中文摘要 β-bungarotoxin(β-Bgt)屬於突觸前phospholipase A2 (PLA2)神經毒素，由台灣雨傘節毒腺純化所得。β-Bgt由兩個不相同的單元體所構成，稱之為A chain 及B chain。A chain 結構相似於哺乳類與蛇類PLA2亦具有PLA2活性，以Cys15與B chain的Cys55構成分子間雙硫鍵形成一個完整的β-Bgt。而B chain的序列相似於Kunitz type 蛋白酶抑制劑與dendrotoxion。β-Bgt除了具有PLA2活性外，經由選擇性的抑制特定電位型鉀離子通道在神經肌肉接合處阻斷神經傳導物質釋放。目前台灣雨傘節已有八個A chain 及三個B chain的cDNA被確認，並可透過任意配對形成完整的β-Bgt異構毒素蛋白。隨著蛋白質層析分離技術的進步至少已分離十六種β-Bgt異構毒素蛋白。根據之前的研究指出A chain與B chain是源自於不同的基因，經各自mRNA轉譯後，A chain與B chain經不明的分子機制構成β-Bgt。此外，我們實驗室之前由台灣眼鏡蛇中選殖一Kunitz type 胰凝乳蛋白酶抑制劑(Naja naja arta chymotrypsin inhibitor，NACI)其基因組成相同於B chain且與B chain序列具有高度相似性。然而，對於B chain及其同源蛋白在基因演化上的研究仍是有限。本論文主要在於探討B chain及其同源蛋白在演化相關性及細胞毒性分子機制。
在此由台灣雨傘節中確認了β-Bgt的B2、B4、B5及B6基因，這些基因具有相同的基因結構皆由三個exon及兩各intron所構成，intron1的長度相近，intron2則明顯的長度不一。相較intron2發現B1及B2在intron2部分有兩個區塊的序列在B4、B5及B6基因中是不存在的。以RT-PCR分析結果指出在B4、B5及B6基因中缺失的兩段序列均可在台灣雨傘節的蛇腺、心臟、肝臟及肌肉組織出現，反映出這兩段的intron的序列可能插入台灣雨傘節的基因中的許多位置。相較於intron的序列，在exon的序列中除了signal peptide的部分之外具有較高的變異性。因此推測B chains在演化的過程中發生了Intron的插入或是剔除的現象；在蛋白質轉譯的序列則屬於accelerated evolution，在蛇的PLA2、neurotoxin及cardiotoxin基因演化過程中也有相同的演化現象發生。
β-Bgt誘導神經瘤SK-N-SH細胞升高細胞內鈣離子及ROS的濃度，並伴隨著p38 MAPK的活化，而導致細胞凋亡。SB202190 (p38 MAPK抑制劑), N-acetylcysteine (抗氧化劑), 1,2-bis (2-amino-phenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) (Ca2+鏊合劑)和抑制細胞內鈣離子升高試劑(ruthenium red and 2-aminoethoxydiphenyl borate)等藥劑，可明顯的降低β-Bgt之細胞毒性。為了解A、B chain在β-Bgt 誘導神經瘤細胞凋亡的過程中所扮演的功能角色，將兩個單元體蛋白分別與神經瘤細胞作用，B chain可造成與β-Bgt相同的細胞毒性效果，而A chain則無此效力，且將β-Bgt的PLA2活性去除後，發現其活性並不影響其細胞毒性。NMDA接受器之拮抗劑(MK801)及鉀離子通道阻斷劑(4-aminopyridine, 4-AP)可明顯的減少β-Bgt、B chain及不具PLA2活性之β-Bgt所造成的細胞毒性。NMDA抗體可拮抗經rhodamin標定的β-Bgt與神經瘤細胞的結合。因此推測β-Bgt是藉由調控NMDA接受器及鉀離子通道使得神經瘤細胞產生細胞凋亡之現象，且B chain在過程中扮演重要的功能角色。
由台灣雨傘節中選殖出三個B chain同源蛋白基因， 因其蛋白質序列與Kuntize type蛋白酶抑制劑相似，稱之為蛋白酶抑制劑類蛋白protease inhibitor-like protein-1 (PILP-1), PILP-2 and PILP-3。這三個基因結構均相同於B chain及NACI基因，均由三個exon及兩個intron所構成。序列經分析後發現與B chain相同在演化上均屬於adaptive evolution。以PCR的方式重組PILPs的cDNA後，利用E.coli獲得重組蛋白並以HPLC進行純化。PILP-1及PILP-2使得人類前髓性白血病U937細胞產生細胞毒性，但PILP-3的效果遠不如前兩者明顯。PILPs造成的細胞死亡現象是屬於細胞凋亡且伴隨著caspase 蛋白水解。除了PILP-3之外，發現PILP-1及PILP-2會促進細胞內p38 MAPK的活化並抑制ERK1/2活化。且PILP-1會改變細胞週期產生sub-G1，傷害粒線體使得細胞發生粒線體膜電位的喪失、細胞色素C的釋放、Bcl-2及caspase 9蛋白的水解。p38 MAPK抑制劑(SB202190)與caspase 8抑制劑，分別可有效降低PILP-1及PILP-2所造成的caspase 8與caspase 3的水解現象。TNF RII受到PILP-1及PILP-2影響其含量有明顯增加之現象，透過SB202190的作用可以有效降低TNF RII的增加。另外在MEK1持續性的活化下，PILP-1及PILP-2所產生的細胞毒性及對ERK1/2和TNF RII的影響性顯著性的降低。在PILP-1及PILP-2誘導TNF RII增加的情形下，額外TNF造成細胞更大幅的死亡。因此推測PILP-1及PILP-2透過增加TNF RII、活化p38 MAPK及抑制ERK1/2誘導細胞發生細胞凋亡。

β-Bungarotoxin (β-Bgt), a presynaptic phospholipase A2 (PLA2) neurotoxin isolated from the venom of Bungarus multicinctus (Taiwan banded krait), consists of A chain and B chain, cross-linked by an interchain disulfide bond. A chain is structurally homologous with phospholipase A2 (PLA2) enzymes, while the sequence of B chain is homologous to the Kunitz-type protease inhibitor and dendrotoxin. In addition to PLA2 activity, β-Bgt blocked the neurotransmission at the neuromuscular junction by selectively inhibiting certain voltage-sensitive potassium channels. The present studies investigated the B chain of β-bungarotoxin and B chain homologous proteins in evolutionary relationship and cytotoxic mechanism.
Eight A chain cDNAs and three B chain cDNAs have been cloned from B. multicinctus venom glands. Random combination of the A and the B chains should produce a number of β-Bgt isotoxins. There are at least 16 isoforms of β-Bgt were been isolated. Previous studies indicate that A and B chains are encoded separately by different genes, and the A and B chain genes do not originate from a common ancestor. These findings suggest that the intact β-Bgt molecules should be derived from the pairing of A and B chains after their mRNAs are translated. And, our recent studies show that B chain genes and Naja naja atra chymotrypsin inhibitor (NACI) share the same genomic organization and high sequence identity. Alternatively, limited studies on the evolutionary divergence of B chain gene and its homologous have been reported.
In the first part, the structural organization of the genes encoding B2, B4, B5 and B6 chains of β-Bgt are reported. These genes shared virtually identical overall organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 and B2 chains were absent in that of B4, B5 and B6 chains. RT-PCR analyses indicated that Bungarus multicinctus venom gland, heart, liver and muscle expressed the RNA transcripts showing sequence similarity with the intronic segment being deleted in B4, B5 and B6 chain genes. This reflects that the ancestral gene of the intronic segment might insert in multiple loci of B. multicinctus genome. Comparative analyses of B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that intron insertions or deletions occur with the evolution of B chains, and that accelerated evolution may diversify the protein-coding sequence of B chain genes same as snake phospholipase A2, neurotoxin and cardiotoxin genes.
The second part is to explore the functional contribution of the two subunits to the toxicity of β-Bgt. β-Bgt was found to induce apoptotic death of SK-N-SH cells via elevating intracellular Ca2+ and intracellular ROS production. Moreover, an activation of p38 MAPK was associated with the cytotoxicity of β-Bgt. SB202190( p38 MAPK inhibitor), N-acetylcysteine (antioxidant reagent), 1,2-bis (2-amino-phenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) (Ca2+ chelator) and the inhibitors of Ca2+ release from intracellular depots (ruthenium red and 2-aminoethoxydiphenyl borate) effectively attenuated the cytotoxicity of β-Bgt. In sharp contrast to the inability of A chain, B chain was able to induce cytotoxic effects on SK-N-SH cells as β-Bgt did. Abolishment of PLA2 activity did not significantly alter the cytotoxic activity of β-Bgt. MK801 (an NMDA receptor antagonist), antibodies against NMDA receptor and 4-aminopyridine (a potassium channel blocker) markedly reduced the cytotoxic effects of β-Bgt, B chain and catalytically inactivated β-Bgt. Moreover, antibodies against NMDA receptor blocked the binding of rhodamine-labeled β-Bgt to SK-N-SH cells. Taken together, our data indicate that B chain is a functional subunit responsible for the cytotoxicity of β-Bgt, and suggest that the cytotoxicity of β-Bgt is mediated by NMDA receptor and potassium conductance.
Homologous proteins of the B chain, namely, protease inhibitor-like protein-1 (PILP-1), PILP-2, and PILP-3, were successfully cloned from the Bungarus multicinctus genome. The 3 cloned genes each comprised 3 exons and 2 introns, similar to the genes of the B chain of β-bungarotoxin and NACI. Based on the Ka/Ks values of the gene sequences of PILPs, the B1 chain, and NACI, the PILPs may have undergone adaptive evolution. The cDNAs of the PILPs were recombined by PCR, and the recombinant proteins were successfully expressed and purified. PILPs induced cytotoxicity in U937 cells, but PILP-3 reduced viability only slightly. Induction of cell death by PILP-1 and PILP-2 was by apoptosis that occurred in a caspases-dependent manner. PILP-1 and PILP-2 activated p38 MAPK and downregulated ERK1/2; however, PILP-3 caused no such effects. PILP-1 increased the population of sub-G1 cells and caused mitochondrial damage. PILP-1-treated cells were observed to demonstrate loss of mitochondrial membrane potential, release of cytochrome C, and a decrease in the level of Bcl-2 and pro-caspase 9. Pretreatment of U937 cells with either SB202190 or a caspase 8 inhibitor significantly prevented the PILP-1- and PILP-2-induced degradation of caspase 8 or 3. PILP-1 and PILP-2 increased the level of TNFRII, which was suppressed by the p38 MAPK inhibitor. The transient transfection of PILP-1- and PILP-2-treated cells with pCMV-MEK1 efficiently increased their survival and phosphorylation of ERK1/2 but reduced the level of TNFRII. After PILP-1 and PILP-2 induced TNFRII, the excess TNF caused a significant reduction in cell viability. TNFRI levels were not significantly changed in PILP-1- and PILP-2-treated U937 cells. These results suggest that PILP-1 and PILP-2 induce apoptosis via an increase in the level of TNFRII and p38 MAPK activity and the suppression of ERK activity.

目錄
頁數
中文摘要…………………………………………………… 1
Abstract…………………………………………………… 5
序論………………………………………………………… 9
第一章
前言………………………………………………………… 18
材料與方法………………………………………………… 20
結果………………………………………………………… 23
圖…………………………………………………………… 28
討論………………………………………………………… 36
第二章
前言………………………………………………………… 38
材料與方法………………………………………………… 40
結果………………………………………………………… 47
圖…………………………………………………………… 51
討論………………………………………………………… 72
第三章
前言………………………………………………………… 75
材料與方法………………………………………………… 77
結果………………………………………………………… 87
圖…………………………………………………………… 97
討論………………………………………………………… 118
參考文獻…………………………………………………… 123

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