探討青黴菌屬過敏原Pen c 13引發人類上皮細胞之緊密連結蛋白質的降解

Pen c 13 Induces Degradation of Tight Junction Proteins of Human Epithelial Cells

蘇育逸(Yu-Yi Su)

指導教授 ： 周綠蘋

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.10417

摘要

黴菌為引起異位性疾病及其他許多過敏疾病的重要致敏物。其中，青黴菌 (Penicillium sp.) 和黃麴菌 (Aspergillus sp.) 為台灣地區室內的重要過敏原，而具有絲胺酸蛋白質水解酶(serine protease) 活性的過敏原已經被鑑定為主要的過敏原群 (group 13) 。當過敏原進入人類呼吸道之後，最先接觸到的就是呼吸道上皮細胞，研究顯示，這些水解酶可能透過活化上皮細胞的膜受器，引發一系列過敏相關之訊息傳導，或是利用本身酵素活性直接攻擊上皮細胞之細胞連結(cell junctions) 而造成局部發炎反應。本實驗室純化得到之具有絲胺酸蛋白質水解酶活性的橘青黴(P. citrinum) 過敏原Pen c 13已知可以活化其細胞膜上的受器(protease-activated receptors) PAR-1、PAR-2而導致發炎反應，因此本篇研究即是探討絲胺酸蛋白質水解酶Pen c 13如何影響肺上皮細胞之細胞連結蛋白質。
本篇研究利用大腸上皮細胞(Caco-2)以及肺上皮細胞(NCI-H441)兩種細胞株來進行實驗。首先，利用測量細胞TEER(transepithelial electrical resistance)值發現，處理Pen c 13的兩種細胞，其TEER值會在24小時之後有明顯下降，初步顯示細胞間的連結可能遭到破壞。接著利用屬於不同細胞連結複合體的蛋白質抗體進行細胞免疫染色，配合共軛焦電子顯微鏡(confocal microscopy)的觀察，發現緊密連結複合體中的蛋白質occludin會因為細胞處理Pen c 13而造成occludin的斷裂。同時，西方點墨法(western blot)的結果再次驗證兩種細胞都會因處理 Pen c 13而造成occludin的斷裂。
另外，利用合成occludin的片斷胜肽配合液相層析串聯質譜(LC-MS/MS)鑑定以確定Pen c 13對於occludin的切點位置。結果切點位於occludin extracellular loop之第88和第91個胺基酸。由於經過Pen c 13處理後的細胞，其細胞型態會改變。因此探討Pen c 13對細胞骨架actin的影響，實驗結果顯示細胞骨架因處理Pen c 13而重新分布。
橘青黴(P. citrinum) 過敏原Pen c 13透過本身水解酶酵素之活性可以破壞大腸上皮細胞Caco-2和肺上皮細胞NCI-H441的緊密連結蛋白occludin，使細胞骨架actin重新分布，增加表皮細胞的通透性，而使得更多過敏原可以越過此屏障進而造成發炎反應。

關鍵字

黴菌過敏原 ； 緊密連結 ； 蛋白質水解酶

並列摘要

The fungi are regarded as the main source of allergens that can cause atopic disease and many other allergic diseases. Penicillium and Aspergillus are the most common indoor fungal species in Taiwan and alkaline serine proteases from them have been identified as a group of major allergens (group 13). When inhaled, allergens initially contact the human airway epithelium. The previously studies showed that the allergens with protease activity may involve in the inflammatory response by proteolytic attack and direct activation of epithelail cells. Pen c 13 allergen is a serine potease we had purified and identified from Penicillium. citrinum, it can cause inflammatory response through the activation of protease-activated receptors 1(PAR1) and PAR2. Thus, the purpose of this study is to investigate the interaction between the Pen c 13 allergen and human lung epithelial cell.
When Caco-2 and NCI-H441 cells were treated with Pen c 13, the TEER value was obviously decreased after 24 hours, it indicated that the junctions between cells may be disrupted. Following immunocytochemistry staining with different types of junctional protein antibodies and using confocal microscopy, we found that the tight junction protein occludin was disrupted. Western blot analysis also confirmed the disruption.
To address whether Pen c 13 cleavage sites within extracellular domain of occludin, we used LC-MS/MS to analyze the products formed by reacting pure Pen c 13 with synthetic peptide segments of the occludin loop sequences. 88AWDRGYGTSLLG99, of the first extracellular loop of occludin, underwent cleavage of AW and RG. Because the cell morphology was changed after treated with Pen c 13, the cytoskeleton actin was investigated. The cytoskeleton actin re-distributed after treated with Pen c 13.
The allergen Pen c 13 from P. citrinum disrupted the tight junction protein occludin of Caco-2 and NCI-H441 cells by its serine protease activity, re-distributed the cytoskeleton actin, and increased the permeability of epithelial cells. These results suggest that Pen c 13 may contribute to inflammatory response by damaging the barrier function of epithelial cells.

並列關鍵字

mold allergen ； tight junctions ； protease

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