Ceni Claire, Muller-Steffner Hélène, Lund Frances, Pochon Nathalie, Schweitzer Annie, De Waard Michel, Schuber Francis, Villaz Michel, Moutin Marie-Jo
Département de Réponse et Dynamique Cellulaires-Commissariat à l'Energie Atomique (DRDC-CEA), 17 avenue des Martyrs, 38054 Grenoble, France.
J Biol Chem. 2003 Oct 17;278(42):40670-8. doi: 10.1074/jbc.M301196200. Epub 2003 Aug 8.
Cyclic ADP-ribose, a metabolite of NAD+, is known to modulate intracellular calcium levels and signaling in various cell types, including neural cells. The enzymes responsible for producing cyclic ADP-ribose in the cytoplasm of mammalian cells remain unknown; however, two mammalian enzymes that are capable of producing cyclic ADP-ribose extracellularly have been identified, CD38 and CD157. The present study investigated whether an ADP-ribosyl cyclase/NAD+-glycohydrolase independent of CD38 is present in brain tissue. To address this question, NAD+ metabolizing activities were accurately examined in developing and adult Cd38-/- mouse brain protein extracts and cells. Low ADP-ribosyl cyclase and NAD+-glycohydrolase activities (in the range of pmol of product formed/mg of protein/min) were detected in Cd38-/- brain at all developmental stages studied. Both activities were found to be associated with cell membranes. The activities were significantly higher in Triton X-100-treated neural cells compared with intact cells, suggesting an intracellular location of the novel cyclase. The cyclase and glycohydrolase activities were optimal at pH 6.0 and were inhibited by zinc, properties which are distinct from those of CD157. Both activities were enhanced by guanosine 5'-O-(3-thiotriphosphate), a result suggesting that the novel enzyme may be regulated by a G protein-dependent mechanism. Altogether our results indicate the presence of an intracellular membrane-bound ADP-ribosyl cyclase/NAD+-glycohydrolase distinct from CD38 and from CD157 in mouse brain. This novel enzyme, which is more active in the developing brain than in the adult tissue, may play an important role in cyclic ADP-ribose-mediated calcium signaling during brain development as well as in adult tissue.
环磷酸腺苷核糖是烟酰胺腺嘌呤二核苷酸（NAD⁺）的一种代谢产物，已知其可调节包括神经细胞在内的多种细胞类型的细胞内钙水平和信号传导。负责在哺乳动物细胞质中产生环磷酸腺苷核糖的酶仍不清楚；然而，已鉴定出两种能够在细胞外产生环磷酸腺苷核糖的哺乳动物酶，即CD38和CD157。本研究调查了脑组织中是否存在一种独立于CD38的ADP核糖基环化酶/NAD⁺糖水解酶。为了解决这个问题，在发育中和成年的Cd38⁻/⁻小鼠脑蛋白提取物和细胞中准确检测了NAD⁺代谢活性。在所有研究的发育阶段，Cd38⁻/⁻脑中均检测到低水平的ADP核糖基环化酶和NAD⁺糖水解酶活性（产物形成的皮摩尔数/毫克蛋白质/分钟范围内）。发现这两种活性均与细胞膜相关。与完整细胞相比，经Triton X - 100处理的神经细胞中的活性显著更高，这表明新型环化酶位于细胞内。环化酶和糖水解酶活性在pH 6.0时最佳，并受到锌的抑制，这些特性与CD157不同。两种活性均被鸟苷5'-O-(3-硫代三磷酸)增强，这一结果表明新型酶可能受G蛋白依赖性机制调节。我们的结果总体表明，小鼠脑中存在一种与CD38和CD157不同的细胞内膜结合ADP核糖基环化酶/NAD⁺糖水解酶。这种新型酶在发育中的脑中比在成年组织中更活跃，可能在脑发育过程以及成年组织中环磷酸腺苷核糖介导的钙信号传导中发挥重要作用。
