利用醣蛋白質體學方式探討第四型岩藻糖轉酶超量表現對肺癌細胞株膜醣蛋白組的改變

Glycoproteomics approaches that depict differential membrane glycoproteome profiles of fucosyltransferase IV-overexpressed A549 cell

紀閔介(Min-Chieh Chi)

指導教授 ： 陳水田

國立臺灣大學/生命科學院/生化科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02141

摘要

醣修飾的改變在癌症生物學上扮演一個重要的角色，其往往會牽涉到後續的癌細胞進程作用，包括：侵犯、轉移等。在本篇論文研究當中，我們利用超量表現第四型岩藻糖轉酶的肺癌細胞株(A549FucT4)及其對照組細胞株(A549Mock)作為實驗的模式細胞，探討該糖轉酶對於細胞膜蛋白糖修飾的影響。在先前的實驗分析結果中：無論是轉移盤移行分析(transwell migration assay)、細胞貼附試驗(adhesion assay)、明膠酶譜法(zymography)或是觀察細胞株在免疫缺陷型小鼠(SCID mice)體中增殖及轉移的情形皆發現岩藻糖轉酶的表現能賦予細胞更惡性的表現。本研究首先利用基質輔助雷射脫附游離飛行式質譜(MALDI-TOF mass spectrometry)法分析膜蛋白岩藻醣基化修飾的情形：不同於A549Mock細胞株的結果，在A549FucT4的醣譜中明顯地發現有些許特異性的寡醣分子被鑑定出，即證明第四型岩藻糖轉酶的表現會促使細胞表面的醣化修飾發生改變。此外我們利用醣蛋白質體學之方式比較並試圖鑑定出具有差異且高度岩藻糖基化修飾的膜蛋白。從嗜醣蛋白墨點(lectin blot)法、西方墨點(western blot)法以及二維電泳的分析結果皆顯示：A549FucT4細胞株有較多膜蛋白種類發生α-1,3岩藻糖基化修飾的現象。進而利用嗜醣蛋白親和性層析(lectin affinity chromatography)分離並富集化帶有岩藻糖之膜蛋白胜肽，經由液相層析串連電灑游離飛行式質譜儀(LC/ESI-TOF-MS)以及非標記質譜定量分析方法鑑定差異性醣修飾之蛋白質。相關被鑑定出具岩藻糖基化差異之膜蛋白多和細胞貼附有關，例如：一型接觸蛋白(contactin-1)、插入素蛋白(integrin)、細胞黏附分子(cell adhesion molecule)及橋粒芯蛋白(desmoglein)等，些許蛋白也同時具調控細胞訊息傳之功能。藉此結果，我們希望能瞭解到第四型岩藻糖轉酶在各種膜醣蛋白進行修飾時，該群蛋白對於肺癌細胞的影響，

關鍵字

肺癌 ； 膜蛋白 ； 第四型岩藻糖轉酶醣蛋白質體學 ； 非標記質譜定量

並列摘要

Aberrant glycosylation play a pivotal role in cancer biology. In this study, we employed A549Mock and A549FucT4 cell lines as the models to comprehend the fucosyltransferase IV affecting glycosylation and cancer progression with a focus on cell membrane proteins by comparative glycoproteomic approaches. All previous data, not only in vitro assay but also in vivo animal experiments, demonstrated that A549FucT4 cell line is more malignant than A549Mock and possesses higher capacity of metastasis. The A549FucT4 over-expresses fucosyltransferase IV, which contribute to Lewis glycoepitopes so that carbohydrates attaching to proteins are likely to be highly fucosylated. The entire set of N-glycan was analyzed by MALDI-TOF mass spectrometry. The MS profile of A549FucT4 N-glycan revealed that additional molecular ion was detected, unique carbohydrate structures from mock transfectant. Lectin-based strategy coupling with LC/ESI-MS/MS was adopted for our purpose of identification of the membrane protsins which were decorated with more fucosyl glycoepitopes. The searching results against MASCOT software by MS/MS data revealed assignment of 19 specific proteins in the result of A549FucT4. All identified peptides containing N-X-S/T sequon were quantitatively analyzed by the automated software, IDEAL-Q. Most of the identified proteins are involved in cell adhesion, such as contactin-1, CD166, and integrin-α 3. In addition, some of them are thought to modulate signaling pathway. We anticipate that our finding can facilitate us to understand the role of the fucosylated membrane proteins in lung cancer.

並列關鍵字

lung cancer ； membrane proteins ； fucosyltransferase IV ； glycoproteomics ； label-free quantitative mass spectrometry

參考文獻

1. Cloos, P. A., and Christgau, S. (2004) Post-translational modifications of proteins: implications for aging, antigen recognition, and autoimmunity, Biogerontology 5, 139-158.

2. Reinders, J., and Sickmann, A. (2007) Modificomics: posttranslational modifications beyond protein phosphorylation and glycosylation, Biomol Eng 24, 169-177.

3. Grunewald, S., Matthijs, G., and Jaeken, J. (2002) Congenital disorders of glycosylation: a review, Pediatr Res 52, 618-624.

4. Marth, J. D., and Grewal, P. K. (2008) Mammalian glycosylation in immunity, Nat Rev Immunol 8, 874-887.

5. Balcan, E., Tuglu, I., Sahin, M., and Toparlak, P. (2008) Cell surface glycosylation diversity of embryonic thymic tissues, Acta Histochem 110, 14-25.