CHARACTERIZATION OF MAMMALIAN ADP-RIBOSLYTRANSFERASES

哺乳动物 ADP-核糖转移酶的表征

• 批准号: 6162666
• 负责人: J MOSS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6162666
• 关键词: ADP ribosylation; adenine phosphoribosyltransferase; arginine; bacterial toxins; biochemical evolution; enzyme activity; enzyme mechanism; enzyme structure; human tissue; laboratory mouse; laboratory rabbit; lymphoma; molecular cloning; protein sequence; striated muscles

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.

单 ADP 核糖基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 造成某些细菌毒素（例如霍乱）的毒性 毒素、百日咳毒素）。 哺乳动物 NAD 的五个成员：精氨酸 从各种组织中克隆了 ADP-核糖基转移酶 (ART1-5)。 这 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) 锚定蛋白可修饰 细胞外粘附分子。 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，但表达 主要分布于睾丸。 ART5 蛋白是膜结合蛋白，但与 ART1 酶似乎不是 GPI 锚定的。 ART1 和 ART5 酶，在大肠杆菌中表达为谷胱甘肽-S-转移酶融合蛋白。 大肠杆菌，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 ADP-核糖基转移酶，而 重组 ART5 的转移酶和 NAD 糖水解酶活性 相等的。 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有区域 毒素转移酶。 这些区域已被证明在一定程度上形成了 细菌毒素转移酶的活性位点。 现场定向 拟议的 ART1 活性位点谷氨酸的诱变被废除 转移酶活性。 此外，当 ART1 的羧基末端一半时， 其中包含被认为对维持一个至关重要的区域 功能催化位点，表达为 GST 融合蛋白， 截短的酶保留了 NAD 糖水解酶活性，与 NAD 结合存在共同机制的假设 ADP-核糖基转移酶之间的催化作用。