https://scholars.lib.ntu.edu.tw/handle/123456789/143474
標題: | 鎖鏈蛇蛇毒的金屬蛋白水解酶及CTLL家族醣蛋白之結構與功能 Structures and functions of venom metalloproteinases and C-type lectin-like (CTLL) glycoproteins families from Russell’s viper |
作者: | 陳鴻森 Chen, Hong-Sen |
關鍵字: | 鎖鏈蛇蛇毒;金屬蛋白水解酶第十凝血因子活化酵素;C型類凝集素;聚醣質譜儀分析;地理變異;親緣分析;Russell’s viper venom;metalloproteinase;factor X activator;C-type lectin-like proteins;glycan mass spectrometry;geographic variation;phylogenetic analysis | 公開日期: | 2008 | 摘要: | 鎖鏈蛇咬傷在許多南亞和東南亞地區的國家仍是嚴重的危害及公衛問題。耗損性凝血功能障礙和血小板減少所造成的系統(或全身)性出血及急性腎功能衰竭,是該毒蛇咬傷的病人中最常見的臨床症狀。而且,不同地理區域的咬傷症狀差異也相當大。為了釐清蛇毒蛋白與這些症狀的關係,在本研究中我們針對二類蛇毒蛋白家族—金屬蛋白水解酶和C型類凝集素(CTLL)分子做進一步純化和特性分析。此外,我們也選殖這些蛇毒蛋白的基因,訂出其蛋白序列。並且利用生物資訊與親緣分析推測它們的演化關係、功能及相關的功能作用位置。 第一部分]了探討在緬甸和東印度鎖鏈蛇咬傷所引起的大量出血臨床症狀,我們從這兩個地區的鎖鏈蛇蛇毒中各純化出類似的P-III 型金屬蛋白水解酶,並將其命名為daborhagin-M和daborhagin-K。它們誘導小鼠皮膚的最低出血劑量都在0.8-0.9毫克。試管中,它們會專一地水解纖維蛋白素原的Aa鏈、纖維連接蛋白與第四型膠原蛋白。分析它們對胰島素B鏈的切位,以及用5個具代表性P-III酵素對發螢光之合成胜肽基質的酵素動力研究,發現daborhagin或其他強出血毒素水解能力高並會選擇性地水解 P1、P1'' 和P2'' 位置為疏水胺基酸殘基的基質。以西方墨點轉漬法分析8個不同地理位置來源的鎖鏈蛇蛇毒樣本,發現只有從緬甸和東印度部的蛇毒含有daborhagin蛋白。接著利用互補DNA選殖、序列分析與胜肽質量指紋技術(PMF),我們得到東印度daborhagin的完整基因及蛋白序列。將27個已知序列的P-III分子以親緣分析進行推演,發現他們演變成兩大類具有不同出血活性的族群,而daborhagin屬於出血活性最強的一族。透過資訊軟體對這兩類不同血性活性族群進行相似度的比對,我們找到了4個可能與P-III分子分類與出血活性相關的功能區塊(motifs)。第二部分]鎖鏈蛇蛇毒第十凝血因子活化酵素(RVV-X)是一種有效促進血液凝結的致命毒素。它是由一條重鏈與二條輕鏈所組成的異三聚體醣蛋白,但其完整的結構和N醣苷化的可能生理意義,仍有待釐清。在本研究中,我利用印尼來源的鎖鏈蛇蛇囊,進行互補DNA選殖,獲得RVV-X的三條鏈的完整基因序列。其中,推導出的重鏈氨基酸序列較先前發表的蛋白序列在羧基端多了四個氨基酸殘基;而這些單元與由Vipera lebetina蛇毒來的第十凝血因子活化酵素的各單元鏈具有77-81 %的相似性。而遠端西方墨點法的分析結果顯示,RVV-X除了作用與第十、九凝血因子外,也會結合蛋白S;一旦這種結合發生,則會促使蛋白S失去作用活性,加劇病患“瀰漫性血管內凝血”症狀。另外,利用雷射基質輔助游離法配合質譜儀鑑定(MALDI-MS)與串聯式質譜儀鑑定(MS/MS)分析由各個蛋白鏈上的N醣苷permethyl衍生物,發現其醣苷是屬於由不同變異之末端岩藻醣基化(fucosylation)和唾液酸醣基化(sialylation)所修飾的複雜且非均質型態;其中最顯著特點是末端Lewis和sialyl-Lewis抗原決定位(epitopes)之存在,我們也利用西方墨點轉漬法證實他們存在於RVV-X。功能方面,以去唾液酸醣基與未處理的RVV-X相比,發現去唾液酸醣基會導致實驗小鼠血液內較慢與較少的纖維蛋白原降解產物形成,因此我們歸納這些醣基抗原決定位可能有助於RVV-X加速進入血管系統。第三部分]了瞭解C型類凝集素(CTLL)的演化關係及在鎖鏈蛇咬傷的臨床症狀所扮演之角色,我們設計了一對引子去選殖並研究來自印度、緬甸和印尼等三個不同地區的鎖鏈蛇蛇囊之CTLL蛋白家族。我們總共選殖了4組CTLL的同源基因(八個不同單元的互補DNA),並分別命名為RVV-X LC、dabocetin、 p31與 p68;其中p31和p68是首次在本研究中被發現。藉由親緣分析A/B鏈各別之演化樹狀圖,可將鎖鏈蛇的CTLL分成三個不同的亞型(subtypes)。我們發現這些CTLL亞型在不同毒蛇地區的樣本相對表現量有差異,但是每個亞型的各別A/B鏈的表現量的調節是較一致的。值得注意的是,除RVV-X LC2蛋白序列有著明顯的差異外,不同鎖鏈蛇樣本的CTLL同源基因幾乎完全相同。而在演化樹狀圖中, p31與由Echis蛇毒得到的EMS16在功能上有著密切關聯。相比之下, p68似乎是一個以二聚異質雙體(a2b2)結構存在的新亞型。我們的研究結果顯示 p68沒有辦法抑制由ADP或膠原蛋白所誘導之血小板凝集,也不造成人類富含血小板血漿(human platelet-rich plasma)之血小板凝集。 Envenomings by Russell’s vipers (Daboia russelii and Daboia siamensis) are one of the major health hazards in many countries of South and Southeast Asia. Systemic bleeding and acute renal failure due to consumptive coagulopathy and thrombocytopenia are rather common clinical manifestations in the snakebite patients. Great geographic variations of their symptoms have also been noted. To better understand the venom proteins contributing to these symptoms, two venom families, metalloproteinase and C-type lectin-like (CTLL) have been purified and characterized in the present study. In addition, cDNA encoding these venom proteins were cloned and sequenced. Their evolutionary relationships and possible functional roles (sites) were also studied based on bioinformatic and phylogenetic analyses.art I. To characterize the highly hemorrhagic symptoms elicited by Myanmar and eastern India Daboia envenoming, two similar P-III metalloproteinases were purified from Daboia venom of both regions, and designated as daborhagin-M and daborhagin-K, respectively. They induced dermal hemorrhage in mice with a minimum hemorrhagic dose of 0.8-0.9 mg. Aa-chain of fibrinogen, fibronectin, and type IV collagen were specifically hydrolyzed by the P-III in vitro. Analyses of its cleavage sites on insulin chain B and the kinetic parameters of five representative P-IIIs toward oligopeptides suggested that daborhagin and other hemorrhagins prefers hydrophobic residues at the P1, P1'', and P2'' positions on the substrates. Of the eight Daboia geographic venom samples analyzed by Western blotting, only those from Myanmar and eastern India showed a strong positive band at 65 kDa. The full sequence of daborhagin-K was determined by cDNA cloning and sequencing, and then confirmed by peptide mass fingerprinting. Furthermore, molecular phylogenetic analyses based on 27 sequences of P-IIIs revealed the co-evolution of two major classes with distinct hemorrhagic potencies, and daborhagin-K belongs to the most hemorrhagic subclass. By comparing the absolute complexity profiles between these two classes, we identified four structural motifs probably responsible for the phylogenetic subtyping and hemorrhagic potencies of P-III SVMPs.art II. RVV-X, the factor X activator from Russell’s viper venom, is a potent pro-coagulating and lethal toxin. As a heterotrimeric glycoprotein, the detailed structures of RVV-X and possible physiological significance of its N-glycans remain to be elucidated. We cloned and sequenced all its three subunits from the cDNAs of Indonesia D. siamensis venom glands. It is found the deduced heavy chain sequence contains a C-terminal extension of four residues more than that previously published. Both light chains showed 77-81% identities to those of a homologus factor X activator from Vipera lebetina venom. Far-western analyses revealed that RVV-X strongly binds protein S besides factor X and IX. If this binding inactivates protein S, it may potentiate the disseminated intravascular coagulation syndrome observed. The N-glycans released from each subunit and the glycopeptides were profiled and sequenced by MALDI-MS and MS/MS analyses of the permethyl derivatives. All the glycans showed a heterogeneous pattern with a combination of variable terminal fucosylation and sialylation on multiantennary complex type of sugars. Among the notable features are the presences of terminal Lewis and sialyl-Lewis epitopes, as confirmed by Western blotting analyses. These glyco-epitopes possibly contributed to rapid homing of RVV-X to vascular system, as supported by the observation that slower and fewer fibrinogen-degradation products were released by desialylated RVV-X as compared with the native RVV-X.art III. To understand the evolution of CTLLs and their roles in the clinical manifestations of Daboia envenoming, we designed a pair of degenerative primers to clone and study the CTLL isoforms. Three set of cDNAs prepared from venom glands of Daboia subspecies from India, Myanmar and Indonesia, respectively, were used. Totally, eight distinct cDNAs encoding the subunits of four paralogous CTLLs—designated as RVV-X LC, dabocetin, p31, and p68—were cloned and sequenced. Among them, p31 and p68 were first identified in the present study. By biochemical analyses of the purified proteins, and cladogram analyses of sequences of both A/B subunits, the venom CTLLs are categorized into three distinct subtypes. We noticed that these CTLL subunits are differentially expressed in the geographic samples, and both subunits of each CTLL appear to be coherently expressed. Remarkably, subunits of orthologous CTLLs of different Daboia subspecies exhibited almost identical sequences, except the LC2 subunits of RVV-X show substantial variations. In the phylogenetic trees, p31 subunits are closely related to those of EMS16 from an Echis venom. In contrast, p68 seemed to be a new subtype, with a dimeric heterodimer (a2b2) structure. Our study showed that p68 does not inhibit the platelet aggregations induced by ADP and collagen, nor agglutinate the platelets in human platelet-rich plasma. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/178855 |
顯示於: | 生化科學研究所 |
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