台灣眼鏡蛇毒5端核苷酸水解酶（V5NTD）cDNA定序與結晶結構分析

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。

關鍵字

蛇毒蛋白； 5端核苷酸酶；蛋白質結晶結構

並列摘要

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.

並列關鍵字

snake venom protein ； 5’-nucleotidase ； protein crystal structure

參考文獻

6. 林意凡、林曉凌、陳慶餘 (2000)。毒蛇咬傷的認識與處置。當代醫學 27，803-808。

1. You, C. W., Poyarkov, N. A., and Lin, S. M. (2015) Diversity of the snail-eating snakes Pareas (Serpentes, Pareatidae) from Taiwan. Zool Scr 44, 349-361

4. Kasturiratne, A., Wickremasinghe, A. R., de Silva, N., Gunawardena, N. K., Pathmeswaran, A., Premaratna, R., Savioli, L., Lalloo, D. G., and de Silva, H. J. (2008) The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med 5, e218

5. Hung, D. Z. (2004) Taiwan's venomous snakebite: epidemiological, evolution and geographic differences. Trans R Soc Trop Med Hyg 98, 96-101

8. Chang, C. C., Wei, J. W., Chuang, S. T., and Lee, C. Y. (1972) Are the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom due to phospholipase A, neurotoxin or cardiotoxin? 台灣醫學會雜誌 71, 323-327

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台灣眼鏡蛇毒5端核苷酸水解酶（V5NTD）cDNA定序與結晶結構分析

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