CHARACTERIZATION OF MAMMALIAN ADP-RIBOSYLTRANSFERASES

哺乳动物 ADP-核糖基转移酶的表征

• 批准号: 6290379
• 负责人: Joel 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/6290379
• 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 post-translational 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). Similar to cholera toxin, some mammalian ADP- ribosyltransferases specifically use the guanidino group of arginine as an ADP-ribose acceptor. Five mammalian NAD: arginine ADP- ribosyltransferases (ART) were cloned from various tissues. ART1 was shown to be a cell surface protein, linked through a glycosylphosphatidylinositol (GPI) anchor. The transferases appear to be selectively expressed in mammalian tissues. ART-1 is found in skeletal and cardiac muscle and lymphoid cells, ART-2 in lymphocytes, ART-4 in spleen andART-5 in testis. ART-5 is primarily an NAD glycohydrolase, with significantly less NAD: arginine ADP- ribosyltransferase. Upon incubation with NAD under standard assay conditions, in the presence or absence of agmatine, an arginine analog, the NAD glycohydrolase activity gradually declines; in contrast, the ADP-ribosyltransferase, activity after a brief delay, increases in velocity, and then, after about one hour, appears to be constant for several hours. The loss of NAD glycohydrolase activity and the increase in ADP-ribosyltransferase activity is associated with the appearance on sodium dodecyl sulfate-polyacrylamide gels of an auto-ADP-ribosylated form of ART-5. One ADP-ribosylation appears to be sufficient for the dramatic changes in NAD glycohydrolase and auto-ADP-ribosyltransferase activities. Incubation for greater than one hour, however, results in the further addition of ADP-ribose moieties. To verify that the auto- ADP-ribosylated ART-5 exhibited differences in enzymatic activities, the transferase was incubated with NAD for one or eight hours, then purified by sequential chromatography over two PD-10 gel permeation columns. The auto-ADP-ribosylated enzyme exhibited an increase in ADP- ribosyltransferase activity and decrease in NAD glycohydrolase when compared to control. The fact that ART-5 incubated with NAD was modified with ADP-ribose was verified by mass spectrometry, which demonstrated an increase of 541 mass units. These data support the hypothesis that the auto-ADP-ribosylation can serve a regulatory role and determine the relative levels of NAD glycohydrolase and ADP- ribosyltransferase activities. - Mono-ADP-ribosylation, ADP- ribosyltransferases, bacterial toxins

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中，并且负责一些细菌毒素的毒性（例如，霍乱毒素、百日咳毒素）。类似于霍乱毒素，一些哺乳动物ADP-核糖基转移酶特异性地使用精氨酸的胍基作为ADP-核糖受体。从不同组织中克隆了5个哺乳动物NAD：精氨酸ADP-核糖基转移酶（ART）. ART 1被证明是一种细胞表面蛋白，通过糖基磷脂酰肌醇（GPI）锚连接。转移酶似乎在哺乳动物组织中选择性表达。ART-1存在于骨骼肌、心肌和淋巴样细胞中，ART-2存在于淋巴细胞中，ART-4存在于脾中，ART-5存在于睾丸中。ART-5主要是NAD糖水解酶，具有显著较少的NAD：精氨酸ADP-核糖基转移酶。在标准测定条件下与NAD孵育后，在存在或不存在胍丁胺（精氨酸类似物）的情况下，NAD糖水解酶活性逐渐下降;相反，ADP-核糖基转移酶活性在短暂延迟后速度增加，然后在约1小时后，似乎在数小时内保持恒定。NAD糖水解酶活性的丧失和ADP-核糖基转移酶活性的增加与ART-5的自ADP-核糖基化形式在十二烷基硫酸钠-聚丙烯酰胺凝胶上的出现有关。一个ADP-核糖基化似乎足以使NAD糖水解酶和自身ADP-核糖基转移酶活性发生显著变化。然而，孵育超过1小时导致ADP-核糖部分的进一步添加。为了验证自ADP-核糖基化的ART-5表现出酶活性的差异，将转移酶与NAD孵育1或8小时，然后通过连续色谱法在两个PD-10凝胶渗透柱上纯化。与对照相比，自身ADP核糖基化酶表现出ADP核糖基转移酶活性的增加和NAD糖水解酶的减少。通过质谱法验证了用NAD孵育的ART-5被ADP-核糖修饰的事实，其证明增加了541质量单位。这些数据支持这样的假设，即自身ADP-核糖基化可以起到调节作用，并决定NAD糖水解酶和ADP-核糖基转移酶活性的相对水平。- 单ADP核糖基化，ADP核糖基转移酶，细菌毒素