第一部份：結合蛋白質體學與生物資訊學工具來分析Pen c 13蛋白水解酶誘導過敏性氣道發炎小鼠模式之肺部蛋白質體差異;第二部份：Pen c 13之B細胞抗原決定位的低過敏性融合蛋白運用於過敏原特異性免疫治療法

Abstract

第一部份 黴菌過敏原與氣喘的病程發展有關，其中有些黴菌過敏原已被鑑定為蛋白水解酶，在此，我們建立一個過敏性氣道發炎動物模式，經由連續支氣管注射具有蛋白水解酶活性之Pen c 13，該蛋白為橘青黴菌所分泌的一種主要過敏原。在功能性的分析中發現，當暴露於Pen c 13會導致肺部組織中發生一些病理性的變化包括：氣道過度反應性增加、發炎細胞浸潤、黏液過度分泌以及膠原蛋白沉積；在血清分析試驗中發現總量IgE以及Pen c 13-特異性IgE和IgG1有顯著上升的情形；另外體外培養暴露於Pen c 13小鼠的脾臟細胞，當給予不具有蛋白水解酶活性的Pen c 13刺激後，會促使第二型輔助性T 細胞的細胞激素IL-4、IL-5和IL-13的分泌增加。 為了探討此致病機制，我們使用差異性螢光標記二維電泳，並結合液相層析串聯質譜儀，接著再利用生物反應路徑資料庫暨分析平台軟體加以進行研究，配對出數個具意義性的功能性生物訊息路徑。其中電腦預測分數最高的訊息路徑，以功能和疾病來分類，是與急性過敏肺部嗜酸性白血球增多症和細胞移動有關，接著將此路徑做為進一步的詳細剖析探討。結果指出許多的典型途徑也牽涉於此路徑中，其中包括：肌動蛋白骨架訊號、白血球外滲訊號、整合素訊號、NRF2-調控的氧化壓力反應、黏著斑激酶訊號、緊密連接訊號以及急性期反應訊號。接著，利用生物反應路徑資料庫暨分析平台軟體的一些搜尋附加工具，找出與過敏性發炎相關的可能蛋白分子，結果發現半乳糖凝集素-3以及層黏蛋白可能參與在此致病機制中。最後我們將研究重點放在細胞間連接蛋白上，因為透過環境中的蛋白水解酶作用下，除了使得上皮屏障打開，可能成為氣喘發展的第一步外，這一些連接蛋白也與肌動蛋白的重新排列有關。總之，當持續暴露於Pen c 13會引起細胞間的連接結構改變和肌動蛋白骨架的重新排列，最後導致上皮細胞通透性的增加和氣道結構的改變。這些影響或許轉變了肺部的微環境並且加速過敏性致敏作用。 第二部份 目前臨床上已被使用的特異性免疫治療主要是給予患者過敏原的萃取物，使患者對於過敏原臨床上的耐受性增加以消除過敏症狀。然而該治療方式會造成副作用風險以至於無法廣泛被運用。最近的研究顯示過敏原萃取物的抗原決定位之複雜性，能夠使用基因重組技術來製造，設計低過敏性的衍生物以增加治療的安全性。在本研究中，我們發展衍生於Pen c 13之B細胞抗原決定位的胜肽，此Pen c 13是由橘青黴菌所分泌的一個人類主要過敏原，並且已被鑑定為一個鹼性絲胺酸蛋白水解酶，能夠當作過敏疾病治療的應用策略。 為了找尋位於Pen c 13的線性抗原決定位，藉由化學或是酵素方式來切割蛋白，產生涵蓋幾乎整個Pen c 13蛋白序列之不同序列的切割胜肽，進一步來繪製過敏性的抗原決定位，研究結果發現，至少有十個不同的線性IgE結合抗原決定位在Pen c 13蛋白的序列之中，其中胜肽S16 (A148-E166) 及胜肽S22 (A243-K274) 能夠分別被90%和100%測試的過敏病人之血清辨識。此外再經由ELISA抑制試驗來對S16、S22胜肽之IgE結合特異性加以驗證。接著本研究挑選胜肽S22來詳細查究其與IgE結合的能力，我們使用分子模擬以及B細胞抗原決定位預測軟體，預測發現有六個胺基酸最可能參與IgE結合。此外，將S22胜肽的N-端 (A243-A260) 與 C-端 (T261-K274) 兩部份分別與GST形成融合蛋白，經過病人血清的篩選之後，實驗結果顯示主要IgE結合能力是位於C-端的胺基酸上。最後在S22胜肽的C-端其最可能參與此IgE結合的胺基酸，經由點突變分別替換成丙胺酸，其中胺基酸274的位置以丙胺酸替換的突變型，有顯著降低與IgE的反應性，實驗推論這個突變型也許能夠設計成為低過敏性的過敏原，以作為未來發展治療人類過敏疾病的安全及有效之治療策略。

Part I Fungal allergens are associated with the development of asthma, and some have been characterized as proteases. Here, we established an animal model of allergic airway inflammation in response to continuous exposure to proteolytically active Pen c 13, a major allergen secreted by Penicillium citrinum. In functional analyses, Pen c 13 exposure led to increased airway hyperresponsiveness, significant inflammatory cell infiltration, mucus overproduction, and collagen deposition in the lung, dramatically elevated serum levels of total IgE and Pen c 13-specific IgE and IgG1, and increased production of the Th2 cytokines IL-4, IL-5, and IL-13 by splenocytes stimulated in vitro with Pen c 13. To examine the mechanisms, we performed two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis combined with nano-LC-MS-MS, followed by Ingenuity Pathways Analysis (IPA) to map significant functional networks. The highest-scoring network that associated with acute allergic pulmonary eosinophilia and cell movement in the Functions and Diseases analysis was selected for further dissection. Additionally, canonical pathways, including actin cytoskeleton, leukocyte extravasation, integrin, NRF2-mediated oxidative stress response, FAK, tight junction, and acute phase response, were also highlighted. Using IPA tools to identify potential targets, galectin-3 and laminin might be involved in novel pathogenic mechanisms. Finally, we focused on junctional proteins, because, in addition to opening of the epithelial barrier by environmental proteases possibly being the initial step in the development of asthma, these proteins are also associates with actin rearrangement. Taken together, Pen c 13 exposure causes junctional structure alterations and actin cytoskeletal rearrangements, resulting in increased permeability and airway structural changes. These effects probably change the lung microenvironment and foster the allergic sensitization. Part II Specific immunotherapy (SIT) that is in use at present involves the administration of allergen extracts to patients leading to the clinical tolerance of the allergens and cure for allergic symptoms. However, the risk of therapy-induced side effects limits its broad application. Recent studies have revealed that the epitope complexity of allergen extracts can be recreated using recombinant allergens, and hypoallergenic derivatives of these can be engineered to increase treatment safety. In present study, we developed the nonanaphylactic peptides derived from B cell epitopes of Pen c 13, an immunodominant human allergen secreted by Penicillium citrinum identified as an alkaline serine protease, to be a generally applicable strategy for the therapy of allergy. To find linear epitopes on Pen c 13, mapping of allergenic epitopes was performed by cleaved peptides which cover most of the protein sequence. The results showed that at least ten different linear IgE-binding epitopes located throughout the Pen c 13. Of these, peptide S16 (A148-E166) and S22 (A243-K274) were recognized by sera from 90% and 100% of the patients tested. In addition, the specificity of IgE binding was confirmed by ELISA inhibition assays. The peptide S22 was selected for dissection of its IgE-binding ability, and therefore we exerted molecular modeling and B-cell epitope predicted server to predict six most possible residues involved in IgE binding. Furthermore, the peptide S22 was split into two parts which comprise N-terminal (A243-A260) or C-terminal (T261-K274) part fused to GST. The result of the serum screening showed that the majority of IgE-binding ability resides indeed in its C-terminal fragment. Final, six most possible residues within C-terminus of the peptide S22 were substituted for alanine individual by point mutations; one of the mutants of Pen c 13 (T261-K274), K274A, had dramatically reduced IgE reactivity and may be designed hypoallergenic forms of the allergen, which develop a safe and efficient therapeutic strategy for treating human allergic diseases in the future.