CHARACTERIZATION OF MAMMALIAN ADP-RIBOSLYTRANSFERASES

单腺苷二磷酸核糖化是蛋白质的翻译后修饰 其中NAD的ADP-核糖部分被转移到蛋白质上并被 对某些细菌毒素(如霍乱)的毒性负责 百日咳毒素)。哺乳动物NAD的五个成员：精氨酸 ADP-核糖基转移酶(Art1-5)是从不同组织中克隆的。这个 来自心肌、骨骼肌和淋巴细胞的Art1蛋白是 糖基磷脂酰肌醇(GPI)修饰的锚定蛋白 细胞外黏附分子。第二个ADP-核糖基转移酶 (ART5)是从小鼠淋巴瘤细胞中克隆出来的，但在 主要是在睾丸。ART5蛋白是膜结合的，但与 Art1酶似乎不是GPI锚定的。Art1和Art5 谷胱甘肽-S-转移酶融合蛋白在E. Coli，用于比较它们的ADP-核糖基转移酶和NAD 糖水解酶活性。使用胍丁胺作为ADP-核糖受体， Art1主要是一种ADP-核糖基转移酶，而 重组ART5的转移酶和NAD糖水解酶活性 等价物。哺乳动物ART的推导氨基酸序列 蛋白质包含几种细菌共有的三个共同区域 毒素转移酶。这些区域已经被证明在一定程度上形成了 细菌毒素转移酶的活性部位。站点定向 Art1活性部位谷氨酸的诱变作用被取消 转移酶活性。此外，当ART1的羧基末端半部分， 它包含假定对维持 功能催化位点，以GST-融合蛋白的形式表达 截短酶保持NAD糖水解酶活性与 假设存在NAD结合的共同机制和 ADP-核糖基转移酶之间的催化作用。

Mono-ADP-ribosylation is a posttranslational modification of proteins in which the ADP-ribose moiety of NAD is transferred to proteins and is responsible for the toxicity of some bacterial toxins (e.g., cholera toxin, pertussis toxin). Five members of the mammalian NAD:arginine ADP-ribosyltransferases (ART1-5) were cloned from various tissues. The ART1 proteins from cardiac and skeletal muscle and lymphocytes are glycosylphosphatidylinositol (GPI)-anchored proteins that modify extracellular adhesion molecules. A second ADP-ribosyltransferase (ART5) was cloned from mouse lymphoma cells, but is expressed predominantly in testis. The ART5 protein is membrane-bound but, unlike the ART1 enzyme, appears not to be GPI-anchored. The ART1 and ART5 enzymes, expressed as glutathione-S-transferase fusion proteins in E. coli, were used to compare their ADP-ribosyltransferase and NAD glycohydrolase activities. Using agmatine as the ADP-ribose acceptor, ART1 was predominantly an ADP-ribosyltransferase, whereas the transferase and NAD glycohydrolase activities of recombinant ART5 were equivalent. The deduced amino acid sequence of the mammalian ART proteins contains three consensus regions common to several bacterial toxin transferases. These regions have been shown to form, in part, the active site of the bacterial toxin transferases. Site-directed mutagenesis of the proposed active-site glutamate of ART1 abolished transferase activity. Further, when the carboxy-terminal half of ART1, which contains the regions postulated to be crucial to maintaining a functional catalytic site, was expressed as a GST-fusion protein, the truncated enzyme retained NAD glycohydrolase activity consistent with the hypothesis that there is a common mechanism of NAD binding and catalysis among ADP-ribosyltransferases.