以蛋白質體及醣質體分析華勒蝮以及眼鏡王蛇兩種蛇毒的地理差異

Proteomic and glycomic analyses of venom geographic variations of Wagler's pit vipers and king cobra

10.6342/NTU.2012.00271

張惠晴

蛇毒地理性差異 ； 磷酯酶A2 ； 聚醣結構 ； 血小板凝集抑制劑 ； 華勒蝮 ； 眼鏡王蛇 ； 三指毒素 ； geographic variation ； venom phospholipases A2 ； glycan structure ； platelet aggregation inhibitor ； Tropidolaemus wagleri ； Ophiophagus hannah ； three-finger toxin

臺灣大學生化科學研究所學位論文

2012年

碩士

蔡蔭和

英文

在後基因體時代，蛇毒的研究在蛋白質體及轉錄體方面都快速進展，許多蝮蛇及眼鏡王蛇蛇毒亦不例外。探討蛇毒地理的或個別的變異性為蛇毒蛋白質體研究的一個層次或面向。在此，我們藉由蛋白質體以及醣質體分析方法探討來自兩個地區的華勒蝮以及四個地區的眼鏡王蛇其蛇毒當中主要的蛋白質的異同以及其生理功能特性。 第一部分 從蘇拉維西以及蘇門答臘的華勒蝮蛇毒中我們分別純化到兩個磷酯酶，其中一個是具活性、第六個胺基酸為榖胺酸的磷酯酶(稱之E6-PLA2)，而另一個則是不具活性、推測其第49個胺基酸可能為賴胺酸的磷酯酶(Lys49-like PLA2)。從這兩個地區分離純化到的磷酯酶無論是在分子量或是N端胺基酸序列都明顯不同，雖然他們在表現物種特有的神經毒素waglerin上具有高度保留性，從結果上看來這兩個地區的華勒蝮可能是兩個不同的種(T.subannulatus 以及 T. wagleri)。這個結果也是與西元2007年發表此蛇分類的結果是相符的。這兩個地區所表現具活性的磷酯酶，在本篇研究中也依照他們可能分屬的種名，命名為Tsu-E6(來自蘇拉維西)以及Twa-E6(來自蘇門答臘)。而後續研究發現，這兩個E6-PLA2在胺基酸序列第14位置的天門醯胺上有醣化的現象。進一步以N-醣醯胺酶(PNGase F)水解去除醣基可發現磷酯酶的分子量明顯從16 kDa減少到14 kDa，並且以質譜(MALDI-TOF)分析水解下來的醣基發現其是一個不具涎酸化(sialylation)且混合型式的寡糖。研究其生理功能，我們發現Tsu-E6對於由ADP引起的小鼠或人類血小板凝集作用都是抑制效果，而Twa-E6對於由ADP或膠原蛋白所引起的人類血小板凝集也是相同的作用，但對小鼠的血小板凝集卻是誘導作用。若進一步去分析這兩個磷酯酶在去除醣基後酵素活性或是血小板凝集作用則是沒有顯著差異。另外，探討醣基是否對蛋白質的熱穩定性有所影響，其結果發現Twa-E6的熱穩定性與其他不具醣基的磷酯酶是一樣的好。從以上結果可知，這個醣基的存在也許有其他的生理意義仍有 待我們進一步的探討。 第二部分 目前文獻上少有眼鏡王蛇毒地理差異的研究。我們買了分別來自印尼、馬來西亞、廣東、海南島等四個不同地區的眼鏡王蛇毒來分析其蛋白成分是否存在地理性差異。從蛇毒的酵素活性、用三種色層柱分析主要蛋白質組成，以及對小鼠毒性等分析結果發現其地理性的差異確實存在。以往文獻指出從中國幾個地區的眼鏡王蛇毒中純化到主要為已知名為OH-APLA2的磷酯酶，但來自印尼以及馬來西亞的蛇毒則表現另一在研究新發現並命名為PLA-3的酵素。三指毒素(3FTx)成分而言，雖然在這四個地區的蛇毒中具有或高或低的oh-55，但其他種神經毒素或3FTxs的組成卻是具有高度變異性的。另外，東南亞的眼鏡王蛇毒不像中國的含有明顯prothrombin活化酵素，我們的研究發現來自印尼的眼鏡王蛇毒含有比較高活性的鹼性磷酸酶(alkaline phosphomonoesterase)，卻有極低的磷酯酶。這些酵素可能彼此在蛇毒毒性上有協同作用，在未來可能值得進一步的探討。

Herein, we have investigated the geographic variations of Tropidolaemus wagleri from two regions and those of O. hannah from four regions by venom protein purifications, identification and biofuntional characterizations using the proteomic and glycomic approaches. Part I. Phospholipases A2 (PLA2s) were purified and characterized from Tropidolaemus wagleri venoms collected from Sulawesi and Sumatra. An active PLA2 and an inactive Lys49-like PLA2 could be isolated from each venom sample. Mass analyses and N-terminal sequences revealed that the PLA2 variants from the two venom samples were different although their neurotoxic waglerins were identical. Thus, the samples were probably derived from two species (T. subannulatus and T. wagleri), consistent with the recent taxonomic study of this genus. Since the Glu6 residue was conserved in both the PLA2s, the PLA2s from Sulawesi and Sumatra were designated as Tsu-E6 and Twa-E6, respectively. Interestingly, both Tsu-E6 and Twa-E6 appeared to be glycosylated at Asn14. Hydrolysis by PNGase F reduced their apparent masses from 16 to 14 kDa. The released glycans were further analyzed by MALDI-TOF and shown to be complex type oligosaccharides without sialylation. Tsu-E6 inhibited ADP-induced aggregation of mouse and human platelets, and Twa-E6 inhibited the ADP- or collagen-induced aggregation of human platelets, but stimulated the aggregation of mouse platelets. However, enzymatic removal of glycans from both the PLA2s did not significantly alter their effects on lipid hydrolysis and platelet aggregation. The thermostability of Twa-E6 was found to be as good as those of other homologous PLA2s. The presence of these oligosaccharides on snake venom PLA2s therefore warrants further analyses. Part II. We profiled and analyzed four geographic venom samples of O. hannah which were obtained from Indonesia, Malaysia, Guangxi, and Hai-Nan. The enzyme activities, toxicities on mice of the four venoms were investigated. The PLA2s and 3FTxs were purified. There are great geographic variations in the enzyme content, protein composition and toxic properties between the Southeast Asian and Chinese king cobra venoms. The OH-APLA2 is the major PLA2 conserved in the venoms from China, and the venom from Indonesia and Malaysia express a PLA2 similar to OH-APLA2 II as judged by PMF analysis. Chinese king cobra venoms appear to have lower metalloprotease or hemorrahagin activities but higher prothrombinase activities. Even though the oh-55 3FTx is conserved in king cobra venoms, it is not the most abundant in all the kign cobra venom. The composition of the other 3FTx variants is highly variable. Finally, the venom from Indonesia is especially performing high alkaline phosphomonoesterase activity, which may present synergistic reaction in the toxic property of venom. This will need a further investigation to resovle.

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