coding for Drosophila CAD (dCAD) was cloned. The polypeptide encoded by the cDNA contained 450 amino acids with a calculated M(r) of 52,057, and showed significant homology with human and mouse CAD (22% identity). Mammalian CADs carry a nuclear localization signal at the C terminus. In contrast, dCAD lacked the corresponding sequence, and the purified dCAD did not cause DNA fragmentation in nuclei in a cell-free system. When dCAD was co-expressed in COS cells with Drosophila inhibitor of CAD (dICAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (drICE), the dICAD was cleaved, and released from dCAD. In addition, dCAD was also cleaved by these caspases, and behaved as a (p32)(2)(p20)(2) complex in gel filtration. When a Drosophila neuronal cell line was induced to apoptosis by treatment with a kinase inhibitor, both dCAD and dICAD were cleaved. These results indicated that unlike mammalian CAD, Drosophila CAD must be cleaved by caspases to be activated.
半胱天冬酶激活的脱氧核糖核酸酶（CAD）是一种在凋亡细胞中切割染色体DNA的酶。在此，我们在果蝇施奈德细胞中鉴定出一种可被半胱天冬酶3激活的脱氧核糖核酸酶，并将其纯化，得到由两个亚基（p32和p20）组成的复合物。基于纯化蛋白质的氨基酸序列设计引物，克隆了编码果蝇CAD（dCAD）的cDNA。该cDNA编码的多肽含有450个氨基酸，计算分子量为52,057，与人和小鼠的CAD具有显著同源性（同一性为22%）。哺乳动物的CAD在C末端带有核定位信号。相比之下，dCAD缺乏相应序列，且纯化的dCAD在无细胞体系中不会导致细胞核中的DNA片段化。当dCAD在COS细胞中与果蝇CAD抑制剂（dICAD）共表达时，会产生一种52 kDa的dCAD，它与dICAD形成异源四聚体复合物。当该复合物用人半胱天冬酶3或果蝇半胱天冬酶（drICE）处理时，dICAD被切割，并从dCAD中释放出来。此外，dCAD也被这些半胱天冬酶切割，在凝胶过滤中表现为（p32）2（p20）2复合物。当用激酶抑制剂处理果蝇神经元细胞系诱导其凋亡时，dCAD和dICAD都会被切割。这些结果表明，与哺乳动物的CAD不同，果蝇的CAD必须被半胱天冬酶切割才能被激活。
