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標題: | 內皮細胞及B細胞多乳糖胺聚醣與雙唾液酸醣質體的有效鑑定 Glycomic mapping of polylactosaminoglycans, terminal disialyl and sialyl sulfo N-acetyllactosamine motifs on mammalian cells |
作者: | Shui-Hua Wang 王穗華 |
指導教授: | 邱繼輝(Kay-Hooi Khoo) |
關鍵字: | 質譜儀,多乳糖胺聚醣,雙唾液酸,硫酸化, Mass spectrometry,polylactosaminoglycans,disialic aicd,sulfation, |
出版年 : | 2011 |
學位: | 博士 |
摘要: | 以質譜為基礎的醣質體及醣蛋白質體大多著重在鑑定醣鏈末端乙酰基乳糖胺上的唾液酸及岩藻醣化,對於N型醣鏈上是否為直鏈或是帶有支鏈的多乳糖胺聚醣則較少著墨,且大多只是憑藉著質譜所得的數據來推算其存在和可能的組成為何,因此,本篇論文主要是利用人類內皮細胞、老鼠及人類的B淋巴球為樣品,鑑定並挑戰分析多乳糖胺聚醣的結構特徵及其末端的唾液酸化和硫酸化修飾。
首先,以人類內皮細胞:EA.hy926和HUVEC為起始原料,利用介質輔助雷射脫附法和電噴灑離子化方法在MS及MS/MS的階段來仔細地探討多乳糖胺聚醣結構,同時並搭配endo-β-galactosidase酵素及Smith降解反應來鑑定其是否為分支醣鏈及長鏈延伸的起始位置。和HUVEC相比,EA.hy926細胞的N型醣鏈帶有較少的唾液酸化和岩藻醣化,但多乳糖胺聚醣的長度較長且具有分支醣鏈,其延伸點並不侷限在目前已被證實具有相當重要生物意義的甘露醣6號碳上。拓展至醣質體方面的研究上,則利用Lycopersicon esculentum凝集素分別在醣、醣蛋白及醣胜肽的階段做純化,較為專一的兩步驟純化方法讓我們得知了至少有40個以上的醣蛋白候補者可能帶有乳糖胺聚醣。 小鼠B細胞株—BCL1上N型醣鏈的修飾主要為核心岩藻醣化,末端α鏈結半乳糖及唾液酸(Neu5Gc)化,且為非分支的多乳糖胺聚醣;相反的是,其O型醣鏈主要為簡單的core 1結構,帶有單唾液酸或雙唾液酸修飾。利用唾液酸酶、 endo-β-galactosidase、MS/MS及化學分析方法可知,雙唾液酸主要靠α2-8鏈結,並同時存在於具有多乳糖胺聚醣延伸或非延伸的N型醣鏈上。以螢光標記唾液酸並搭配高效液相層析儀顯示雙唾液酸結構的含量在N型醣鏈和O型醣鏈是相當的,且CD45為其攜帶者之ㄧ。透過小鼠α2,8-sialyltransferase VI(ST8sia VI)基因抑制實驗可知,此酵素同時參與N型醣鏈和O型醣鏈上雙唾液酸的生合成,且ST8Sia VI和雙唾液酸結構的表現量皆會隨著B細胞分化而增加。 有趣的是,儘管其生理功能目前還不是很清楚,BCL1的N型醣鏈末端的單唾液酸、雙唾液酸化乙酰基乳糖胺,及N型醣鏈核心結構皆可被硫酸化修飾。另外,在人類活化的B淋巴球上也鑑定到了α2,6-sialyated 6-sulfo-LacNAc結構,單和α2-6唾液酸化乙酰基乳糖胺相比,其為目前已知CD22更好的配體,這些B細胞上多乳糖胺聚醣鏈附加修飾的鑑定,使得Galectin和Siglec對B細胞分化的調控可以更為複雜精緻。總而言之,質譜分析技術的發展和進步,對於我們詳細地鑑定多乳糖胺聚醣結構來講,為一個相當重要的基礎,有助於我們對醣生物學及其它生理功能更近一步的了解。 Most mass spectrometry (MS)-based glycomic and glycoproteomic analyses focus on identifying changes in terminal glyco-epitopes represented by sialylation and fucosylation at specific positions of the terminal N-acetyllactosamine units. Much less attention was accorded to the underlying linear or branched poly-N-acetyllactosamine (polyLacNAc) extension from the N-glycan trimannosyl core other than a simple inference of its presence due to mass data and hence glycosyl compositional assignment. To advance the frontiers of glycomics, this thesis work aims primarily to address the analytical challenges in structural characterization of polylactosaminoglycans and associated terminal modifications such as sialylation and sulfation decorating the human endothelial cells, mouse and human B cells. Using the human endothelial cells, EA.hy926 and HUVEC, as starting materials, we have systematically investigated the MALDI- and ESI-MS-based methodologies for probing the structural details of polyLacNAc at both MS and MS/MS levels in conjunction with the use of endo-β-galactosidase and Smith degradation to identify branching motifs and initiation sites. N-glycans in EA.hy926 were found to be less sialylated and fucosylated but more extended and branched than those of HUVEC, thus demonstrating a fundamental glycomic difference. For EA.hy926, its polyLacNAc chains were shown to be not restricted to extending from a specific antenna including the biologically important 6-arm position. Extending to glycoproteomics, the Lycopersicon esculentum lectin based enrichment strategy was optimized at glycan, glycoprotein, and glycopeptide levels, leading to identification of over 40 protein carriers utilizing a two-step enrichment workflow. For mouse B cells, the N-glycans of a B lymphoma cell line, BCL1, were found to be mostly core-fucosylated, capped with α-Gal or Neu5Gc sialic acid, and carry non-branched polyLacNAcs. In contrast, its O-glycans were based on simple core 1 structures, mono- or disialylated on both arms. Sialidase digestion, in conjunction with further MS/MS and chemical analyses, established the identity of the terminal disialyl motif as Neu5Gcα2-8Neu5Gc-, which was shown by endo-β-galatosidase digestion to be additionally present on both polyLacNAc extended and non-extended N-glycans. Fluorescent-labeling of released sialic acids coupled with fluorometric high performance liquid chromatography analysis revealed that the amount of the disialyl motif was comparable for both N- and O-glycans, and CD45 is one of the protein carriers. Gene knockdown studies provided positive correlation indicative of mouse α2,8-sialyltransferase VI (ST8sia VI) being involved in the biosynthesis of disialic acid on both N- and O-glycans. Importantly, both the expression level of ST8sia VI and the total amount of disialic acids increase during B cell differentiation. Interestingly, sulfation was additionally found on the terminal mono- and disialylated LacNAc of the polyLAcNAc chains, as well as on the LacNAc proximal to the trimannosyl core in BCL1 although its biological relevance is at present unclear. On the other hand, similar analysis led to identification of α2,6-sialylated 6-sulfo-LacNAc epitope on both the N- and O-glycans of activated human B cells, which is known to constitute a better ligand than the non-sulfated α2,6-sialylated LacNAc for human CD22. These additional modifications of polyLacNAcs apparently complicate the simplistic interpretation of the modulating roles of galectins and Siglecs in the B cell differentiation model. The development of enabling analytical techniques sensitive enough to identify and characterize the fine structural details of the underlying polyLAcNAc is an important step towards a better understanding of the glycobiology of this and many other physiological processes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10103 |
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