霍氏格里蒙菌熱穩定溶血素之功能性探討及其於腫瘤治療藥物應用

Abstract

本研究針對霍氏格里蒙菌之熱穩定溶血素（Thermostable direct hemolysin, TDH）其胺基酸-酪胺酸53、蘇胺酸59以及絲胺酸63號位置分別進行其單點、雙點以及三點突變以探討其不同之生理活性。我們利用熱微差掃描分析儀(Differential Scanning Calorimetry, DSC)探討熱穩定溶血素的相轉變溫度並輔以圆二色谱 (Circular Dichroism,CD) 觀察其在升溫過程中所導致相轉變之二級結構變化。並利用紅血球溶血試驗及細胞毒性測試觀察各個熱穩定溶血素蛋白質突變株對溶血效應及其對細胞生長的影響。由結果呈現單一點突變的蛋白質會使得蛋白質結構較為不穩定，但其依然保有阿累尼亞斯效應(Arrhenius effect)；而含有雙點突變之熱穩定溶血素(組胺酸53/異白胺酸59 (Gh-TDHY53H/T59I)或異白胺酸59/蘇胺酸63(Gh-TDHT59I/S63T)) 蛋白質突變株會使得蛋白質喪失原本具有阿累尼亞斯效應的現象，而三點突變的蛋白質(組胺酸53/異白胺酸59/蘇胺酸63 (Gh-TDHY53H/T59I/S63T))亦會具有相同的結果。上述結果顯示阿累尼亞斯效應與霍氏格里蒙菌熱穩定溶血素其蛋白質結構的穩定度具有一定的關聯性。此外由圆二色谱的結果得知，擁有阿累尼亞斯效應的蛋白質在高溫狀況下其二級結構相較於喪失阿累尼亞斯效應的蛋白質還要鬆散，所以經由快速降溫過程中蛋白質重新摺疊以形成具有溶血功能的構形；而在喪失阿累尼亞斯效應的蛋白質中我們發現在加熱60℃以上時，其二級結構的構形皆會維持在-螺旋且其含量不受溫度的影響。我們認為這些蛋白質結構在高溫的狀態下並沒有被打開，所以在冷卻後其構形依然維持在60℃的纖維化型態(fibril form)。 而在細胞毒性以及溶血活性方面，這些霍氏格里蒙菌熱穩定溶血素對於細胞及紅血球具有不同程度的傷害性,由流式細胞儀的實驗我們證實熱穩定溶血素與膜結合的首步是由其N-末端胜肽或芳香族殘基色胺酸65和酪胺酸87負責，而色胺酸39和酪胺酸107殘基則負責綁定過程後的相關溶血和細胞毒性。綜合上結果得知，就霍氏格里蒙菌熱穩定溶血素而言，當其酪胺酸經突變效應後，確實對其生化特性或結構方面造成不同程度的影響。同時也進行霍氏格里蒙菌其熱穩定溶血素之高度純化，並培養成晶體，利用X射線繞射法得到其三維結構。進而探討腸炎弧菌與霍氏格里蒙菌之熱穩定溶血素在結構上的差異，相較於腸炎弧菌之熱穩定溶血素的C4四聚體結構，霍氏格里蒙菌之熱穩定溶血素形式以二聚體-二聚體的P21212晶體結構對稱。我們成功地利用結構學找出其四聚體、二聚體及單體熱穩定溶血素的構象存在並以突變方式分別得到四聚體、二聚體及單體熱穩定溶血素，由實驗證實二聚體為其生理活性的最小單位結構基礎。 另一方面利用突變技術形成毒性減弱之單體並融合上表皮生長因子之接受體辨識胜肽(EB)將熱穩定溶血素改造成針對腫瘤细胞具有導向性的先驅藥物。分別以不同表皮生長因子接受體表現量之細胞株對於融合受體辨識胜肽之谷胺酸46熱穩定溶血素突變株(Gh-TDHR46E-EB)進行其毒性測試，結果顯示對於高表現量的表皮生長因子接受體細胞株 (A431細胞株) 在每西西50微克(50 g/ml)接受體融合溶血素突變株有明顯致毒殺性。螢光免疫及細胞生長抑制實驗結果可確認接受體融合溶血素突變株確實有結合上A431細胞株並抑制癌細胞生長。在動物實驗靜脈注射給予之熱穩定溶血素突變株，有效抑制其腫瘤生長。總而言之，本論文的研究結果解釋熱穩定性溶血素作用並提供了新的見解，更是為熱穩定性溶血素開創新的藥物應用上的發展。

The TDH of G. hollisae (Gh-TDH) exhibits a similar Arrhenius effect to that of Vp-TDH and was implied as a pathogenic factor responsible for causing disease from G. hollisae infection. In order to understand the roles of critical amino acids participating in the protein Arrhenius effect and varied functional activities from Gh-TDH and to determine the intrinsic difference between Gh-TDH and Vp-TDH, a series of biophysical studies on wild type and various mutants of TDH were carried out. Among these mutated strains, , a 2- to 4-fold decrease in hemolytic activity and altered Arrhenius effect were detected from the Gh-TDHY53H/T59I and Gh-TDHT59I/S63T double-mutants and the Gh-TDHY53H/T59I/S63T triple-mutant, respectively, in contrast to Gh-TDH wild-type (Gh-TDHWT) protein. In parallel, results from circular dichroism (CD) and differential scanning calorimetry (DSC) experiments showed a conspicuous change from a -sheet to -helix structure around the endothermic transition temperature among these mutated proteins. To elucidate the structural basis of characteristic pore formation, the three-dimensional structure of the TDH from Grimontia hollisae (Gh-TDH) has been solved at 2.1 Å resolutions. In contrast to the symmetrical C4 tetramer structure of the TDH from V. parahaemolyticus, the Gh-TDH forms a dimer of dimers in overall structure and crystallizes in the orthorhombic space group P21212. The Gh-TDH exhibits full, partial, and inactive pore-forming toxicity when it exists in tetramer, dimer, and monomer conformations, respectively, supporting the dimer as a minimal unit of structural basis for physiological activity. In parallel, the flow cytometry analyses on both the N-terminal truncation and the solvent-exposed aromatic residue mutants showed that initial binding of the Gh-TDH to the membranes is promoted by Trp65 and Tyr87 and the N-terminal helix, while both Trp39 and Tyr107 residues are related to the post-binding process of hemolytic and cytotoxic activities. Taken together, these findings provide new insights into TDH’s actions in membrane recognition/binding, insertion, hemolytic activity, and cytotoxicity. In order to further investigate the toxic TDH protein for application in pharmatheutical anti-cancer drugs, the scaffold-modified TDH mutant with Epidermal growth factor receptor binding peptide (EB), Gh-TDHR46E-EB, was designed and its binding affinity to recombinant human EGFR increased. The in vitro cytotoxic activity and the selectivity of Gh-TDHR46E-EB to induce cellular death on A431 cells were demonstrated. In vivo experiments also revealed that Gh-TDHR46E-EB displayed significant anti-tumor effects. Thus, Gh-TDHR46E-EB can be used as a possible candidate for therapeutic use and opens a new avenue for the design of anti-tumor drugs in the future.