CHARACTERIZATION OF MAMMALIAN ADP-RIBOSLYTRANSFERASES

单腺苷二磷酸核糖基化是蛋白质翻译后的修饰 其中NAD的ADP-核糖部分被转移到蛋白质， 导致某些细菌毒素的毒性（例如，霍乱 毒素、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（ART 1 -5）是从各种组织中克隆的。 的 来自心肌、骨骼肌和淋巴细胞的ART 1蛋白是 糖基磷脂酰肌醇（GPI）锚定蛋白， 细胞外粘附分子。 第二ADP-核糖基转移酶 （ART 5）是从小鼠淋巴瘤细胞克隆的，但表达于 主要在睾丸中。 ART 5蛋白是膜结合的，但与 ART 1酶似乎不是GPI锚定的。 ART 1和ART 5 在大肠杆菌中表达谷胱甘肽-S-转移酶融合蛋白。 coli中的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， ART 1主要是ADP-核糖基转移酶，而 重组ART 5的转移酶和NAD糖水解酶活性 相当于 哺乳动物ART的推导氨基酸序列 蛋白质包含三个共有区，这些共有区是几种细菌 毒素转移酶 这些区域已被证明形成，部分， 细菌毒素转移酶的活性位点。 定点 ART 1的拟议活性位点谷氨酸的诱变被废除 转移酶活性 此外，当ART 1的羧基末端一半， 它包含了被认为对维持一个 功能性催化位点，表达为GST融合蛋白， 截短的酶保留NAD糖水解酶活性， 假设存在NAD结合的共同机制， 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.