探討突變效應影響霍氏格里蒙菌之熱穩定性溶血素的溶血活性、膜結合能力及其蛋白質四級結構之研究

Abstract

熱穩定溶血素（Thermostable direct hemolysin, TDH）已被證實是腸炎弧菌中最主要的毒性因子，並以同源四聚體的型式存在於溶液中。霍氏格里蒙菌所產生的熱穩定溶血素與腸炎弧菌中的熱穩定溶血素在胺基酸序列上具有87% 的一致性。在之前的研究中，我們發現霍氏格里蒙菌其熱穩定溶血素的酪胺酸-53 (Tyr-53) 和苯丙胺酸-159 (Phe-159) 這兩個胺基酸位置會使其原聚體(protomer)與原聚體之間產生兩種不同的介面(interface)。而精胺酸-46 (Arg-46) 位置則會與鄰近原聚體上的數個胺基酸位置相互作用，因此其擁有最主要的作用力以維持原聚體與原聚體的結合。我們利用分生技術將酪胺酸-53和苯丙胺酸-159這兩個胺基酸位置分別進行單點突變和雙點突變成使其形成天門冬胺酸 (Asp)，我們證實野生型熱穩定溶血素 (Gh-TDHWT) 與單點突變天門冬酸胺-53突變株(Gh-TDHY53D) 在非變性凝膠上會分別形成四聚體和二聚體的構形，而單點突變天門冬酸胺-159突變株 (Gh-TDHF159D) 在非變性凝膠上的位置則是相近於單一點突變天門冬酸胺-53突變株之位置，並且兩者皆具有相當的溶血活性。此外我們將精胺酸-46進行單點突變成麩胺酸 (Glu) 後，在非變性凝膠上則呈現單聚體的構形，且不具有溶血活性，此結果和腸炎弧菌熱穩定溶血素的精胺酸-46改變至麩胺酸-46之突變株 (Vp-TDHR46E) 之結果相同。我們也發現雙點突變在53和159位置之天門冬酸胺雙突變株 (Gh-TDHY53D/F159D) 在非變性凝膠上其所呈現的位置與單點突變胺基酸46突變株相近，溶血實驗中也幾乎完全失去活性。另一方面，由於腸炎弧菌其熱穩定溶血素和霍氏格里蒙菌之熱穩定溶血素的溶血機制尚未清楚，因此我們根據DALI程式的比對，發現來自海葵中的細胞毒蛋白(Eqt II)與霍氏格里蒙菌中熱穩定溶血素的結構具有高度相似性，並且參考相關文獻與利用PyMol軟體推測出幾個可能參與和細胞膜作用的位置。我們利用溶血活性實驗證實當色胺酸39號位置 (Trp-39)、色胺酸65號位置 (Trp-65)、酪胺酸87號位置 (Tyr-87)和酪胺酸107號位置 (Tyr-107)分別進行丙氨酸(Ala)掃描式單點突變後幾乎不具有溶血活性。為了進一步了解這四個胺基酸位置對於紅血球細胞膜之結合能力，我們進行流式細胞儀實驗，發現單點突變65和87號丙氨酸突變株(Gh-TDHW65A or Y87A) 無法和紅血球作用，然而單點突變39和107號丙氨酸突變株(Gh-TDHW39A or Y107A) 卻明顯有和紅血球結合之能力，此表示胺基酸65和87兩個位置可能參與和紅血球細胞膜結合之過程，而胺基酸39和107則可能參與熱穩定溶血素與紅血球細胞膜結合後之相關過程。在細胞實驗中，也指出單點突變65和87丙氨酸突變株無法和海拉細胞 (HeLa cells) 作用，而單點突變39和107丙氨酸突變株不僅擁有和海拉細胞結合之能力也具有細胞毒性，因此推測霍氏格里蒙菌其熱穩定溶血素進入紅血球與細胞之機制不同。先前利用X射線晶體繞射方法證實霍氏格里蒙菌其熱穩定溶血素是以兩個二聚體為骨架而產生四聚體的蛋白質結構，而我們也從分生實驗中間接證實此結果。並且從上述實驗結果推論胺基酸位置酪胺酸-53和苯丙胺酸-159在此蛋白之四級結構中扮演重要性的角色，而胺基酸位置色胺酸65和酪胺酸87號位置則參與和紅血球結合之作用，色胺酸39和酪胺酸107號位置這兩個胺基酸位置則可能在霍氏格里蒙菌其熱穩定溶血素和紅血球結合後的過程(post-binding)相關。

Thermostable direct hemolysin (TDH), a pore forming toxin, has been recognized as a virulent factor in Vibrio parahaemolyticus. It exists as a homo-tetramer in solution. The homologous TDH from Grimontia hollisae shows 87% identity to Vp-TDH, but displays a distinct interface structure between protomers. Notably, the Gh-TDH and equinatoxin II (Eqt II) from sea anemone exhibit a highly structural similarity, whereas Eqt II has been shown to interact with the lipid membrane via specific regions. In order to understand the roles of functional residues that located in the distinct protomer-protomer interface between Gh-TDH and Vp-TDH and to figure out the critical amino acids involved in the putative membrane binding region, a series of biophysical studies on various mutants of TDH were carried out. The protomer-protomer interface related Gh-TDHY53D and Gh-TDHF159D mutants formed dimer conformation, as observed from the Native PAGE. Furthermore, the Gh-TDHY53D and Gh-TDHF159D mutants exhibited slightly lower hemolytic activities as compared to that of Gh-TDHWT. Thus, the Gh-TDH proteins might form dimer-based tetramer and display variant extent of hemolytic activities, depending on different conformational structures. In parallel, no hemolytic activity was observed from the Gh-TDHW39A, Gh-TDHW65A, Gh-TDHY87A, Gh-TDHY107A and Gh-TDHY87A/Y107A mutants. Moreover, neither fluorescent signal on the cell surface nor fluorescence intensity in flow cytometry analysis was observed while Gh-TDHW65A and Gh-TDHY87A conjugated with FITC was treated with erythrocytes or HeLa cells, indicating that the Trp-65 and Tyr-87 might play critical roles in the process of Gh-TDH initial binding membrane. Interestingly, the flow cytometry analysis and cell experiments indicated that Gh-TDHW39A and Gh-TDHY107A possessed the abilities to bind with cell membrane as that of Gh-TDHWT, in spite of the loss of their hemolytic activity. These results indicated that the Trp-39 and Tyr-107 may assist in the post-binding process of Gh-TDH during its hemolytic process. In summary, both the Tyr-53 and Phe-159 showed their importance in directing protein quaternary structure formation. The Trp-65 and Tyr-87 residues might play a role participating in the protein-membranes binding whereas Trp-39 and Tyr-107 were related to the post-binding process in Gh-TDH hemolytic process.