免疫相關蛋白與目標分子結合之鑑定及功能探討

Abstract

免疫系統是人體內相當嚴密調控的防禦機制，經由先天免疫與後天免疫調節而保護我們不受外來抗原入侵，此論文著述兩種免疫調控方式，一為外源性分子Maltoheptaose對後天免疫的訓練與強化；第二部分為內源性的免疫受體CD69於配體結合後之免疫反應的平衡與調控機制。小麥草為一常見的天然營養輔助品，被認為具有許多功效與療效，包括免疫調節，因此長期被作為預防以及治療疾病。我們先前發現小麥草中的寡醣成分具有活化人類周邊血單核細胞的活性，並且鑑定其活性物質為Maltoheptaose，利用細胞體篩選技術證明其直接活化單核細胞，增加CD69、CD80、CD86、IL-12以及TNF-α表現，而進一步影響自然殺手細胞和T細胞的活化。在此研究中，我們使用一合成的Maltoheptaose-probe來執行其目標結合分子的鑑定，Maltoheptaose-probe的結構包括Diazirine和Alkyne，利用其光致交聯特性而與目標分子產生共價鍵結，並經由點擊化學反應標定生物素以利純化。應用此Maltoheptaose-probe和質譜儀分析，以及利用外加不同受體的阻斷性抗體於細胞表面進行篩選，我們於單核細胞株模式中鑑定Toll-like receptor 2 (TLR-2)為Maltoheptaose的目標結合受體，並藉由NF-κB螢光素酶報告基因的活性證明Maltoheptaose活化TLR-2的下游分子NF-κB進入細胞核內調節基因表現，達到免疫調控的作用。 CD69為一免疫細胞表面受體蛋白，大多於免疫活化時大量表現，多數研究認為其具備調節免疫平衡的能力，與調節性T細胞的分化和功能有重要關聯性，但關於CD69如何調控調節性T細胞以及生物體內的CD69配體之了解仍然不足。利用免疫沉澱法以及質譜儀分析，我們從人類周邊血單核細胞中鑑定到兩個結合蛋白質，分別是S100A8和S100A9，此兩個結合蛋白彼此會以單體、二聚體和四聚體存在，我們透過結合分析及阻斷性試驗證明CD69分別與S100A8和S100A9有結合性及專一性，而核糖核酸干擾實驗結果說明了S100A8與S100A9同時存在能夠與CD69達到最佳的結合。我們運用奈米級液相層析串聯質譜儀分析CD69胜肽上的 N聯聚醣之準確修飾位置和結構組成，並發現此N聯聚醣決定了與S100A9的結合性，且顯著地影響了人類初始T細胞分化成調節性T細胞的能力。在功能性分析中，我們觀察到CD69與S100A8/S100A9結合後調節人類周邊血單核細胞的細胞激素TGF-β與IL-4，也透過影響SOCS3-STAT3分子路徑而促進人類調節性T細胞的形成。本研究定義了一對新的受體-配體結合關係為CD69-S100A8/S100A9，並證實他們於調節性T細胞分化過程中的影響與調節機制。

The immune system mounts robust immune response against invading microorganisms while retaining the capacity to keep natural autoimmunity under control. This thesis contains two parts to study the immune modulation through different manners including a novel herbal derived immunostimulator “maltoheptaose” and the identification of CD69 receptor-ligand mediated immune regulation. Wheatgrass is one of the most widely used health foods, but its functional components and mechanisms remain unexplored. The wheatgrass-derived oligosaccharides (WG-PS3) were previously isolated and found to induce CD69 and TH1 cytokine expression in human peripheral blood mononuclear cells (hPBMC). In particular, WG-PS3 directly activated the purified monocytes by inducing the expression of CD69, CD80, CD86, IL-12, and TNF-α but affected NK and T cells only in the presence of monocytes. After further purification and structural analysis, maltoheptaose was identified from WG-PS3 as an immunomodulator. Herein, maltoheptaose was demonstrated to activate monocytes via Toll-like receptor 2 (TLR-2) signaling, which was discovered by pretreatment of blocking antibodies against Toll-like receptors (TLRs) and by use of alkyl diazirine-based photo-affinity probes and click chemistry. The TLR-2 downstream signaling NF-κB activation was also observed in maltoheptaose-treated THP-1 cells, as determined by NF-κB-driven luciferase activity. This study is the first to reveal the immunostimulatory component of wheatgrass with well defined molecular structures and mechanisms. The second part describes the identification of endogenous ligands for the CD69 receptor and its biological function. CD69 is a leukocyte activation receptor that functions as the regulatory factor in the maintenance of immune homeostasis and is positively selected on the surface of activated regulatory T cell (Treg) lineage. However, whether and how CD69 is involved in the generation of Treg cells has yet to be elucidated. Here we show that CD69 per se is not sufficient to support the switch of the CD4+ naive T cells into Tregs in the absence of antigen-presenting cells, suggesting that additional ligand-receptor interaction is likely required for the stimulation. Using immunoprecipitation and mass spectrometry, we identified the S100A8/S100A9 complex as the natural ligand of CD69 receptor in hPBMC. The in vitro binding assay and competition assay further confirmed a specific interaction existing between CD69 and S100A8/S100A9 complex. Mechanistically, as peptide N-glycosidase treatment of CD69 abolished its interaction with S100A8/S100A9, the ligand-receptor binding of CD69 most likely requires N-linked glycans of CD69. To be more precise, we also determined the glycosylation site and the N-glycan compositions of CD69 through the nanoflow liquid chromatography coupled with tandem mass spectrometry. Functionally speaking, we showed that interaction between CD69 and S100A8/S100A9 is required for the up-regulation of SOCS3 expression leading to inhibited STAT3 signaling during conversion into Treg cells. In addition, CD69-S100A8/S100A9 association increases the secretion of functional cytokines of Treg cells TGF-β leading to reduced IL-4 production in hPBMC. Overall, our results not only define a novel ligand-receptor binging between CD69 and S100A8/S100A9 but also provide their functional and mechanistic interplays involved in Treg cell differentiation.