シアル酸代謝酵素の逆遺伝学的および生化学的解析によるシアル酸の機能探索

通过唾液酸代谢酶的反向遗传和生化分析探索唾液酸功能

• 批准号: 13J03566
• 负责人: 呉 迪
• 金额: $ 1.73万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2013
• 资助国家: 日本
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13J03566/
• 关键词: sialic acid; TILLING; CMP-Sia synthetase; insect; Sialic acid; localization; シアル酸; CMP-シアル酸合成酵素; 昆虫; 細胞内局在; メダカ

Sialic acid (Sia) has a variety of natural modifications, which are often regulated developmentally and organ-specificically. However, the significance of the structural diversity and the spatiotemporally regulated expression of Sia have remained largely mysterious up to now. Thus, our long goal is to give an insight into biological function and its molecular mechanisms of Sia. For this purpose, we focus on CMP-Sia synthetase (CSS): (1) To reveal the significance of the structural diversity and the spatiotemporally regulated expression of Sia-glycoconjugates at an animal level; (2) To understand how important CSS is in invertebrates, focusing on the intracellular localization and the role of C-terminal half domain.For Objective (1), CSS knock-down (KD) medakas were generated using TILLING methods. Two mutant medaka strains I230N and L303Q, which had very low in vitro activity, were established. CSS knock-out (KO) medaka was also made by TALEN methods.For Objective-(2), the CSS cDNAs were cloned from Tribolium castaneum (red flour beetle), Aedes aegypti (yellow fever mosquito), and Drosophila melanogaster (fruit fly) to analyze kinetics for Sia species and intracellular localization using the recombinant enzymes.

唾液酸 (Sia) 具有多种天然修饰，通常在发育和器官特异性方面受到调节。然而，Sia 的结构多样性和时空调控表达的意义迄今为止仍然很神秘。因此，我们的长期目标是深入了解 Sia 的生物学功能及其分子机制。为此，我们重点研究CMP-Sia合成酶（CSS）：（1）揭示Sia-糖缀合物在动物水平上的结构多样性和时空调控表达的意义； (2)了解CSS在无脊椎动物中的重要性，重点关注细胞内定位和C端半结构域的作用。对于目标(1)，使用TILLING方法生成CSS敲低(KD)青鳉。建立了两个体外活性极低的青鳉突变菌株I230N和L303Q。 CSS 敲除 (KO) 青鳉也是通过 TALEN 方法制备的。对于目标 (2)，从赤拟谷盗 (红面粉甲虫)、埃及伊蚊 (黄热病蚊子) 和果蝇 (果蝇) 中克隆 CSS cDNA ）使用重组酶分析 Sia 物种的动力学和细胞内定位。

项目成果

Sialic acid is an indispensable sugar for early development: Establishment of CMP-Sia synthetase-mutant medaka by TILLING method

唾液酸是早期发育不可缺少的糖：TILLING法建立CMP-Sia合成酶突变青鳉

• 发表时间: 2014
• 作者: 村上 久;郡司ペギオ幸夫;呉 迪
• 通讯作者: 呉 迪

The insect CMP-sialic acid synthetases : New features in the enzymeactivity and intracellular localization

昆虫 CMP-唾液酸合成酶：酶活性和细胞内定位的新特征

• 发表时间: 2013
• 作者: Kyosuke Kojima;Kanae Sasaki;Wei Zhang;Takeshi Takahashi;Terunao Takahara;Hideki Shibata and Masatoshi Maki;呉 迪
• 通讯作者: 呉 迪

Sufficient provision of CMP-sialic acid is necessary for growth and development of medaka fish

充足的CMP-唾液酸对于青鳉鱼的生长发育是必要的

• 发表时间: 2015
• 作者: 河合(久保田)寿子;武藤梨沙;Pierre SETIF;Marc Nowaczyk;Matthias Rogner;池上貴久;田中 秀明;栗栖源嗣;山本 孝子;呉 迪
• 通讯作者: 呉 迪

The study on gene structure and intracellular localization of the insect CMP-sialic acid synthetases.

昆虫CMP-唾液酸合成酶基因结构及细胞内定位研究。

• 发表时间: 2013
• 作者: Di Wu;Akiko Fujita;Kayo Hamaguchi;Anne Harduin-Lepers;Philippe Delannoy;Vladislav M. Panin;Chihiro Sato;Ken Kitajima.
• 通讯作者: Ken Kitajima.

The first comparative study of enzyme properties of the insect CMP-sialic acid svnthetases.

昆虫 CMP-唾液酸合成酶的酶特性的首次比较研究。

• 发表时间: 2013
• 作者: T. Ishiwata;Y. Furukawa;K. Sugikawa;K. Kokado;K. Sada;呉 迪
• 通讯作者: 呉 迪