2014 Fiscal Year Annual Research Report
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
| Project/Area Number |
13J03566
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| Research Institution | Nagoya University |
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Principal Investigator |
呉 迪 名古屋大学, 生命農学研究科, 特別研究員(DC1)
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| Project Period (FY) |
2013-04-01 – 2016-03-31
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| Keywords | Sialic acid / TILLING / CMP-Sia synthetase / localization / insect |
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| Outline of Annual Research Achievements |
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.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
After I obtained two interesting CSS-KD medaka strains, I observed the phenotype of them and also measured the sialic acid amount in CSS-KD fish. All the results indicated that sialic acid was important for medaka embryonic development.
To understanding how important CSS in invertebrates, I not only studied the in vitro and in vivo activity of three insect CSSs, but also intracellular localization of them.
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| Strategy for Future Research Activity |
To understand the importance of Sia-glycoconjugates at an animal level, it is not enough to generate the CSS-KD medaka fish because CSS-KD medaka is embryonically lethal. I plan to generate conditional KO fish using TALEN method and Cre-LoxP system. Now, the CSS-KO medaka strain has already completed. Next, I will continue to establish Cre-loxP system in CSS-KO fish.
For the insect CSSs study, no in vitro activity to Neu5Ac, Neu5Gc, or Kdn was detected for any of the insect CSSs, when they expressed in E. coli system. However, the insect CSSs showed in vivo and in vitro activity when they expressed in LEC29.Lec32 cells, which are deficient in CSS activity. I would like to answer the reason for this phenomenon. Furthermore, I found that mosquito and beetle CSSs were localized in ER, differently than Drosophila CSS, which was localized in the Golgi. Thus, the intracellular localization of CSS is not the same between the insect species. I would like to answer how and why insect CSSs showed different localization from mammalian CSSs.
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