行政院國家科學委員會專題研究計畫期中進度報告:草菇毒蛋白A2與細胞膜作用機制之探討(2/3)

草菇毒蛋A2 (Vovatoxin A2; VVA2)是由草菇子實體純化出來的一種溶血毒素 (hemolysin)。溶血毒素對細胞膜的作用機制可分為三種：形成孔洞型 (pore-forming)、酵素 型 (enzymatic)，及表面作用劑型 (surfactant)。形成孔洞的溶血毒素結構單位是由親水性蛋 白單體 (monomer) 聚集成嵌膜寡聚合體 (oligomer)，其嵌入細胞膜之蛋白結構主要可分為 兩種：amphipathic α-helix 及amphipathic β-sheet，而後者之孔洞形成作用機制可統整為三 步驟：細胞膜結合 (membrane binding)、寡聚合物形成 (oligomerization)及細胞膜嵌入 (membrane insertion)，至目前為止有二種模式被提出來解釋形成孔洞毒素的作用機制。 先前的研究成果得知VVA2 共有199 個胺基酸組成，但不具cysteine 殘基。VVA2 可 在紅血球細胞膜及微脂體 (liposomes)表面產生孔洞並與其寡聚合體之形成有關。 patch-clamp 電生理實驗，顯示VVA2 的作用機制是可穿透細胞膜，其孔洞可讓分子量為70 kDa 的dextran 進出。此外，我們亦分析出VVA2 由單體型式轉變成寡聚合體的過程中，會 產生二級及三級蛋白質結構變化，從以上的實驗可鑑定出VVA2 造成細胞溶解機制應是藉 由孔洞形成型式造成的。 為了進ㄧ步分析VVA2 與細胞膜作用機制，以期鑑定其嵌入細胞膜功能區域 (membrane insertion domain)及了解VVA2 結構與功能之關係，本年度我們運用surface plasmon resonance (SPR)、stop-flow fluorescence 及NuPAGE技術分析VVA2 與細胞膜作用之 動力學特徵，結果顯示VVA2 與細胞膜作用後，大部分不解離，我們亦發現VVA2 在寡聚合 形成發生之時間是在其穿透細胞膜發生之前且與其構型之改變 (conformational change)是 相伴發生的。由於VVA2 在有微脂體存在時不管在4 oC或37 oC環境下，均可形成寡聚合體， 然而只有在37 oC環境下才具有溶血作用，因此我們認為VVA2 形成孔洞的模式較支持 pre-pore模式學說。此外，利用限制性水解 (limited proteolysis)及electrospray ionization mass spectrometry (ESI-MS)，我們亦鑑定出一段N端片段(N-terminal fragment; NTF)(1-127 殘基)， 可能是VVA2 的ㄧ個功能性區域 (functional domain)。經由proteolytic digestion方法，則確認 VVA2 嵌入細胞膜功能區域 (membrane insertion domain)位置乃包含其C端β-6, 7, 8 strands。Volvatoxin A2 (VVA2) is a hemolytic toxin that was isolated from the edible mushroom Volvariella volvacea. The hemolytic toxic proteins can be classified based on their mechanism of action to lyse red blood cells: pore forming, enzymatic degradation of membrane lipids and solubilizing cell membranes by a surfactant action. All pores are formed through aggregation of water-soluble monomers to oligomeric integral membrane protein. A pore-forming toxin (PFT) is classified as an α-PFT or a β-PFT according to their structure of insertion, amphipathic α-helices or β-sheet. The general mechanism of pore formation by β-PFTs can be summarized as three steps: membrane binding, oligomerization, and membrane insertion. Until now, two model of pore formation by the toxic proteins have been proposed. Our previously studies showed that the primary structure of VVA2 was found to be comprised of 199 amino acid residues without cysteine residues. It formed pores in human erythrocyte membranes and liposomes in response to their formation of oligomers. This toxic protein also permeabilized the cell membrane and allowed the passage of 70-kDa dextran rhodamine B into cultured Xenopus myocytes. Furthermore, conformational change occurred when VVA2 interacted with liposomes. To further characterize the mechanism of VVA2 interacting with phospholipids, identify its membrane insertion domain and realized the relationship of its structure and function, we use surface plasmon resonance (SPR), stop-flow fluorescence and NuPAGE technologies to address these issues this year. The irreversible binding of VVA2 to the membrane was demonstrated by SPR analysis. Furthermore, the molecular events of VVA2 oligomerization accompanied by the conformational change and preceded its membrane permeabilization were shown by stopped-flow fluorescence and NuPAGE system. Because VVA2 oligomerization occurred at 4 or 37 oC in the presence of liposomes, while VVA2 lysed red blood cells at 37 oC but not 4 oC, it is likely that VVA2 oligomerization precedes membrane insertion, a step that is temperature-dependent, favoring pre-pore model. In addition, we identified an N-terminal fragment (NTF) (1-127 residues) of VVA2 as a domain by limited proteolytic digestion. We also found a fragment localized at the C-terminal half of VVA2 containing β-6, 7, and 8, which is protected from protease digestion due to its insertion of a membrane