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

唾液酸（Sia）具有多种天然修饰，这些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至今仍是一个谜。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，我们对CMP-Sia合成酶（CSS）进行了以下研究：（1）在动物水平上揭示Sia-糖复合物的结构多样性和时空表达调控的意义;（2）了解CSS在无脊椎动物中的重要性，重点关注CSS的细胞内定位和C端半结构域的作用。使用TILLING方法生成CSS敲除（KD）medakas。筛选出两株体外活性极低的青鳉突变株I230 N和L303 Q。目的（2）克隆赤拟谷盗（Tribolium castaneum）、埃及伊蚊（Aedes aegypti）和黑腹果蝇（Drosophila melanogaster）的CSS cDNA，分析其在Sia种内的表达动力学和胞内定位。

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.