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標題: | 醣質體分析應用於探討醣轉移酵素與特定癌症相關抗原表現之相關聯性 Systematic and Targeted Glycomic Analysis to Map the Expression of Specific Terminal Glycotopes in Relation to Over-expression of β1,3-Galactosyltransferase V |
作者: | Yen-Ying Chen 陳彥穎 |
指導教授: | 邱繼輝(Kay-Hooi Khoo) |
關鍵字: | 醣質體學,質譜,醣化現象,醣轉移酵素, glycomics,mass spectrometry,glycosylation,glycosyltransferase, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | 細胞表面的醣化現象,被認為在細胞間辨識、溝通、和黏結等方面扮演著重要角色,而機制上,通常是經由特定的末端醣結構所調控。因此,了解細胞如何形成特定醣類結構,是醣生物學中相當重要的一環。哺乳類動物細胞的醣類結構,主要是一基本的雙醣單元,聚合延伸形成醣鏈骨架,其上可添加不同的修飾而成為各式各樣的末端醣結構。
擔任骨架的雙醣單元以第二型醣鏈(Galβ1-4GlcNAc, 也稱為LacNAc)最為常見。另外一種雙醣單元則為第一型醣鏈(Galβ1-3GlcNAc),兩者的不同只在於單元內兩單醣間的鍵結方位不同。一般來說,細胞內的第二型醣鏈可繼續延伸出去,形成多乳糖胺聚醣(polylactosaminoglycans);而第一型醣鏈則普遍被認為無法再被延伸出去。但有極少數的例子,如1990年代初期,在一大腸癌細胞株Colo 205的表面醣脂質即發現帶有第一型醣鏈的延伸結構,並有高度的岩藻醣修飾現象。探討其生合成機制,在已知的醣轉移酵素中,以第五號β1,3-半乳糖轉移酵素最有可能負責第一型醣鏈的延伸,不僅因在發現延伸型第一型醣鏈的Colo 205細胞中此酵素有相當高的表現量,此醣轉移酵素亦被證實對於第一型醣鏈單元的形成,有著重要的影響。 為證明第五號β1,3-半乳糖轉移酵素所扮演的角色,此酵素被轉殖且穩定表現於另一大腸癌細胞株 DLD-1。利用辨認特殊醣抗原的抗體所進行的免疫染色法和串聯式質譜分析,包括雷射輔助脫附游離(MALDI)的Q/TOF(低能量斷裂模式)和TOF/TOF(高能量斷裂模式),與配達液相層析的LTQ-Orbitrap所進行的product ion-dependent MS3等方法,對轉殖細胞的醣質體進行全面的分析。首先,確定了轉殖第五號β1,3-半乳糖轉移酵素的DLD-1細胞大量表現第一型醣鏈相關的岩藻醣化抗原,如Lewis a、Lewis b和sialyl Lewis a,並且抑制了某些原本在DLD-1裡高表現量的第二型醣鏈相關抗原,如Lewis y。接著確立轉殖細胞的醣脂質和醣蛋白上,出現了延伸型第一型醣鏈結構。最後,藉由把醣質體圖譜上帶有不同末端修飾分子的訊號加以量化而進行比較,發現轉殖第五號β1,3-半乳糖轉移酵素的DLD-1,其表面醣化中的唾液酸修飾(sialylation)和polylactosaminoglycans被明顯抑制,而岩藻醣化(fucosylation)則有提升的現象,說明了第五號β1,3-半乳糖轉移酵素在大腸癌細胞的醣化作用裡,不僅提供了延伸型第一型醣類結構的生成,亦影響到其他的醣類末端修飾作用,對於細胞的醣質體生成,有著重要影響。 Glycosylation plays essential roles in cell-cell recognition, communication and adhesion by means of specific glycotopes. Among the important glycobiology issues to be addressed is the specific elongation of core structures, leading to terminal decoration with bioactive glycotopes. Type II chain (Galβ1-4GlcNAc), N-acetyllactosamine (LacNAc), is a ubiquitous disaccharide building block of mammalian glycoconjugates, which may be further elongated with additional LacNAc units into the so-called polylactosaminoglycans. In contrast, anther basic unit which differs only in linkage, namely the type I chain, or Galβ1-3GlcNAc, is generally not extended. Among the rare exceptions are the multi-fucosylated extended type I chains carried on the lactosylceramides of a colorectal cancer cell line, Colo205. β1,3-galactosyltransferase V (β3Gal-T5), a crucial enzyme for type I chain formation, was suggested to be involved with in vivo biosynthesis of extended type I chains. In pursuit of this, β3Gal-T5 was over-expressed in another colorectal cell line, DLD-1, which did not synthesize extended type I chain naturally. A concerted strategy combing antibody-dependent detection and tandem mass spectrometry (MS), which would distinguish the subtle linkage variation between type I and II chains, was developed and implemented to delineate the induced glycomic alteration. We showed that DLD-1 transfected with β3Gal-T5 (DLD3GT5-1) strongly expressed type I glycotopes whereas type II glycotopes were significantly decreased. A complementary low and high energy CID MALDI-MS/MS analysis validated the presence of extended type I chains and associated fucosylated glycotopes on glycolipids and N-glycans of DLD3GT5-1. Furthermore, the comprehensive survey through product ion-dependent MS3 in LC-MS/MS analysis performed on LTQ-Orbitrap showed that type I-derived Lea was extensively biosynthesized on N-glycans upon β3Gal-T5 over-expression, in contrast with predominant type II-derived Lex in mock transfectant. Finally, semi-quantitative analysis based on MALDI-MS profiles revealed a significantly suppressed expression of sialylation and polylactosaminoglycans but elevated fucosylation in N-glycans and O-glycans, thus suggesting the determining roles of β3Gal-T5 in glycome shaping in colorectal cancer cell lines. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37128 |
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顯示於系所單位: | 生化科學研究所 |
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