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研究生:吳柏龍
研究生(外文):Po-Long Wu
論文名稱:台灣眼鏡蛇毒與整合素作用之非RGD毒蛋白的鑑定與生物活性探討
論文名稱(外文):Identification of Non-RGD Integrin-Binding Proteins in Taiwan Cobra Venom and Their Biological Implication
指導教授:吳文桂
指導教授(外文):Wen-guey Wu
學位類別:博士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:119
中文關鍵詞:心臟毒蛋白整合素噬骨細胞的分化蛇毒金屬蛋白酵素骨再吸收作用黏著蛋白受體
外文關鍵詞:CardiotoxinintegrinOsteoclast differentiationSnake venom metalloproteasebone resorptioncell adhesion receptor
相關次數:
  • 被引用被引用:2
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  • 下載下載:20
  • 收藏至我的研究室書目清單書目收藏:2
被台灣眼鏡蛇咬傷的主要症狀之ㄧ為傷口癒合速度緩慢且造成嚴重的組織壞死。一般認為心臟毒蛋白在眼鏡蛇毒性中扮演重要的角色。心臟毒蛋白是眼鏡蛇毒中最主要的蛋白,大約占蛇毒總重的百分之五十。但對於心臟毒蛋白是否在細胞上有蛋白受體並未有清楚的結果。
在此論文中,我們運用了包括分子生物學、生物物理、細胞實驗鑑定出眼鏡蛇毒內幾個不同且不具有精胺酸-甘胺酸-天冬胺酸的胺基酸序列的整合素結合蛋白,同時也探討它們於傷口癒合上可能的生物意義及治療學上的運用。我們發現了蛇毒中主要的成分心臟毒蛋白會專一性的和{alpha}v{beta}3整合素結合,利用表面電漿共振決定出心臟毒蛋白A5和{alpha}v{beta}3整合素的解離常數為0.3微莫爾濃度,同時在微莫爾濃度範圍內,會抑制噬骨細胞的分化及噬骨細胞的骨質再吸收作用,將來具有潛力發展成整合素的拮抗物。
雖然心臟毒蛋白A3也會與{alpha}v{beta}3整合素結合,但心臟毒蛋白A3所導致細胞死亡的現象,不論是用抗{alpha}v{beta}3整合素抗體LM609無法完全的抑制,或是表現{beta}3的中國倉鼠卵巢細胞也並未對心臟毒蛋白A3所造成的毒性更為靈敏,此結果顯示心臟毒蛋白A3和{alpha}v{beta}3整合素結合對於毒性這件事並未有明顯的相關性。除了{alpha}v{beta}3整合素之外,我們量測到心臟毒蛋白也會以微莫爾濃度範圍的解離常數與{alpha}v{beta}1整合素結合。相對於野生種及B2變異株的中國倉鼠卵巢細胞而言,大量表現{alpha}5整合素的中國倉鼠卵巢細胞對於心臟毒蛋白A3更為靈敏,顯示出心臟毒蛋白A3所導致細胞死亡機制和{alpha}5整合素表現量有關連。Focal adhesion kinase (FAK)是整合素下游的訊號傳遞分子,有FAK缺陷的胚胎纖維母細胞對於心臟毒蛋白A3的靈敏度降低,顯示其他整合素可能和心臟毒蛋白A3所導致細胞死亡機制有關。然而我們並未找到一個可完全抑制心臟毒蛋白A3所導致細胞死亡的方法,顯示心臟毒蛋白A3所導致細胞死亡是複雜且具多方面性。
蛇毒金屬蛋白酵素一般廣泛的存在於腹蛇科及響尾蛇科。我們是第一次從台灣眼鏡蛇毒中純化到此蛇毒金屬蛋白酵素-atragin,同時也利用分子生物學得到此酵素的核苷酸序列。結果顯示此一蛇毒金屬蛋白酵素是屬於第三類型的蛋白,和印度眼鏡蛇中的金屬蛋白酵素cobrin有著極高的相似度(97%),隱含著來自同一祖先的可能性,且再度的證實金屬蛋白酵素存在於神經性毒蛇中。其中位於像去整合素區域中也具有大家認為會與整合素結合的XXCD序列。Atragin會造成內皮細胞的凋亡現象,而此過程可能是藉由分解細胞外間質或內皮細胞上的{alpha}v{beta}3整合素蛋白,而導致細胞失去附著。Atragin 也會抑制胚胎纖維母細胞的移動,與心臟毒蛋白A5也有協同性的作用。而這抑制細胞移動的主要原因可能來自於金屬蛋白酵素區域的酵素活性,且與整合素的交互作用伴演重要角色。這顯示蛇毒金屬蛋白酵素於人類被蛇毒咬傷後,傷口癒合速度緩慢有關。
最後,藉由比較不同序列的心臟毒蛋白與{alpha}v{beta}3整合素結合能力,我們認為心臟毒蛋白A5指環一的疏水區域和指環二附近帶電的胺基酸扮演著和整合素結合的重要位置。
Severe tissue necrosis with a retarded wound healing process is a major symptom of the cobra snakebite. Cobra cardiotoxins (CTXs) are the major toxin peptides and constitute ~ 50% weight of the cobra venom. CTXs are believed to play a critical role in cobra venom toxicity. It is not clear whether there are cellular receptors for CTXs.
In this study, we perform experiments spaning molecular biology, biophysical, cell-based and bone resorption in vitro studies to identify several Non-RGD integrin binding protein in Taiwan cobra venom and their biological implication on wound healing or the therapeutic potentials. Non-cytotoxic cobra CTX A5 binds to integrin {alpha}v{beta}3 with an apparent ~ 0.3 microM and inhibits both osteoclast differentiation and bone resorption in microM range with therapeutic potentials as an integrin antagonist.
Although CTX A3 bind to {alpha}v{beta}3 integrin. Anti-{alpha}v{beta}3 integrin antibody LM609 can barely protect cell from CTX-induced cytotoxicity and neither {beta}3-CHO cells are more susceptible to CTX action. These results show that {alpha}v{beta}3 integrin are not involved well in CTX A3-induced cytotoxicity. SPR studies showed CTX A3 and CTX A5 also bind to {alpha}v{beta}1 integrin with an apparent dissociation constant of 1.8~2.5 microM. {alpha}v -CHO cells are more susceptible to CTX action compared with wildtype- and B2- variants. It implies that {alpha}v-integrins are involved in CTX-induced cytotoxicity. Fibroblasts lacking focal adhesion kinase (FAK), which is directly downstream of integrins, are much less sensitive to CTX A3 than those with FAK. These results establish that integrins are involved in CTX-induced cytotoxicity.
Snake venom metalloproteases (SVMPs) are widely distributed in most viperid and crotalid venoms. We first cloned and purified a novol metalloprotease atragin from Naja atra venom. cDNA sequence analysis revealed that atragin as a novel PIII-type SVMPs, sharing highly sequence homolog (~97 %) with cobrin purified from Naja naja with conserved integrin binding motif XXCD compared with other SVMPs. It implied that atragin and cobrin come form the same ancestor. Atragin induced apoptosis of endothelium cells through digestion of extracellular matrix or cell surface receptor, therefore, lead to lost of cell adhesion. Atragin also inhibited fibroblast migration and showed synergistically inhibition effect with CTXA5. Apoatragin, which lacks metalloprotease activity, is unable to inhibit fibroblast migration. It suggested that SVMP might play an important role in the wound region with a retarded wound healing process.
Finally, by comparing the integrin binding affinity among CTX homologues, we propose that the hydrophobic domain near the tip of loop I and the charged residues flanking the other hydrophobic loop II region may be involved in CTXs-integrin interaction.
CHINESE ABSTRACT………………………………………………… I
ENGLISH ABSTRACT………………………………………………… III
INTRODUCTION……………………………………………………… V
ABBREVIATION……………………………………………………… XVI
TABLE CONTENT…………………………………………………… XIX

CHAPTER 1 Non-cytotoxic cobra cardiotoxin A5 binds to integrin {alpha}v{beta}3 and inhibits bone resorption: Identification of cardiotoxins as non-RGD integrin- binding proteins of the Ly-6 family

SUMMARY…………………………………………………………… 1
INTRODUCTION…………………………………………………… 2
EXPERIMENTAL PROCEDURES……………………………… 4
RESULTS & DISCUSSION…………………………………………… 9
FIGURES & FIGURE LEGENDS…………………………………… 14

CHAPTER 2 Relationship between cardiotoxin A3-integrin binding and cytotoxicity

SUMMARY…………………………………………………………… 25
INTRODUCTION…………………………………………………… 26
EXPERIMENTAL PROCEDURES………………………………… 28
RESULTS & DISCUSSION………………………………………… 30
FIGURES & FIGURE LEGENDS…………………………………… 36

CHAPTER 3 A novel metalloprotease from Taiwan cobra venom induced endothelial cell apoptosis and inhibited fibroblast migration through interacting with {alpha}v{beta}3 integrin : Imply the role of SVMP on snake bitten wound healing process

SUMMARY………………………………………………………… 45
INTRODUCTION………………………………………………… 46
EXPERIMENTAL PROCEDURES………………………………… 48
RESULTS & DISCUSSION……………………………………… 52
FIGURES & FIGURE LEGENDS………………………………… 60

APPENDEX I Cobra cardiotoxin-induced membrane pore formation in sulfatide-containing membrane vesicles : specificity & kinetics & relationship with phase separation.

SUMMARY………………………………………………………… 81
INTRODUCTION…………………………………………………… 82
EXPERIMENTAL PROCEDURES………………………………… 84
RESULTS & DISCUSSION………………………………………… 86
FIGURES & FIGURE LEGENDS…………………………………… 94

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