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Title: | 斑馬魚多重唾液酸化醣質體在胚胎發育過程的
表現調控與結構分析 Developmentally regulated oligosialylation pattern in Zebrafish embryogenesis |
Authors: | Lan-Yi CHANG 張蘭頤 |
Advisor: | 邱繼輝(Kay-Hooi Khoo) |
Co-Advisor: | 高楊(Yann Guerardel),阿朵安(Anne Harduin-Lepers) |
Keyword: | 唾液酸,斑馬魚,胚胎發育,質譜儀,唾液酸傳遞酶, sialic acid,oligosialylation,Zebrafish,glycomic,developmental,mass spectrometry, |
Publication Year : | 2008 |
Degree: | 博士 |
Abstract: | 九碳糖唾液酸是位於生物體醣質末端的修飾分子,它是細胞與細胞間的溝通橋樑,也是傳達訊息的細胞表面辨識分子。位於細胞表面的醣分子常常可以反應細胞的生理狀態,因此也被當作腫瘤生成或是胚胎發育時期的階段性指標抗原。在本篇論文,我們以斑馬魚為模式動物,去探討負責合成α2,8 鍵結的多重唾液酸鏈的多重唾液酸傳遞酶(oligo/polysialyltransferase)家族(ST8Sia)其基因表現的部位和時間,如何在胚胎發育過程中被影響和調控,並且結合化學方法和質譜儀分析去定出唾液酸化的醣質結構。首先,利用先進的質譜作醣質分子定序,我們比較不同胚胎發育階段的斑馬魚之受精卵醣質,這些唾液酸化的醣質有別於一般在哺乳類的醣質結構,像是連接在天門冬素(Asparagine)氮分子上的唾液酸化複合醣(sialylated complex type N-glycans)為4-galactosylated sialyl Lewis X; 連在絲氨酸(Serine)或羥丁氨酸(Threonine)的氧分子上的醣(O-glycans)為雙唾液酸化的O-glycans,Fucα1-3GalNAcβ1-4NeuAc/NeuGcα2-3Galβ1-3GalNAc,及多重唾液酸化的醣脂質。以質譜觀察到多重唾液酸可能只出現在特定發育階段之後,我們進一步用螢光標示液象層析去更精準的偵測及量化不同發育階段的多重唾液酸。值得注意的是,在醣蛋白上的多重唾液酸,不論在量上或鏈長都會隨著胚胎發育而漸減,而在醣脂質上的反而會漸增。為了進一步去了解多重唾液酸化的消長現象是如何發生,我們利用
定量及時聚合酶連鎖反應(quantitative real-time PCR)去檢視多重唾液酸傳遞酶的基因表現層次,和內源性唾液酸水解酶的活性在不同發育階段的調控情形。結果所有的ST8Sia的表現,皆隨著胚胎發育而增加。內源性的唾液酸水解酶活性,為醣蛋白上多重唾液酸 的降解提供了一個可能的解釋,即大多數的多重唾液酸化的醣蛋白是來自母體,他們在受精前就已經存在於卵子中,而在受精後被釋放到胚胎與卵膜的間隙,再被內源性的唾液酸水解酶降解。進一步分離授精卵的卵膜、胚胎、和介於兩著之間的間質液,並分析這三部分的多重唾液酸的鏈長和含量,以及唾液酸水解酶活性,我們初步證實了大多數重唾液酸化的醣蛋白的確是來自母體。而位於醣脂質上的多重唾液酸因為在胚胎發育後期才逐漸出現,與負責其生合成的多重唾液酸傳遞酶表現量成正相關,因此推測是由胚胎新合成,而非源自母體。經由核糖核酸整體原位雜交技術(whole-mount in situ hybridization),顯示多重唾液酸傳遞酶在發育早期都表現在胎兒的神經系統。總結本篇論文,我們利用高感度的串聯式質譜,解開了斑馬魚受精卵醣質在發育過程的全貌及改變,以及精確量化多重唾液酸化醣質的結構的消長,並且分析其調控因子,其中包括多重唾液酸傳遞酶的基因表現量和唾液酸水解梅的活性。這些數據交叉證實了多重唾液酸化在胚胎發育期間複雜的調控機制,也進一步為神經發育學家提供了唾液酸醣生物學層面的研究線索與觀點。 The nine-carbon sugar, sialic acid, plays important roles as the recognition epitopes in cell-cell communications. Its regulated expression often reflects the status of a cell, and has been reported as stage-specific antigen for tumorigenesis or embryogenesis. In this thesis work, the biological functions of developmentally regulated sialylation, particularly in relation to the spatial and temporal regulated expression of α2-8 sialyltransferases (ST8sia) during the embryogenesis of Zebrafish, was investigated using a combination of chemical and mass spectrometry (MS) analyses. Glycomic survey mapping followed by advanced glycan sequencing were first performed across the different developmental stages of fertilized Zebrafish eggs. Among the unusual structures identified are the complex type N-glycans that carry β4-galactosylated sialyl Lewis X, the disialylated O-glycans, Fucα1-3GalNAcβ1-4NeuAc/NeuGcα2-3Galβ1-3GalNAc, and the oligosialylated glycolipids. The stage-specific, distinctive Neu5Ac/Neu5Gc sialylation patterns observed then prompted a more precise quantification of the respective oligosialylation by fluorescent chromatography after chemical derivatization. Interestingly, the oligosialic acids of glycoproteins were found to decrease whereas those of glycolipids increased along the embryogenesis. To determine if the observed profiles are due to the regulation of biosynthesis or catabolism, or both, the transcripts of all inferred Zebrafish ST8Sia genes responsible for the biosynthesis of oligosialylation were quantitatively mapped by real-time PCR, and the activity of endogenous sialidases were assayed, for each of the developmental stages. The presence of sialidases and an increased level of expression of all the ST8Sia genes led to a working model, which proposes that the oligosialic acids from glycoproteins are maternally inherited (synthesized before fertilization) and exocytosis out of the embryo into the perivitelline space where it undergoes catabolism, whereas oligosialic acids on the glycolipids are de novo synthesized by the increasingly expressed ST8Sia in the embryos. Furthermore, through whole mount in-situ hybridization, most of the ST8Sia genes were shown to be expressed in the nervous system during early embryogenesis, with different onset and locations. In summary, the precise MS and MS/MS-based glycomic profiles, together with detailed structural determination, quantitative analysis of the developmentally regulated oligosialylation pattern, and the spatial and temporal expression profiling of ST8Sia of Zebrafish, collectively provides new sialoglycobiology insights for neuroscientists at the molecular level and highlighted the significance and complicated regulation of oligosialylation in early neuronal development. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41854 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生化科學研究所 |
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