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研究生:黃泓翔
研究生(外文):Hung-Hsiang Huang
論文名稱:微量醣質的高感度結構分析與血吸蟲醣生物學的探討
論文名稱(外文):Highly Sensitive Structural Characterization of Complex Glycans and the Glycobiology of Schistosomes
指導教授:邱繼輝邱繼輝引用關係
指導教授(外文):Kay-Hooi Khoo
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:89
中文關鍵詞:血吸蟲
外文關鍵詞:Schistosome
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現在世界上約有二十分之一,即兩億人口感染血吸蟲病,並有更多人口因貧窮,居住環境不良及衛生設備缺乏而受到此種寄生蟲的威脅。經由以前的研究發現,隨著不同的發育階段,其血吸蟲體表上的醣化作用會因此而改變,而此種隨著發育階段而改變的醣質結構被認為與血吸蟲病的免疫病理有很大的相關性。
本論文研究主要是藉由質譜與串聯式質譜分析、二維高效率液相層析分析、酵素水解、氣相層析質譜儀分析、偏過碘酸氧化反應、三氧化鉻氧化反應及其他化學方法,將曼氏血吸蟲尾蚴時期所表現的醣蛋白上的醣類結構予以完整定序。除此之外,並用質譜分析技術及其他相同方法,將日本式血吸蟲尾蚴醣蛋白上醣類結構與曼氏尾蚴所發現的結構加以比較,進而探討此兩血吸蟲從尾蚴發育到成蟲時醣蛋白在不同階段及不同物種的特性醣化作用。
研究中發現曼氏血吸蟲尾蚴N-linked醣類,在其a6-core fucosylated N-inked醣類所延伸出來的支鏈帶有Lewis x的結構,其中有的含有b2-core xylosylated修飾。在曼氏血吸蟲尾蚴的O-linked醣類上也發現有相同的Lewis x的結構。特殊的是此種醣化修飾是在一種新的O-linked醣類核心結構上,Galb1→3(Galb1→6)GalNAc。利用此種特殊O-linked醣結構核心可以衍生出一序列帶有Lewis x的醣質。此外經比對,日本式血吸蟲尾蚴的N-linked醣類上也可以發現Lewis x的存在,但此種N-linked醣類並沒有經過b2-xylosylation.
和曼氏血吸蟲尾蚴一樣,Lewis x的結構也存在於曼氏血吸蟲尾蚴感染宿主及蛻變發育後的成蟲階段,但血吸蟲成蟲的醣類並無b2-xylosylation的修飾。從這些實驗來看,b2-xylosylation在血吸蟲會隨著不同物種、階段而有不同的修飾現象,而Lewis x則在每一個發育階段都有不同level的表現。這是否是為了感染宿主及在宿主寄生所產生的一種機制還不得而知,但這現象對於未來研究血吸蟲與宿主之間的交互作用及研發疫苗提供一個方向。
Schistosomiasis is a helminthic parasitic disease caused by blood flukes of the genus Schistosoma, afflicting about 200 million individuals in the tropics. The glycoconjugates from the various developmental stages of schistosomes have been implicated in the immnuopathogenesis of schistosomiasis. Based primarily on mass spectrometry (MS) and CID-MS/MS analysis, coupled with 2D-HPLC separation and mapping, sequential exoglycosidase digestions, GC-MS linkage analysis, periodate oxidation, chromium trioxide oxidation and other chemical derivatization, complete structural characterization of the major N- and O-glycans of S. mansoni cercariae were accomplished and presented in this thesis. In addition, the protein glycosylation profile of S. japonicum cercariae and the adults of both schistosome species were mapped by MS analysis to evaluate developmental stage-specific regulation and/or species-specificity.
The N-glycans of S. mansoni cercariae were found to contain a high abundance of Lewis x (Lex) structure on mono- and biantennary, a6-core fucosylated complex type structures, with and without core b2-xylosylation. The same Lex structure was also found to be the main terminal sequence on the major O-glycans of S. mansoni cercariae which are based on a novel core structure Galb1→3 (Galb1→6) GalNAc→Ser/Thr. Extension on the 3-arm or both 3- and 6-arms of the GalNAc gives a mono- or biantennary like structures, very similar to the N-glycans. In addition, the C6 position of the 3-linked Gal on this novel core can be further extended with one Gal residue to give a series of O-glycans which can be represented as R→3(±Gal1→6)Galb1→3(±R→3Galb1→6)GalNAc, where R= ±Galb1→4(±Fuca1→3)GlcNAc.
The characterization of one to two Lex on the mono- and biantennary N- and O-glycans of S. mansoni cercariae, provided a firm chemical evidence for the expression of Lex epitope on the glycoproteins of the invading larvae. Following infection, transformation and development into adult, the Lex structure could still be detected on the N-glycans, along with other non-fucosylated N-acetyllactosamine (lacNAc), N-N’-diacetyllactosdiamine (lacdiNAc), and fucosylated lacdiNAc termini, but does not constitute the predominant epitope as in the cercariae stage.
In comparison, the protein glycosylation profiles of both the S. japonicum cercariae and adult are very similar to those of S. mansoni with one notable exception, namely the absence of b2-xylosylation on the cercarial N-glycans. Thus, N-glycan core b2-xylosylation is a species-specific modification, as well as developmentally regulated. On the other hand, Lex appears to be commonly found in all stages of both schistosome species, although its expression level relative to other terminal sequences may vary from stage to stage.
1. Introduction 5
2. Material and Methods 19
3. Results 26
4. Discussion 78
5. References 85
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