幽門桿菌烷基-過氧化物還原酶：氧壓力環境下的蛋白質體分析及其功能的研究

Alkylhydroperoxide Reductase (AhpC) of Helicobacter pylori：Proteomic Analysis and Functional Characterization under Oxidative Stress

莊明恆(Ming-Hong Chuang)

指導教授 ： 邱式鴻

國立臺灣大學/生命科學院/生化科學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.01696

摘要

幽門桿菌是一種主要為螺旋狀形態且生長緩慢的微耗氧性(5 % O2)革蘭氏陰性桿菌。由於諸多的研究已闡明了此菌是造成部份重要的腸胃疾病如胃炎、胃潰瘍、十二指腸潰瘍甚至是胃癌的主要元兇，所以世界衛生組織已將此菌列為第一類的致癌物。於2005年更將諾貝爾醫學獎頒給「揭示胃炎及消化性潰瘍係感染幽門螺旋桿菌引發的」的兩位澳洲籍科學家巴利．馬歇爾和羅賓．華倫，所以瞭解幽門桿菌對世人的重要性可見一斑。它感染於人體的胃部後引發人體免疫系統的活化而大量釋放出活性氧分子(ROS)，而這樣的反應似乎在此菌的殘存寄生與相關腸胃疾病的致病性上扮演十分重要的角色。
本研究的目的就是要探討過氧化環境對此菌的生理影響並進一步闡明相關蛋白質體表現的變化與作用機制。經由蛋白質體學方法的研究，我們已鑑定出11個蛋白質體，其中AhpC與UreE這兩種蛋白質於長時期曝露在高氧濃度(20% O2)的環境下其原有的特性會大幅地改變。此外，同源性研究的比對結果有趣地顯示出此AhpC蛋白與哺乳動物(包含人類) Prx無論在氨基酸序列、結構與酵素作用特性的相似度上皆遠高於與其他革蘭氏陰性菌的AhpC的比較結果。除此之外，在功能變化上的研究，我們也首次發現它是一種具有兩種功能的蛋白，在正常微耗氧或曝露在空氣中短時期(8小時以內)的狀況時，此蛋白保有一般正常的過氧化物還原酵素的功能，在這樣的狀態下它的結構以主要為dimer的LMW oligomers形式存在。可是，當曝露的時間過長(16小時以上)或細胞本身受傷的程度擴大時，此蛋白會藉由Trx的作用由多個oxidized-AhpC monomer分子聚合而成分子量大於660 kDa，外表呈現為立體球狀的HMW complexes，更重要的是它的功能會由原來的酵素功能完全轉變成可保護受損蛋白避免進一步變性沉積的保護子(molecular chaperone)。另外，從RT-PCR與二維電泳觀察AhpC蛋白於氧壓力下的調控與蛋白結合能力上的研究結果，發現在胃癌組織環境生存的幽門桿菌其AhpC於氧化壓力下的蛋白活性、基因表現的穩定性或是聚合後的HMW complexes的保護能力都比十二指腸潰瘍菌株好；同時由電顯之切片影像結果也顯示出胃癌組織環境的幽門桿菌較能抗氧化，且在一遇高氧壓力後會大幅表現於胞內以達到轉變為chaperone的高效率能力。所以長時期感染人體胃部並經過反覆發炎導致的ROS攻擊環境似乎不僅讓幽門桿菌發展出一套接近人體本身的基因且練就出高度抗氧化的潛力。
以此蛋白針對幽門桿菌感染相關的腸胃疾病進行血液抗體的鑑定研究，從Western- blotting與ELISA的結果顯示出胃炎與十二指腸潰瘍組織是屬於較弱的被ROS攻擊的組織，所以其所引發的抗體含量較低且辨認的AhpC蛋白為具正常構形的LMW oligomers。然而，胃潰瘍或胃癌組織其引發ROS攻擊的程度就相對很高，產生的抗體含量不僅較多外，其所辨認的AhpC蛋白的確就是HMW complexes。因此，綜合本論文的研究成果，我們相信AhpC這個在幽門桿菌內含量最多的抗氧化蛋白不僅可作為區分胃癌與十二指腸潰瘍菌株的研究起始標的以進行此二類菌株的生理特性差異的研究外，也具有在未來發展為可辨別不同腸胃疾病的醫療輔助用診斷試劑的應用潛力與價值。

關鍵字

幽門桿菌 ； 活性氧 ； 蛋白質體學 ； 同源性 ； 蛋白保護子 ； 電顯 ； 胃癌 ； 十二指腸潰瘍 ； 診斷試劑

並列摘要

Helicobacter pylori (Hp) is a spiral, slow growing gram-negative microaerophilic bacterium. Many studies have documented Hp is the etiological agent of gastro-duodenal diseases, such as chronic gastritis, gastric and duodenal ulcers, and gastric cancer. Furthermore, World Health Organization (WHO) has classified Hp as a class 1 carcinogen. The colonization of Hp in human stomach can induce inflammatory and immune reaction to release reactive oxygen species (ROS). Such responses appear to play a pivotal role in the survival of Hp and pathogenesis of Hp-related diseases. Therefore, this study is aimed to elucidate the influence of oxidative stress on H. pylori and characterize its underlying mechanisms and protein-expression profiles. By proteomic approaches, we have identified more than 10 proteins. Among these, AhpC and UreE are greatly reduced under long-term oxidative stress. Moreover, the sequence-homology comparison showed that the alkylhydroperoxide reductase (AhpC), an abundant antioxidant protein in Hp, is more homologous to mammalian peroxiredoxins than to eubacterial AhpC. We further demonstrated that this protein can switch its structure and function from low-molecular-weight (LMW) dimer or oligomers with peroxide reductase activity under normal microaerobic conditions or short-term (< 8 h) oxidative shock to high-molecular-weight (HMW) complexes with molecular-chaperone function after severe long-term (> 16 h) oxidative stress (20% O2) in the presence of thioredoxin (Trx).
As regards the structural and functional properties of AhpC under oxidative stress, we found that AhpC isolated from the gastric-cancerous strains possesses higher protein stability and chaperone activity than that from the duodenal-ulcerous strains under stress conditions. The expression of AhpC in different strains of H. pylori generally decreases under long-term oxidative stress as revealed by 2D-PAGE and RT-PCR. AhpC expression under short-term shock actually increases, being localized in the interior of H. pylori from strains isolated from patients of gastric cancer in contrast to its distribution on the cell surface of the microorganism from strains isolated from patients of duodenal ulcer.
Based on the significant difference between AhpC isolated from strains of gastric cancer and duodenal ulcer, we have applied ELISA and Western blotting to study the cross-reactivity of AhpC to antisera of H. pylori-infected patients with different pathological outcomes. The immunoassay revealed that antisera from tissues of patients with more extensive inflammation like that in patients of gastric ulcer or cancer react strongly with the HMW complexes of AhpC, whereas those from less extensive inflammation like patients of gastritis react weakly with the LMW oligomeric form of AhpC. Therefore it is conceivable that the antioxidant protein AhpC of H. pylori may prove to be useful as a diagnostic protein marker to monitor extents of tissue damages from patients of gastro-duodenal diseases infected by H. pylori.

並列關鍵字

Helicobacter pylori ； Proteomics ； oxidative stress ； 2D-PAGE ； RT-PCR ； TEM ； Western blotting ； ELISA ； gastric cancer ； duodenal ulcer ； molecular chaperone ； homology

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