蛇毒5端核苷酸酶（snake venom 5’-nucleotidase, V5NTD）常現於各種毒蛇的毒液之中。然而由於其在眼鏡蛇毒液內的含量相對稀少（約佔整體眼鏡蛇毒蛋白質重量之0.38%），因此過去的眼鏡蛇毒咬傷研究缺少對V5NTD生理功能的討論。近年來的研究指出，V5NTD能夠藉由水解血液中單磷酸腺苷（adenosine monophosphate, AMP）引發下游嘌呤傳訊路徑來抑制凝血作用。為了探討V5NTD代謝AMP的機制，我們建立了一套蛋白質純化機制，從中華眼鏡蛇（Naja atra）分離出V5NTD蛋白質並結晶，成功得到了1.9 Å高解析度的蛋白質結晶結構數據。透過結構上的比較，我們發現V5NTD與其在人類的同源蛋白——CD73有高度的相似，而CD73被認為是參與調控免疫反應的胞外酵素（ecto-enzyme）。在完成用cDNA定序V5NTD一級結構之後，我們利用分子置換的方式建構V5NTD的結構——V5NTD單一分子為60 kD並以靜電力相吸形成二聚體。這份研究不僅提供了從粗蛇毒內純化V5NTD的方法，也提出了第一個脊椎動物天然的5端核苷酸酶的結構，解釋了在自然界中此酵素的二聚體介面的交互作用，以及其與人類同源蛋白不同的的醣化修飾，為往後探討V5NTD於蛇毒咬傷時的生理功能提供分子結構基礎。

Venom 5’-nucleotidases (V5NTD) are widely represented in venomous snakes. Its biological function was less extensively investigated in the past cobra envenomation research owing to its scarcity in cobra venom (0.38% of Taiwan cobra venom proteins). In recent studies, it has been implied to conduct anti-coagulation by liberating of extracellular adenosine through purinergic pathways. To understand how V5NTD catalysed AMP, we purified V5NTD from Taiwan cobra (Naja atra) crude venom to determine its 3D structure at 1.9 Å by X-ray crystallography. By structural analysis, we found the crystal structure of V5NTD is similar to its human homologous protein, ecto-5’-nucleotidase (e5NT, a.k.a. CD73), an ecto-enzyme known for its immune regulatory activity. Through decoding V5NTD protein sequence from Naja atra venom gland cDNA, we use molecular replacement to exhibit the homodimeric isoform consists of monomers with 60 kD. This research not only provides a method to purify V5NTD from crude cobra venom, but also build the first native vertebrate ecto-5’-nucleotidase structure which elucidates the ionic-interaction in V5NTD dimerization interface and different glycosylation sites from human CD73. This molecular structural-based study can assist to clarify the biological functions of V5NTD after envenomation.

壹、 緒論 1
貳、 材料與方法 6
一、 蛇毒5端核苷酸酶cDNA定序 6
（一） 蛇毒囊解剖與組織研磨 6
（二） RNA萃取與cDNA製備 6
（三） 序列比對與引子（primer）設計 7
二、 純化蛇毒5端核苷酸酶 8
（一） 快速蛋白液相層析（FPLC） 8
（二） 反相高壓液相層析鑑定（RP-HPLC） 8
（三） 蛋白質N端定序 9
三、 快速篩選生長晶體條件 9
四、 X光繞射資料收集與數據解析（data processing） 10
（一） 數據收集（data collection） 10
（二） 數據解析（data processing） 10
（三） 以分子置換的方式解析立體結構 11
（四） 立體結構模型之建立與修正 12
參、 結果 13
一、 蛇毒5端核苷酸酶序列在物種之間有高同源性 13
二、 蛇毒5端核苷酸酶結晶型為二聚體的開放態構型 15
三、 蛇毒5端核苷酸酶金屬離子螯合位與人類CD73相同 17
四、 蛇毒5端核苷酸酶醣化修飾位與人類CD73不同 18
肆、 討論 20
一、 V5NTD一級結構討論與方法優化 20
二、 蛋白質純化方式之改良 21
三、 V5NTD與hCD73蛋白質性質與結構比較 22
四、 靜電力為形成V5NTD二聚體之主要作用力 22
五、 V5NTD與hCD73的醣化修飾 23
六、 V5NTD可能有與胞膜相連的狀態 26
七、 V5NTD可能生理功能探討 28
伍、 圖表 30
圖1、V5NTD片段氨基酸序列BLAST的結果。 30
圖2、蛇毒液內各蛋白質的純化步驟。 31
圖3、V5NTD純度分析。 32
圖4、中華眼鏡蛇V5NTD cDNA與對應之氨基酸序列。 33
圖5、中華眼鏡蛇、金黃珊瑚蛇、東部菱背響尾蛇與短尾蝮之V5NTD與小鼠、人類之CD73一級結構比較與二級結構預測。 34
圖6、中華眼鏡蛇V5NTD全長之氨基酸序列與其他近似物種的親緣關係。 35
圖7、中華眼鏡蛇V5NTD結晶型態與高解析度1.9Å之電子雲密度圖。 36
圖8、中華眼鏡蛇V5NTD單體構型與細部特徵。 37
圖9、中華眼鏡蛇V5NTD藉由CTD區域之靜電力吸引形成二聚體。 38
圖10、中華眼鏡蛇V5NTD與hCD73之金屬離子螯合位。 39
圖11、中華眼鏡蛇V5NTD與hCD73之N-glycosylation位置。 40
表1、以FPLC純化蛇毒蛋白質時使用之緩衝溶液 41
表2、中華眼鏡蛇V5NTD蛋白結晶條件與X光繞射數據資訊 42
表3、中華眼鏡蛇V5NTD與人類CD73的蛋白質性質比較 43
陸、 附錄 44
附圖一、蛇毒液內各蛋白質的組成比例。 44
附圖二、東部菱背響尾蛇與短尾蝮之蛇毒5端核苷酸酶cDNA序列比對 45
附表一、英文縮寫與中文譯名對照 46
附表二、 蛇毒液內主要酵素* 47
附表三、 引子列表 48
柒、 參考資料 48

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