Mechanism of cyclic ADP-ribose hydrolysis in mammalian cells.

哺乳动物细胞中环状 ADP-核糖水解机制。

• 批准号: 06454165
• 负责人: TAKASAWA Shin
• 金额: $ 4.61万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454165/
• 关键词: CD38; cyclic ADP-ribose; ADP-ribosyl cyclase; cyclic ADP-ribose hydrolase; second messenger; transgenic mouse; insulin secretion; molecular cloning; 部位特異的点突然変異導入法

1.We isolated a cDNA for CD38, which has been reported to be a human leukocyte atigen, from a human insulinoma and expressed the cDNA in COS-7 cells. When we incubated the plasma membrane with fraction NAD^+ or cyclic ADP-ribose (cADPR) and analyzed the reaction products by HPLC,the formation and hydrolysis of cADPR (ADP-ribosyl cyclase and cADPR hydrolase activities) were detected. Moreover, we found that ATP (2-10 mM), generated in the glucose metabolism in beta-cells, inhibited the cADPR-hydrolyzing activity, resulting in the accumulation of cADPR.2.We isolated rat CD38 cDNA from islets of Langerhans and determined its primary structure. Rat CD38 is composed of 303 amino acids and shares 89% homology with human CD38. The membrane fraction of the COS-7 cells into which rat CD38 expression vector had been introduced exhibited both ADP-ribosyl cyclase and cADPR hydrolase activities. Rat CD38 mRNA is expressed in various tissues including pancreatic islets but not in RINm5F cells.3.We p … More roduced transgenic mice overexpressing human CD38 in pancreatic beta cells. The enzymatic activity of CD38 in transgenic islets was greatly increased, and ATP efficiently inhibited the cADPR hydrolase activity. Glucose- and ketoisocaproate (which, like glucose, generates ATP during the metabolism) -induced but not tolbutamide- nor KCI-induced insulin secretion from transgenic islets were 1.7-2.3-fold higher than that of control. In glucose-tolerance tests, the transgenic serum insulin level was higher than that of control. Thus, the cADPR-CD38 signaling system has a regulatory role in insulin secretion by glucose in beta-cells, suggesting that not only Ca^<2+> from extracellular sources (Ca^<2+> influx through voltage-dependent Ca^<2+> channels evoked by glucose-induced cell membrane depolarization) but also Ca^<2+> release from intracellular stores (cADPR-induced Ca^<2+> release from the endoplasmic reticulum) play important roles in insulin secretion.4.We introduced site-directed mutations to CD38 and found that C119K- and/or C202E-CD38 exhibited only ADP-ribosyl cyclase activity. Furthermore, Aplysia ADP-ribosyl cyclase into which we introduced the mutations K95C and E176C,which correspond to residues 119 and 201 of human CD38, exhibited not only ADP-ribosyl cyclase activity but also cADPR hydrolase.5.We isolated human CD38 gene and determined its structure. The CD38 is consists of 8 exons that extending -100 kbp on the human genome, and is mapped to human chromosome 4p 15 by fluorescent in situ hybridization. Less

1.从人胰岛素瘤组织中分离出CD 38的cDNA，并在COS-7细胞中表达。将细胞质膜与NAD^+或cADPR共孵育，用HPLC分析反应产物，检测cADPR的形成和水解（ADP-核糖环化酶和cADPR水解酶活性）。此外，我们还发现β细胞葡萄糖代谢过程中产生的ATP（2-10 mM）抑制cADPR的水解活性，导致cADPR的积累。2.从大鼠胰岛中分离CD 38 cDNA，并测定其一级结构。大鼠CD 38由303个氨基酸组成，与人CD 38具有89%的同源性。导入大鼠CD 38表达载体的COS-7细胞的膜部分表现出ADP-核糖基环化酶和cADPR水解酶活性。大鼠CD 38 mRNA在包括胰岛在内的多种组织中表达，但在RINm 5 F细胞中不表达。 ...更多信息 在胰腺β细胞中产生过表达人CD 38的转基因小鼠。转基因胰岛中CD 38的酶活性显著升高，ATP能有效抑制cADPR水解酶的活性。葡萄糖和酮异己酸（与葡萄糖一样，在代谢过程中产生ATP）诱导的转基因胰岛胰岛素分泌比对照高1.7-2.3倍，但甲苯磺丁脲和KCl诱导的胰岛素分泌均无明显差异。在糖耐量试验中，转基因小鼠血清胰岛素水平高于对照组。因此，cADPR-CD 38信号系统在β细胞中葡萄糖的胰岛素分泌中具有调节作用，这表明不仅来自细胞外的Ca^<2+>（葡萄糖诱导的细胞膜去极化引起的电压依赖性Ca^2+通道的Ca^2+内流）以及细胞内储存的Ca^2+释放（cADPR诱导的内质网Ca^<2+>释放）在胰岛素分泌中起重要作用。4.我们对CD 38进行了定点突变，发现C119 K-和/或C202 E-CD 38仅具有ADP-核糖基环化酶活性。此外，我们将人CD 38的119和201位氨基酸残基K95 C和E176 C突变导入到拟南芥ADP-核糖环化酶中，使其不仅具有ADP-核糖环化酶的活性，而且还具有cADPR水解酶的活性。CD 38基因由8个外显子组成，在人类基因组上延伸约100 kbp，并通过荧光原位杂交定位于人类染色体4p 15。少

项目成果

Takuo Watanabe: "Pancretic beta-cell replication and amelioration of surgical diabetes by Reg protein." Proceedings of the National Academy of Sciences of USA. 91. 3589-3592 (1994)

Takuo Watanabe：“Reg 蛋白的胰腺 β 细胞复制和手术糖尿病的改善。”

Hiroshi Okamoto: "A role cyclic ADP-ribose in stimulus-secretion coupling in pancreatic beta-cells." 15th International Diabetes Federation Congress Abstractss. 15-15 (1994)

Hiroshi Okamoto：“环状 ADP-核糖在胰腺 β 细胞刺激-分泌耦合中的作用。”

東郷暁: "CD38とアメフラシADP-ribosyl cyclaseのアミノ酸置換による酵素活性の変換" 生化学. 67. 540-540 (1995)

Akira Togo：“通过 CD38 和海兔 ADP-核糖基环化酶的氨基酸取代来转换酶活性”生物化学 67. 540-540 (1995)。

Kato, I.: "Enhancement of glucose-induced insulin secretion in transgenic mice overexpressing human VIP gene in pancreatic beta -cells." Annal. N.Y.Acad. Sci.(in press). (1996)

Kato, I.：“在胰腺 β 细胞中过度表达人类 VIP 基因的转基因小鼠中，葡萄糖诱导的胰岛素分泌得到增强。”

東郷 曉: "セカンドメッセンジャーcyclic ADP-ribose 合成・分解反応に必須なCD38のCys119とCys201" 糖尿病. 38(in press). (1995)

Akira Togo：“CD38 的 Cys119 和 Cys201 对于第二信使循环 ADP-核糖合成和降解反应至关重要”，糖尿病 38（出版中）。