臺灣博碩士論文加值系統

心臟毒素是眼鏡蛇毒液中的主要成分，眼鏡蛇心臟毒素在生理條件下是個鹼性蛋白，由60~62個胺基酸所組成。它是一個由β摺疊形成的三指環蛋白結構。當眼鏡蛇咬到動物或是人時，在組織表面心臟毒素會造成發炎。然而心臟毒素的主要作用目標至今仍然不清楚。經由之前的研究發現，細胞膜表面的肝素硫酸是心臟毒素最有可能的作用標地。我們已經成功的解出心臟毒素A3和高硫酸化六醣肝素複合物的晶體結構 (解析度3.4Å)。由這個複合物結構，我們發現了檸檬酸在幫助心臟毒素A3形成二元體上扮演了重要的角色。檸檬酸主要是與心臟毒素A3的Lys23和Lys31形成作用，進而穩固了心臟毒素A3的二元體。這個發現的重要性在於天然的蛇毒中含有大約50mM的檸檬酸。除此之外六醣肝素主要是藉著與Lys12、 Lys18和 Lys35作用來和心臟毒素A3結合。在之前實驗室利用核磁共振技術解出的心臟毒素A3和二醣肝素複合物結構，顯示二醣肝素的結合位置是和X光技術解出的六醣肝素結合位置是相同的。另外一個值得討論的地方是在心臟毒素A3所形成的二元體中，一個有和六醣肝素結合，但是另一個卻是沒有。這個研究提供了一個天然蛇毒中檸檬酸的可能生物活性，並且模擬出細胞表面的肝素硫酸與心臟毒素A3的可能作用模式。

Cardiotoxin (CTX) is a major component of cobra toxin. Cobra cardiotoxins (CTXs) are basic proteins, composed of 60-62 amino acids, in which β-sheets form three finger-loop structures. When cobra bites animals or human, CTX can induce tissue inflammation. However, the CTX major target on cell membrane is still unclear now. Our previous studies have proved that heparan sulfate is the most potential target of CTX on cell membrane. We have determined the crystal structure of CTX A3 and hexasaccharide complex at 3.4 Å resolution using synchrotron radiation X-ray. Through the complex structure, we found that citrate anion plays an important role in prompting CTX A3 to form dimmer by interacting with Lys31 and Lys23. The finding is important because anionic citrate is a major component (~50mM) of venom, but the specific role of citrate is still poorly understood. Besides, Hexasaccharide heparin bind to CTX A3 by interacting with positively charged residue Lys12, Lys18 and Lys35. The binding site of hexasaccharide heparin from X-ray is similar to that of the disaccharide from NMR study. It is also worthy to discuss that one of CTX A3 dimmer is heparin bound form and the other is heparin non-bound form. This study suggests a novel role for venom citrate activity and identify specific sulfation pattern of heparan sulfate in binding to CTX A3.

壹、 緒論
1-1 蛇毒簡介 ----------------------------------------------------------------1
1-2眼鏡蛇蛇毒介紹-------------------------------------------------------- 2
1-3 醣胺素的簡介 --------------------------------------------------------- 5
1-4 醣胺素在身體中的分布 ----------------------------------------------7
1-5 為何要做心臟蛇毒蛋白和醣胺素複合物的結構----------------- 8

貳、 材料與方法
2-1 心臟蛇毒蛋白的純化 ------------------------------------------------ 13
2-2 高硫酸化六醣肝素的純化--------------------------------------------15
2-3 晶體培養 ----------------------------------------------------------------17
2-4 晶體繞射數據的收集與處理-----------------------------------------18
2-5 心臟毒素A3和高硫酸化六醣肝素複合物結構的決定 ------- 20

參、 結果
3-1 心臟毒素A3 的分子結構 -------------------------------------------27
3-2 心臟毒素A3和高硫酸化六醣肝素的晶體堆疊 -----------------27
3-3 Lys23-31所形成的陰離子結合口袋區(anionic pocket) ---------28
3-4高硫酸化六醣肝素和心臟毒素A3的結合位置 ------------------28



肆、 討論
4-1 心臟毒素A3和高硫酸化六醣肝素結合位置的探討 ----------30
4-2 檸檬酸幫助心臟毒素A3形成二元體的探討 -------------------31
4-3 心臟毒素A3形成二元體的個別構型探討-----------------------33

伍、 附錄
5-1 心臟毒素A5和二醣肝素的結構探討-----------------------------37
5-2 心臟毒素A5的結構比較-----------------------------39
5-3 心臟毒素A5和六醣肝素的晶體-----------------------------------40

陸、 附圖表------------------------------------------------------------------42

柒、 參考資料--------------------------------------------------------------84

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