2013-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/708733摘要：金針菇免疫調節蛋白 (fungal immunomodulatory protein from Flammulina veluptipes, 簡稱 FIP-fve) 是我國新發現之免疫調節活性物質，其分子量為 12.7 KDa 含 114 個胺基酸，已完成分子選殖，在自然狀態為雙元體 (homodimer) 構造。先前國內多個研究團隊的研究發現，FIP-fve 於體外可活化抗原呈獻細胞 (antigen presenting cells, APCs) 或輔助細胞，在死亡的 APCs 存在下可活化T 淋巴球細胞；於動物模式中，FIP-fve 具有活化 TH1 免疫反應、抗過敏、抗過敏原、抗腫瘤、作為抗癌疫苗佐劑之作用。作為具開發潛力的生醫保健對象而言，FIP-fve 活化 APCs 與T 淋巴球細胞的關鍵細胞受器仍不清楚，且FIP-fve對於不同免疫細胞協同作用的機轉、受FIP-fve誘導後 APCs 與 T 細胞間的互動關係，以及 FIP-fve 雙元體結構與其作用的關連，至今仍無不夠明瞭。 由於 FIP-fve 已知的作用方式，類似於超抗原 (super antigen) 之模式，且預試驗的電腦結構模擬比對結果，顯示FIP-fve之雙元體構造，與 MHC class II β chain 之結構高度相似，可做為支持FIP-fve具備類似超抗原作用之依據；本計畫即擬以「FIP-fve之雙元體為超抗原」作為假說，設計實驗深入研究 FIP-fve 活化APCs與 T 細胞的分子機制，並著重於探討多重免疫細胞間的相互作用。第一年度將探討 FIP-fve 是否不必經由 antigen processing 之過程便可活化 T 淋巴細胞，評估FIP-fve 活化T 細胞時 APCs 的角色，以及探討FIP-fve活化 T 細胞後, T 細胞的 TCR V-β and V-α usage, 此皆為超抗原作用之重要指標；同時將將選殖及表現製備八種 truncates FIP-fve缺失蛋白，作為研究 FIP-fve 雙元體作用之材料。第二年度將研究 FIP-fve 與 MHC class I, II 分子的親和能力，以及FIP-fve對 APCs 的作用，探討FIP-fve對APCs之活化作用、FIP-fve對MHC分子之親和能力，以及利用anti-MHC 抗體中和方式反證MHC分子之親和能力。第三年度將探討FIP-fve雙元體結構與其活化 T 細胞的分子機制，利用不同FIP-fve truncates為材料，正向與反向驗證FIP-fve的雙元體構造確為其具備類似超抗原作用之關鍵，評估不同 Truncated FIP-fve 蛋白活化免疫時，T 細胞與 APCs 交互作用之差異，以及 FIP-fve 雙元體結構與免疫細胞經由 MHC 分子活化之關係。本計畫之研究成果預期將能闡明與解釋 FIP-fve 分別活化 T 淋巴球細胞以及 APCs 的細胞受器與作用機制，說明 FIP-fve 之雙元體結構與類超抗原作用之關連，可提供未來更廣泛、更深化應用 FIP-fve 之關鍵資訊。 <br> Abstract: The fungal immunomodulatory protein from Flammulina velutipes, designated FIP-fve, is a novel mushroom protein isolated and cloned by Taiwan researchers. FIP-fve, having a molecular weight of 12.7 kDa with 114 animo acids, is a homodimer in its natural structure. Within in vitro experiments, FIP-fve has been reported to activate antigen presenting cells (APCs) and accessory cells as well as to induce T lymphocyte activation in the presence of irradiated APCs. Moreover, in various animal models, FIP-fve exhibited the capability of arousing TH1 immune responses and showed anti-hypersensitivity, against allergen-induced hypersensitivity, anti-hepatoma and anti-cancer functions, and it could be used as an adjuvant for mouse EP-1 virus induced breast cancer. However, the primary receptors responding for FIP-fve signaling remains unclear. Neither the diverse roles of FIP-fve directly toward the different immune cells nor the further synergistic cooperation of both APCs and T cells are far from understood. The relationship between homodimeric structure of FIP-fve and further immune-regulating activities have not been extensively studied. It was known that the functions of FIP-fve were similar to those of super antigens. On the other hand, the structure of FIP-fve, estimated by bioinformative investigation in preliminary experiments, was highly resembling that of MHC class II β-chain, which gives the structural evidence to support that FIP-fve could possibly be a super antigen. Based on our hypothesis that FIP-fve is a super antigen, we will explore the molecular mechanisms involving the cell to cell interaction among APCs and T cells after FIP-fve induction. In the first year, we will evaluate the involvement of APCs and antigen processing on FIP-fve induced T cell activation. The T cell receptor (TCR) Vβ and Vα usage in induced cells, which is a major evidence of super antigen, will be determined using FACS and quantitative PCR methods. After that, eight truncated FIP-fve clones and prepare truncated FIP-fve proteins will be constructed for further investigation of its homodimeric functions. In the second year, the direct binding and affinity between FIP-fve and MHC molecules will be studied. The mAbs against MHC molecules will be applied to neutralize the APC activation and to prove the involvement of MHC molecules. In the third year, we will use truncated FIP-fve proteins to compare the necessity of homodimeric structure on the activation of both APCs and T cells and to research the relationship between its homodimeric structure and the functions. Furthermore, the cell to cell interaction between APCs and T cells will also be investigated and clarified while the truncated FIP-fve proteins were applied. The expected contributions of this project are 1) to identify the membrane receptors of FIP-fve within APCs and T cells, respectively, 2) to explain the molecular mechanism of how FIP-fve interacts with APCs and T cells, to each other and both together, 3) to clarify the relationship between the homodimeric structure and the super antigen-mimic function of FIP-fve, and 4) to provide the key and strong information for extensive utilization of FIP-fve in the future.金針菇免疫調節蛋白抗原呈現細胞蛋白間交互作用Flammulina veluptipesimmunomodulatory proteinantigen presenting cellsprotein-protein interaction