探討IL-15選擇性剪接異構體對於IL-15受體結合與IL-15調控反應之影響

Abstract

介白質-15 (Interleukin-15, IL-15)是四α螺旋細胞激素家族的一員，能夠調節記憶性CD8 T細胞 (Memory CD8+ T cell)、自然殺手細胞 (Natural killer cell, NK cell)、自然殺手T細胞 (Natural killer T cell, NKT cell) 以及腸道上皮內淋巴細胞 (Intestinal intraepithelial lymphocyte, IEL) 的複製和存活。介白質-15同時也是一個前發炎性細胞激素 (Proinflammatory cytokine)，能夠加重人類發炎性疾病如類風濕性關節炎、乾癬、乳糜瀉等的病理狀態。本實驗室過去的研究發現，一種缺少了部分第七外顯子的IL-15選擇性剪接異構體 (以下簡稱IL-15ΔE7)，具有調節皮膚免疫反應的功能，而這個效果不是因為缺少IL-15所導致。然而，這個調節作用的機制尚不清楚。為了尋找IL-15ΔE7可能的調控機制，在本論文研究中，我利用台灣國立交通大學所設計的「(PS)2蛋白質結構預測伺服器」模擬建構出IL-15ΔE7的蛋白質結構，並且用PyMOL蛋白質結構分析軟體比較IL-15和IL-15ΔE7對於IL-15受體結合的異同。結構分析結果顯示因為IL-15ΔE7缺少了對IL-15Rα的結合重要的麩胺酸 (Glutamate) 殘基，可能因此失去對IL-15Rα的結合。另外，IL-15ΔE7可能保留對於IL-2/IL-15Rβ的結合。然而，IL-15ΔE7蛋白質的構象改變 (Conformational change) 可能使其失去對於IL-2/IL-15Rγc的結合，甚至阻礙IL-15ΔE7的生物活性。得利於實驗室先前建立的酵母菌蛋白表現與純化系統，我們可以製造IL-15以及IL-15ΔE7重組蛋白，藉此在IL-2/IL-15依賴性HT-2細胞株研究IL-15ΔE7的生物活性以及其對IL-15調控反應之影響，驗證從蛋白質結構分析所獲得的結果。用飽和濃度下的IL-15ΔE7處理HT-2細胞無法引起STAT5 (IL-2/IL-15Rγc訊息傳遞途徑) 和ERK1/2 (IL-2/IL-15Rβ訊息傳遞途徑) 的磷酸化，而這跟先前的研究是一致的。另外，以IL-15ΔE7預處理接著以IL-15處理或者同時以IL-15ΔE7和IL-15共處理的實驗顯示IL-15ΔE7對IL-15媒介的訊息傳遞途徑似乎沒有拮抗效果。許多免疫學上重要的分子例如IL-2、IL-4、IL-6以及IL-15，他們的選擇性剪接異構體被認為是細胞激素訊息傳遞的天然抑制劑，會競爭原型細胞激素對於受體的結合。然而，我們尚未知道IL-15ΔE7是否為IL-15的競爭型抑制劑。為了測試這個可能性，我修改並設計了一個配體競爭性結合實驗 (Ligand competition assay)。以固定濃度的IL-15 (EC50) 和十倍序列稀釋的IL-15ΔE7共同處理HT-2細胞，再利用即時聚合酶鏈鎖反應 (Real-time PCR)檢測IL-15調控之細胞分裂和下游基因表現 (細胞週期調控基因Cdk1、抗細胞凋亡基因Bcl2) 的變化。結果顯示即使以100倍濃度的IL-15ΔE7共處理也無法抑制IL-15引起之Cdk1和Bcl2表現，這暗示IL-15ΔE7可能不會跟IL-15競爭IL-15受體結合。本論文的蛋白質結構分析預測IL-15ΔE7對於IL-15受體的結合可能會改變，因此，未來值得測量IL-15ΔE7對於各別IL-15受體次單元的結合常數 (binding constant)，這有助於更精確的設計實驗以及深入了解IL-15ΔE7生物功能的分子機制。另外，IL-15ΔE7是否會透過IL-15受體引起不同的訊息傳遞途徑藉此調控IL-15的功能，尚須實驗證實。

Interleukin-15 (IL-15) is a member of the four α-helix bundle cytokine family, which is best known to support the proliferation and survival of memory CD8+ T cells, natural killer (NK) cells, NKT cells, and intestinal intraepithelial lymphocytes (IELs). IL-15 also functions as a proinflammatory cytokine that promotes pathophysiologic state for human inflammatory diseases such as rheumatoid arthritis, psoriasis, and coeliac disease, etc. Previous study has shown that IL-15ΔE7, one variant of IL-15 generated by alternative splicing, has immune modulatory function in skin, and the effect is not caused by the lack of IL-15. However, the mechanism underlying this modulatory effect is still unknown. To find the possible mechanism underlying the IL-15ΔE7 modulation, in this thesis, I used (PS)2 (Protein Structure Prediction Server) web portal to predict the protein structure of IL-15ΔE7 and comparatively analyzed the interactions between IL-15 receptor subunits with IL-15 versus IL-15ΔE7 by PyMOL software. The results from structural analysis revealed that IL-15ΔE7 might lose its binding to IL-15Rα because it lost key glutamate residues that were responsible for receptor interaction. While the binding with IL-2/IL-15Rβ might be preserved, impaired interaction with IL-2/IL-15Rγc caused by protein conformational change could severely impede the bioactivity for IL-15ΔE7 as was well known for IL-15. Aided by the capability to generate recombinant IL-15ΔE7 protein in better quality using yeast recombinant system, the functional effects of IL-15ΔE7 on IL-15-mediated responses in IL-2/IL-15-dependent HT-2 cell line were further investigated to confirm the information derived from structural analysis. In consistent with the previous study, I demonstrated that IL-15ΔE7 treatment at saturating doses did not activate the phosphorylation of STAT5 (IL-2/IL-15Rγc signaling) and ERK1/2 (IL-2/IL-15Rβ signaling) as were induced by IL-15 in HT-2 cell line. Also, cotreatment and pretreatment experiments showed that IL-15ΔE7 had no antagonistic effect on IL-15-mediated signaling transduction. Variants of many immunologically important molecules such as IL-2, IL-4, IL-6, IL-15, generated by alternative splicing, are believed to be natural inhibitors of cytokine signaling, essentially acting as dominant negative forms of the cytokine that compete with the full-length cytokine for receptor binding. However, whether IL-15ΔE7 can also act as a competitive inhibitor of IL-15 function is unknown. To test this possibility, a “modified” ligand competition assay was performed by cotreating HT-2 cell line with fixed concentration of IL-15 (around EC50) and 10-fold serial titrated concentrations of IL-15ΔE7. IL-15-mediated responses of cell proliferation and downstream gene expression (cell cycle regulation gene Cdk1 and anti-apoptotic gene Bcl2) were measured by real-time PCR analysis. The results showed that cotreatment of IL-15ΔE7 did not block Cdk1 and Bcl2 expression even at 100 times of concentration than IL-15, which suggested that IL-15ΔE7 might not compete IL-15 for IL-15 receptor binding. Since the structural analysis have predicted the change in IL-15 receptor binding of IL-15ΔE7, it is worth to further measure the binding constant of IL-15ΔE7 to each IL-15 receptor subunits, which helps to better design the experiments and to understand the molecular mechanism of IL-15ΔE7 biological function. Also, whether IL-15ΔE7 transduces differential signaling through IL-15 receptors or that may have interfering effect on IL-15 remain to be elucidated.