CHARACTERIZATION OF MAMMALIAN ADP-RIBOSYLTRANSFERASES

单腺苷二磷酸核糖化是一种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，并与某些细菌毒素(如霍乱毒素、百日咳毒素)的毒性有关。像霍乱毒素一样，一些哺乳动物的ADP核糖转移酶专门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)。ART1是一种细胞表面蛋白，通过糖基磷脂酰肌醇(GPI)锚点连接。这些转移酶似乎选择性地在哺乳动物组织中表达。ART-1存在于骨骼、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。ADP-核糖基转移酶具有一个在哺乳动物和细菌中保守的核心催化结构域。为了确定对ADP-核糖基转移酶活性的要求，从ART-1的小鼠同源物中获得截断突变体。该转移酶催化精氨酸和类似的小分子胍基化合物以及含有精氨酸残基的多肽和蛋白质的ADP-核糖化。在缺乏ADP-核糖的胍基受体的情况下，转移酶将NAD水解为ADP-核糖和烟酰胺，但比精氨酸ADP-核糖化反应慢得多。去掉氨基末端和羧基末端的信号序列，导致NAD：精氨酸ADP-核糖基转移酶的活性。氨基末端是输出到内质网所必需的，而羧基末端是添加GPI锚所必需的。氨基酸末端序列的进一步缺失导致转移酶活性的显著丧失，通过蛋白质和游离精氨酸的ADP-核糖化来衡量。相反，蛋白质氨基末端区域的缺失增加了NAD糖水解酶的活性，表明氨基末端对NAD的流产水解酶有抑制作用。相反，去掉离假定催化位点更近的羧基末端的氨基酸，会导致转移酶和糖水解酶活性的丧失。

Mono-ADP-ribosylation is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to protein and is responsible for the toxicity of some bacterial toxins (e.g., cholera toxin, pertussis toxin). Like 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 is 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 and ART-5 in testis.The ADP-ribosyltransferases are characterized by a core catalytic domain that is conserved among the mammalian and bacterial enzymes. To define the requirements for ADP-ribosyltransferase activity, truncation mutants were made from the murine homologue of ART-1. This transferase catalyzes the ADP-ribosylation of arginine, and similar small guanidino compounds, as well as peptides and proteins containing arginine residues. In the absence of a guanidino acceptor for ADP-ribose, the transferase hydrolyzes NAD to ADP-ribose and nicotinamide, but at a much slower rate than arginine ADP-ribosylation. Removal of the signal sequences at the amino terminus, which is required for export into the endoplasmic reticulum, and at the carboxy terminus, which is necessary for addition of the GPI anchor, resulted in an active NAD:arginine ADP-ribosyltransferase. Further deletion of sequence from the amino terminus resulted in a significant loss of transferase activity, measured by ADP-ribosylation of both proteins and free arginine. In contrast, deletion of the amino terminal region of the protein increased NAD glycohydrolase activity, suggesting that the amino terminus has an inhibitory effect on abortive hydrolysis of NAD. In contrast, removal of amino acids at the carboxy terminus, which are closer to the putative catalytic site, resulted in a loss of both transferase and glycohydrolase activities.