标题: | 探讨突变效应影响霍氏格里蒙菌之热稳定性溶血素的溶血活性、膜结合能力及其蛋白质四级结构之研究 A Study of Mutational Effect on Hemolytic Activity, Membrane Binding Ability, and Protein Quaternary Structure of Thermostable Direct Hemolysin (TDH) from Grimontia hollisae |
作者: | 黃婉婷 吳東昆 Wu, Tung-Kung 生物科技學系 |
关键字: | 热稳定溶血素;霍氏格里蒙菌;溶血活性;细胞毒性;肠炎弧菌;TDH;Grimontia hollisae;Hemolytic activity;cytotoxicity;Vibrio parahaemolyticus |
公开日期: | 2011 |
摘要: | 热稳定溶血素(Thermostable direct hemolysin, TDH)已被证实是肠炎弧菌中最主要的毒性因子,并以同源四聚体的型式存在于溶液中。霍氏格里蒙菌所产生的热稳定溶血素与肠炎弧菌中的热稳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,我们发现霍氏格里蒙菌其热稳定溶血素的酪胺酸-53 (Tyr-53) 和苯丙胺酸-159 (Phe-159) 这两个胺基酸位置会使其原聚体(protomer)与原聚体之间产生两种不同的介面(interface)。而精胺酸-46 (Arg-46) 位置则会与邻近原聚体上的数个胺基酸位置相互作用,因此其拥有最主要的作用力以维持原聚体与原聚体的结合。我们利用分生技术将酪胺酸-53和苯丙胺酸-159这两个胺基酸位置分别进行单点突变和双点突变成使其形成天门冬胺酸 (Asp),我们证实野生型热稳定溶血素 (Gh-TDHWT) 与单点突变天门冬酸胺-53突变株(Gh-TDHY53D) 在非变性凝胶上会分别形成四聚体和二聚体的构形,而单点突变天门冬酸胺-159突变株 (Gh-TDHF159D) 在非变性凝胶上的位置则是相近于单一点突变天门冬酸胺-53突变株之位置,并且两者皆具有相当的溶血活性。此外我们将精胺酸-46进行单点突变成麸胺酸 (Glu) 后,在非变性凝胶上则呈现单聚体的构形,且不具有溶血活性,此结果和肠炎弧菌热稳定溶血素的精胺酸-46改变至麸胺酸-46之突变株 (Vp-TDHR46E) 之结果相同。我们也发现双点突变在53和159位置之天门冬酸胺双突变株 (Gh-TDHY53D/F159D) 在非变性凝胶上其所呈现的位置与单点突变胺基酸46突变株相近,溶血实验中也几乎完全失去活性。另一方面,由于肠炎弧菌其热稳定溶血素和霍氏格里蒙菌之热稳定溶血素的溶血机制尚未清楚,因此我们根据DALI程式的比对,发现来自海葵中的细胞毒蛋白(Eqt II)与霍氏格里蒙菌中热稳定溶血素的结构具有高度相似性,并且参考相关文献与利用PyMol软体推测出几个可能参与和细胞膜作用的位置。我们利用溶血活性实验证实当色胺酸39号位置 (Trp-39)、色胺酸65号位置 (Trp-65)、酪胺酸87号位置 (Tyr-87)和酪胺酸107号位置 (Tyr-107)分别进行丙氨酸(Ala)扫描式单点突变后几乎不具有溶血活性。为了进一步了解这四个胺基酸位置对于红血球细胞膜之结合能力,我们进行流式细胞仪实验,发现单点突变65和87号丙氨酸突变株(Gh-TDHW65A or Y87A) 无法和红血球作用,然而单点突变39和107号丙氨酸突变株(Gh-TDHW39A or Y107A) 却明显有和红血球结合之能力,此表示胺基酸65和87两个位置可能参与和红血球细胞膜结合之过程,而胺基酸39和107则可能参与热稳定溶血素与红血球细胞膜结合后之相关过程。在细胞实验中,也指出单点突变65和87丙氨酸突变株无法和海拉细胞 (HeLa cells) 作用,而单点突变39和107丙氨酸突变株不仅拥有和海拉细胞结合之能力也具有细胞毒性,因此推测霍氏格里蒙菌其热稳定溶血素进入红血球与细胞之机制不同。先前利用X射线晶体绕射方法证实霍氏格里蒙菌其热稳定溶血素是以两个二聚体为骨架而产生四聚体的蛋白质结构,而我们也从分生实验中间接证实此结果。并且从上述实验结果推论胺基酸位置酪胺酸-53和苯丙胺酸-159在此蛋白之四级结构中扮演重要性的角色,而胺基酸位置色胺酸65和酪胺酸87号位置则参与和红血球结合之作用,色胺酸39和酪胺酸107号位置这两个胺基酸位置则可能在霍氏格里蒙菌其热稳定溶血素和红血球结合后的过程(post-binding)相关。 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. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT079928505 http://hdl.handle.net/11536/49955 |
显示于类别: | Thesis |