以蛋白質體學鑑定及免疫分析橘青黴菌分泌性過敏原，並探討 Pen c 13 誘導人類肺上皮細胞的發炎反應

Abstract

摘要 青黴菌是引起室內過敏最主要的過敏原之一，若吸入此黴菌的孢子則會引發呼吸性的過敏症狀。然而在文獻上卻僅有少部分青黴菌過敏原曾經被報導過。而在台北都會地區，橘青黴菌 (Penicillium citrinum) 則是具有高度盛行率和致病性的過敏性黴菌，但卻也如同一般的青黴菌一樣，在學術界中未曾被深入的探討與研究。因此，本論文的研究主題主要是以橘青黴菌分泌性過敏原為研究對象，並分為以下兩大研究主軸來進行。第ㄧ部份是利用蛋白質體相關技術，來分析並鑑定橘青黴菌培養分泌物中的可能致敏分子，並探討其免疫特性。第二部份則是研究其中ㄧ個主要的橘青黴菌過敏原 Pen c 13， 在誘導人類肺上皮細胞釋放化學激素或是相關細胞激素的可能機制。 分析並鑑定過敏原分子的身份及免疫特性，是研究過敏機制最基本也是最困難的首要工作。而基於蛋白質體學相關技術的蓬勃發展，相形之下，這些研究工作也較過去來的準確許多。我們首先是以蛋白二維電泳技術，嘗試解析橘青黴菌中所有分泌性蛋白質，並建立這些蛋白質與黴菌過敏病人的 IgE 反應圖譜。我們結合液相層析串聯式質譜和蛋白質 N-端定序技術，則成功地鑑定出一個已被報導過的重要過敏原 Pen c 13 以及四個全新定義的過敏原;分別是 Cu/Zn superoxide dismutase、pectate lyase (Pen c 32)、glucanosyltransferase (GEL1) 以及 catalase (Pen c 30)。其中我們將針對 Pen c 30 來進行免疫特性分析，因為我們發現 Pen c 30 會與 48% 患有黴菌過敏病人血清中的 IgE 結合。而透過純化、cDNA 選殖、生化特性以及免疫分析等方法，我們也確實證明 Pen c 30 與 IgE 結合的高活性和致敏特性。同時也發現 Pen c 30 為ㄧ經過醣分子後修飾的醣蛋白，而且這些醣基分子對 Pen c 30 的致敏特性則扮演了決定性的角色。而至於另ㄧ個過敏原 Pen c 13 ，我們也發現它除了會與 76% 黴菌過敏病人血清中的 IgE 結合外，同時也會與 Asp f 13 有 IgE 交叉反應，並且強烈誘導黴菌過敏病人引發立即性過敏反應。另一方面，經由 Pen c 13 的 IgE 抗原決定點之圖譜分析，我們在大腸桿菌中表現ㄧ段重組的 Pen c 13 最主要 IgE 抗原決定點的胜肽片段， Pen c 13 (T261~K274) 外，也證明此胜肽對 Pen c 13 過敏病人之 IgE 的強烈反應。更進ㄧ步發現 ，Pen c 13 (T261~K274)(T272A) 或 (K274A) 的單點突變，將會減少或完全地喪失此胜肽的 IgE 結合能力。這個結果顯示 T272 和 K274，在 Pen c 13 與 IgE 結合構形中是非常重要的胺基酸殘基。 從文獻中我們清楚地知道，某些已知的致敏性過敏原，本身即具有絲胺酸蛋白酶的酵素活性，並且是造成氣喘的重要致病機制之一。而至於其相關的活化機制則仍然不是很清楚。相對地，我們也發現 Pen c 13 亦為一鹼性的絲胺酸蛋白酶。因此，在本論文的第二部份，我們將進一步探討，Pen c 13 是否同樣會利用其酵素活性，透過活化肺上皮細胞的蛋白酶活化膜受器 (protease-activated receptors，PARs) ，來誘發化學激素 IL-8 的釋放。首先我們發現， Pen c 13 在刺激 A549 細胞後，會誘導 IL-8 的釋放外，也會立即誘導細胞內鈣離子移動至細胞質中；並因而使得細胞對其他 PAR-1 和 PAR-2 活化物的作用喪失敏感性 (desensitization)。但若 A549 細胞是處在無鈣離子存在的培養基中，則 Pen c 13 便無法誘導細胞 IL-8 的釋放。更進ㄧ步的實驗亦證實，當肺上皮細胞先被處理抗 PAR-1 和 PAR-2 切位的抗體後，則會抑制 Pen c 13 誘導細胞 IL-8 的產生。相反地，另一抗 PAR-4 的抗體卻無法造成此一抑制現象。這個結果清楚地說明了 Pen c 13 透過 PAR-1 和 PAR-2 而非 PAR-4 膜受器的選擇性作用機制。 同時我們也明確地證實 Pen c 13 的確會正確地切在 PAR-1 與 PAR-2 的蛋白酶切位上。而磷脂酶 C (phospholipase C) 的抑制劑也可達到與此類似的抑制效果，更強化了 Pen c 13 與鈣離子作用的緊密關係。 除此之外，Pen c 13 誘導 A549 細胞 IL-8 的表現則是經由 ERK ½ 的活化 ，而不是透過 p38 和 JNK 的活化。並且 Pen c 13 增加 ERK1/2 的磷酸化是經由鈣離子相關路徑。這些結果顯示 Pen c 13 誘導人類肺上皮細胞 IL-8 的釋放，是透過 PAR-1 和 PAR-2 的活化機制，並且和細胞內鈣離子相關路徑有密切的關係。 總結本篇論文的結論，我們是首次以蛋白質體方法，鑑定出橘青黴菌四個新分泌性過敏原。而透過這些新過敏原的發現，將可應用於過敏診斷以及黴菌過敏疾病之治療。並且在 Pen c 13 最主要 IgE 抗原決定點上的研究結果，將有助於設計此過敏原的低過敏性構形的研發。除此之外，Pen c 13 誘導人類肺上皮細胞 IL-8 的釋放機轉，則被證實是經由 PAR-1 和 PAR-2 的活化以及細胞內鈣離子相關路徑有關。這些成果，將可促進我們對過敏性呼吸道疾病的病理機制，提供一個新的研究與過敏治療方向。

Penicillium species are important indoor allergens and it is well known that the inhalation of fungal spores can produce respiratory allergic symptoms. However, only a limited number of allergens have been reported. In the Taipei urban area, Penicillium citrinum is very common and, because of their high prevalence and allergenic properties, P. citrinum allergens deserve, but have not yet undergone, extensive investigation. Here, in this thesis, we focused our studies in secreted allergens of P. citrinum and fulfilled our researches through two separate approaches. First of all, we identified and immunologically characterized those secreted allergens of P. citrinum and secondly, to elucidate the possible mechanism(s) of IL-8 induction toward human airway epithelial cells activated by one of the important allergens, Pen c 13. The molecular characterization of allergens is essential for a better understanding of the pathogenesis of allergic diseases. In this regard, we established a two-dimensional (2-D) protein and 2-D IgE-reactive profiles respectively for an overview of the IgE-reactive proteins. By combining to use the methods of nanoLC-MS/MS and N-terminal sequencing, one known allergen, Pen c 13, and four novel allergens, namely Cu/Zn superoxide dismutase, pectate lyase (Pen c 32), glucanosyltransferase GEL1 and catalase (Pen c 30) were successfully identified. And following the findings, we then focused our attention on one secreted IgE-reactive protein, Pen c 30, which was found to bind to serum IgE from 48% of mould-sensitized allergic patients. Its IgE-binding activity and allergenicity were confirmed by purification, molecular cloning, biochemical characterization, and immunological analyses. In addition, Pen c 30 was also characterized as a glycoprotein and its glycan epitope was proved to be involved in the allergenicity. On the other hand, the well-known allergen, Pen c 13, was shown in this study to react with sera IgE in 76% of sera from mold allergic patients as well as IgE-inhibition with Asp f 13 in vitro and induced immediate type skin reactions in sensitized patients. Another IgE-binding epitope candidate of Pen c 13, Pen c 13 (T261~K274) was constructed and also shown positive reactivity to the Pen c 13-sensitized individuals. To our surprising, a significant reduction or complete loss in IgE binding of T272A or K274A mutant of Pen c 13 (T261~K274) indicated the involvement of T272 and K274 in IgE binding conformation of Pen c 13. It had long been characterized that the allergenic serine proteases involved in the pathogenesis of asthma although the activation mechanism was still uncertain. The Pen c 13, also demonstrated to be an alkaline serine protease, was further elucidated in the second part of this study to see whether Pen c 13 caused IL-8 release and activated via protease-activated receptors (PARs) in human airway epithelial cells. Pen c 13 induced IL-8 release in human airway epithelial cells. Moreover, treatment with Pen c 13 induced intracellular Ca2+ mobilization and desensitized A549 cells to the action of other proteases as well as PAR-1 and PAR-2 agonists. Pen c 13-mediated IL-8 release was significantly decreased in Ca2+-free medium. Furthermore, blocking antibodies against the cleavage sites of PAR-1 and PAR-2, but not PAR-4, inhibited Pen c 13-induced IL-8 production, as did inhibition of phospholipase C. It was proved unambiguously that Pen c 13 cleaved PAR-1 and PAR-2 at their cleavage sites. Surprisingly, Pen c 13 induced IL-8 expression via activation of ERK 1/2 but not of p38 and JNK. Treatment of human airway epithelial cells with Pen c 13 increased phosphorylation of ERK 1/2 was also shown via a Ca2+-dependent pathway. These finding showed that Pen c 13 induced IL-8 releases in airway epithelial cells and depended on PAR-1 and PAR-2 activation through the process of intracellular calcium. To the end, the main purpose of this study was to identify human IgE-reactive proteins in the culture filtrate of P. citrinum by using a proteomic approach. These novel allergens might be useful in allergy diagnosis and in the treatment of mould allergic disorders. And the study of the most major IgE-binding epitope of Pen c 13 could help to design hypoallergenic forms of the allergen. Additionally, Pen c 13 induces IL-8 release on human airway epithelial cells by PAR-1 and PAR-2 activation and intracellular calcium. Taking together, these studies promote our understanding of the pathogenesis of allergenic respiratory diseases and point the new direction for effective therapies.