Functional Proteomic Studies on Galectin-1 Induced Glycosylation Involved in Lung Cancer Metastasis

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基本資料

Project title

Code/計畫編號

NSC98-2311-B019-004-MY2

Translated Name/計畫中文名

半乳糖凝集素誘導醣化作用在肺癌惡性轉移之功能性蛋白質體學研究

Project Coordinator/計畫主持人

Tai-Yuan Chen

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

陳水田
余忠仁
楊崑德

Department/Unit

Chang Gung Medical Foundation

Website

https://www.grb.gov.tw/search/planDetail?id=1902336

Year

2009

Start date/計畫起

01-08-2009

Expected Completion/計畫迄

31-07-2010

Bugetid/研究經費

1400千元

ResearchField/研究領域

基礎醫學
生物技術(醫)

背景：肺癌屬於高死亡率的癌症，其中超過一半的病患在診斷一年內死亡，第四期病人的五年存活率低於5%。腫瘤惡性轉移會導致癌症急速惡化並加速死亡。腫瘤惡化和轉移已被證實和腫瘤細胞的細胞膜上醣蛋白之醣殘基結構有關，且岩藻醣化跟癌症進程息息相關。我們從第四型岩藻糖基轉移酶過量表現的肺癌細胞中發現peroxiredoxin-1 (PRDX-1), heat-shock protein 27 (HSPB1) 和 galectin-1 半乳糖凝集素(Gal-1)大量表現。半乳糖凝集素已知可調控細胞貼附和運動並和細胞增生、細胞凋亡、血管新生和mRNA剪接作用有關。特定目標：主要探討半乳糖凝集素在肺癌細胞中是否有誘導高度醣化現象及與腫瘤惡性轉移的相關性。分析醣蛋白中蛋白質與醣殘基的主成分、結構、鍵結與功能性或致惡性轉移原因之相關性。進一步對醣蛋白中蛋白質與醣殘基進行定量分析以驗證和評估生物標誌物的功能性。另外也試著以不同蛋白質體學技術建構半乳糖凝集素高度表現或誘導醣化後所調控的蛋白質交互作用網絡。實驗設計與方法：分別以固相(MALDI-TOF/TOF)和液相層析(ESI-LC-MS/MS) 串聯質譜儀鑑定半乳糖凝集素誘導的醣化現象。先以共同免疫沈澱法大量收集半乳糖凝集素和其作用之醣化蛋白質後，再以酵素分離醣殘基和蛋白質部份，以固相串聯質譜儀分析。或者將和半乳糖凝集素作用之醣化蛋白質以液相層析串聯質譜儀搭配二種離子碰撞方法(CID&ETD)進行醣蛋白分析。另一部份分別以半乳糖凝集素功能增加(過量表現)和功能喪失(RNA干擾)以及外源性重組半乳糖凝集素添加培養去探討半乳糖凝集素在肺癌細胞運動、侵襲和惡性轉移的角色。同時以小鼠腫瘤惡性轉移模式觀察半乳糖凝集素在活體上對腫瘤惡性轉移的影響。預期結果：此計畫成果可幫助釐清半乳糖凝集素的高度表現或誘導之醣化現象可能造成肺癌細胞惡性轉移的分子機制，並可提供未來臨床轉譯研究的基礎。 Background: Lung cancer is a leading disease worldwide, and more than 50% patients die within one year of diagnosis. Metastasis is a critical hallmark which makes cancers much more difficult to treat and greatly increases mortality. Fucosylation is closely associated with process of cancer metastasis. We have discovered three proteins: peroxiredoxin-1 (PRDX-1), heat-shock protein 27 (HSPB1) and galectin-1 (Gal-1) were significantly higher expression in α-1,3-fucosyltransferase IV (FUT-4) overexpressed A549 cells. Galectin-1 mediates cell adhesion and migration, and is involved in several processes, including proliferation, apoptosis, angiogenesis and mRNA splicing. Specific aims: The specific aim of this study is to investigate whether galectin-1 induces higher glycosylation in lung cancer cells to promote cancer mobility and metastasis. The abundant component, structure, linkage, and functions of glycans and proteins, will be achieved. Meanwhile, the quantitative analyses of targeted glycoproteins will be conducted for validating and assessing the biomarkers. The protein network derived by galectin-1 induced glycosylation or hyper-expression will be constructed by proteomic and bioinformatic strategies. Design and methodology: Two approaches will be adopted. Galectin-1 interacting glycoprotein from wild type A549 and FUT4 overexpressed A549 will be initially separated into glycan and protein moieties before applying to MALDI-TOF/TOF analysis. Alternatively, galectin-1 interacting glycoprotein obtained from coimmunoprecipitation is directly sent to LC-MS/MS couple with CID and ETD. On the other hand, gain-of-function and loss-of-function studies will be also performed. The transient and stable transfection of Gal-1 into A549 cells will be created. In contrast, RNAi approach will be conducted to validate galectin-1 function in lung cancer metastatic power. Moreover, recombinant galectin-1 expressed in E. coli and CHO cell systems will be created and extracellular added to culture for investigating functional characteristics including proliferation, migration, invasion, and metastatic potentials. The mice metastasis model will also be monitored. Anticipated results: This study will decipher significance of glycosylation status induced by galectin-1 that possibly mediates lung cancer metastatic capability. Specific identification of the galectin-1-induced glycosylation that is involved in proliferation and/or migration may be translated into clinical application.

DSpace CRIS

Functional Proteomic Studies on Galectin-1 Induced Glycosylation Involved in Lung Cancer Metastasis

基本資料

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