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研究生:陳玉貞
研究生(外文):Yu-chen Chen
論文名稱:單核球細胞lipid rafts的醣蛋白質體研究
論文名稱(外文):The glycoproteome of lipid rafts in monocytes
指導教授:吳烘
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
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:2010
畢業學年度:98
語文別:中文
論文頁數:104
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蛋白質醣基化作用在調控各種不同的細胞生化反應過程中扮演相當重要的角色。在之前的研究指出lipid rafts 是屬於細胞膜上特化結構,其特性富含了glycogangliosides、訊息傳遞分子和一些囊泡運輸的蛋白質。雖然位在細胞內和細胞膜上醣化蛋白質過去已經被研究探討, 但是分佈在細胞膜上lipid rafts glycoproteome 的研究還需要更深入了解。因此本論文的研究目標主要是利用density gradient centrifugation 結合lectin affinity chromatography 的方式,將存在THP- 1 單核球細胞lipid raft domains 中的醣蛋白分離出來。在一維電泳分離後利用串聯式質譜儀總共鑑定出65 個不同的蛋白質,分別存在於wheat germ agglutinin(WGA)和concanavalin A(Con A)-enriched fraction 中。除此之外,我們還進一步預測蛋白質醣基化修飾的位置,分別利用不同的蛋白質資料庫以及電腦軟體進行交叉分析,發現共有55 個蛋白質具有潛在N-linked 醣基化修飾的位置。另外,有33 個蛋白質是屬於O-linked 醣基化修飾的類型,和5 個具有C-linked 醣基化修飾的蛋白質,其中有3 個蛋白質是同時含有N-linked、O-linked 和C-linked 醣基
化修飾的位置。將這些蛋白質再藉由文獻搜尋和各種蛋白質資料庫比對分析,結果顯示lipid rafts 醣蛋白除了位在細胞表面之外,在其它的胞器也有被分析出。而且在THP-1 細胞膜上lipid rafts glycoproteome 的成份大部份富含receptor activity、
cell adhesion molecule、protein transporter、enzyme 和其他的分子,表示這些lipid rafts醣蛋白參與許多不同的生物功能和活性,例如細胞與細胞之間的互動和訊息傳遞作用等。利用lectin affinity chromatography 的分離策略,本論文已成功鑑定出分佈
在lipid raft domains 中的醣蛋白。未來可以更進一步的研究lipid rafts 蛋白質醣化修飾正確位置,並且還可以探討醣化對lipid rafts 蛋白質之間的功能,以及蛋白質之間的互動關係。
Glycosylation of proteins plays a significant role in controlling various cellular processes. Lipid rafts are specialized microdomains of membrane compartments characterized by its enrichment of gangliosides, ignalling molecules and proteins involved in vesicular traffic. Although protein glycosylation at the cellular and membraneous level have been studied previously, the glycoproteome in lipid rafts remains to be elucidated. Using density gradient centrifugation and lectin affinity
chromatography, the aim of this study is to conduct a omprehensive analysis of glycoproteins in lipid rafts isolated from THP-1 monocytes. Here we report that there
are 58 proteins identified by mass spectrometry in lipid rafts fractions captured by wheat germ agglutinin affinity column, whereas 13 are found in concanavalin A-enriched fraction. Further bioinformatic analysis on the glycosylation sites reveals that 59 proteins exhibit potential N-linked glycosylation sites. In addition, there are 37 proteins listed in the category of O-linked glycosylation and 31 of these contain both types of glycosylation. receptor activity, cell adhesion molecules, protein transporter, enzyme and other molecules a significant portion of the raft glycoprotome. These findings are consistent with the roles of lipid rafts in biological functions, such as cell-cell interaction and transmembrane signaling. In summary, the lectin-based separation strategy has successfully revealed the glycoprotein components in lipid rafts domains. Further studies are required to elucidate the role of these raft proteins and the functional significance of glycosylation.
摘要iv
目次vi
表目錄ix
圖目錄x
縮寫對照表xi
第一章 研究動機與目的1
第二章 文獻探討3
2-1 醣蛋白之簡介3
2-1.1 醣基化的介紹3
2-1.2 醣蛋白的結構與分類4
2-1.2.1 N-linked 醣蛋白4
2-1.2.2 O-linked 醣蛋白8
2-1.2.3 C-linked 醣蛋白11
2-1.2.4 GPI-anchored proteins 13
2-1.3 醣蛋白的功能15
2-2 Lectin 之簡介16
2-2.1 Lectin 的分類與生理功能之研究16
2-2.2 Lectin 的應用17
2-2.3 Lectin affinity chromatography 19
2-3 Lipid rafts 之簡介22
2-3.1 Lipid rafts 的結構22
2-3.2 Caveolae 的結構23
2-3.3 Lipid rafts markers 26
2-3.4 Lipid rafts 的功能26
2-4 蛋白質體學28
2-4.1 蛋白質分離28
2-4.2 蛋白質鑑定29
2-4.3 醣蛋白質體學31
2-4.4 Lipid rafts 蛋白質體學32
2-5 預測醣基化位置軟體之簡介33
2-6 THP-1 monocytic cell line 35
第三章 材料與方法37
3-1 材料37
3-1.1 細胞株37
3-1.2 藥品試劑37
3-1.2.1 細胞培養試劑37
3-1.2.2 ㄧ般藥品與試劑37
3-1.2.3 抗體38
3-2 研究方法38
3-2.1 細胞培養38
3-2.2 Lipid rafts 的萃取39
3-2.3 西方墨點法(western blotting) 39
3-2.4 醣蛋白的純化39
3-2.5 Instant blue staining 40
3-2.6 蛋白質鑑定40
第四章 結果與討論41
4-1 THP-1 細胞lipid rafts 的萃取42
4-2 Lipid rafts 醣蛋白的分離45
4-3 鑑定Con A 和WGA 純化蛋白身份47
4-3.1 預測lipid rafts 蛋白質醣基化位置47
4-3.2 Lipid rafts 醣蛋白分佈在細胞中的位置68
4-4 比較過去蛋白質體學之研究70
4-4.1 膜醣蛋白質體學之研究70
4-4.2 Lipid rafts 蛋白質體學之研究71
4-5 Lipid rafts 醣蛋白生物功能分類與介紹72
4-5.1 Receptor activity 72
4-5.2 Cell adhesion molecule 74
4-5.3 Enzyme 76
4-5.4 Protein transporter 77
4-5.5 其它的分子功能78
4-6 結論80
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