臺灣博碩士論文加值系統

位於非還原端醣抗原決定位(glyco-epitopes)上的硫酸化(sulfation)被指出調控多種重要的生物機制，其中包含人類淋巴球周邊淋巴結歸巢行為(homing)。由於O 與N 型硫酸化醣鏈在細胞內含量極少，以質譜儀分析技術來檢測尚未純化的硫酸化醣鏈是有一定的困難度。為了針對淋巴球的多樣性功能提出不同觀點，並且能利於更準確地呈現淋巴球中硫酸化醣抗原決定位真實情況，建立一個靈敏並且有效率的硫酸化醣質體檢測流程是目前首當其衝的要項，尤其針對同時鑑別硫酸基在於醣鏈上修飾的確切位置以及分離不同醣鏈的同質異構物，雖然在科學理論基礎上可行，然而，現階段僅利用單一檢測方式困難度大為提高。本論文的研究重旨在於利用MALDI-MS/MS 與nanoLC-MS/MS 結合了反相式(reverse phase)與多孔石墨碳(porous graphitized carbon)管柱分析技術，有系統性地檢測淋巴球全面性的醣質體，以及針對特定表現於N 型醣鏈上的硫酸化醣抗原決定位加以深入探討，並進一步以二種方式進行相對定量：一、定量整體硫酸化醣在整體複雜式N 型醣鏈的相對比例，二、僅針對α-2,6-disialylated biantennary N 型醣鏈上的單硫酸化醣與此特定醣鏈的相對比例。
利用互補系統性的分析，本論文提供了第一份以質譜分析為基礎的證據，確立6-sulfo-α-2,6-sialyl LacNAc 化學結構，並證實此KN343 抗原的表現在人類NALM-6和MEC-1 細胞株以及人類CD19+與CD4+淋巴球和慢性淋巴細胞白血病樣品。在這些樣品中，非硫酸化與硫酸化的主要N 型醣鏈末端皆以α-2,6 唾液酸鍵結為主要結構；在慢性淋巴細胞白血病樣品中，主要的α-2,6-disialyl biantennary N型醣鏈上所檢測到的KN343 相對量，與白血病樣品中呈現CD19 陽性的細胞比例是成正相關的。另一方面，利用同樣的質譜分析流程也一併鑑定老鼠naive T, Th1,Th2, Th17 和iTreg T 淋巴球的醣質體特徵。各別T 淋巴球的次細胞族群所表現的硫酸化N 型醣鏈特徵與表現量有所不同，但仍然足以偵測，而在老鼠B 淋巴球所表現的硫酸化醣鏈往往遠低於偵測極限。此外，naive T 與Th1 以及Th2 的O 與N型醣質體特性，主要分別以di-sialyl 與α-Gal 以及Neu5Ac 的末端修飾各為其特徵。本篇論文報導了T 淋巴球的次細胞族群表現特定醣質體資訊以及多面相N 型醣質體的檢測流程，提供未來探討人類與老鼠免疫淋巴球之醣生物學的重要依據。

Sulfate modification (sulfation) on terminal glyco-epitopes (glycotopes) has been demonstrated as a critical element that mediates many important biological processes including human lymphocyte homing to peripheral lymph node. Yet, sulfated O- and N-glycans are generally of very low abundance and barely detectable by mass spectrometry (MS) analysis without enrichment. To gain better insights into lymphocyte functional diversity, a highly sensitive and efficient analytical workflow is required for precision mapping of the occurrence of sulfated sialyl glycotopes on these immune cells. In particular, it should be capable of resolving different sulfated sialylated isomers by defining both the location of sulfate and the sialyl linkages, which is currently difficult to achieve by any single analytical platform. In this thesis work, the global glycomic profile and the specific sulfo-, sialyl glycotopes carried on the N-glycans of various sources of lymphocytes were systematically defined by a combination of MS-based methods, including MALDI-MS/MS and nanoLC-MS/MS analyses on both reverse phase and porous graphitized carbon (PGC) columns. Moreover, the degree of sulfation was determined at two levels, either as % total of complex type N-glycans that was sulfated or, more specifically, as relative amount of α-2,6-disialylated biantennary N-glycans that was mono-sulfated.
Using such concerted analytical approach, this thesis work provided the first MS-based evidence for the occurrence of the target glycotope defined by monoclonal antibody KN343, namely 6-sulfo-α-2,6-sialyl LacNAc, on the human B cell precursor leukemia cells (NALM-6) and chronic B cell leukemia cells (MEC-1), human CD19+ and CD4+ lymphocytes isolated from peripheral blood of healthy donors, and peripheral blood mononuclear cells (PBMC) collected from 10 chronic lymphocytic leukemia (CLL) patients. In each case, α-2,6-sialyl linkage was found to be predominant and the relative abundance of the total sulfated N-glycan fractions, as well as that of the major α-2,6-disialyl biantennary N-glycans carrying the KN343 sulfated glycotope, were quantified and correlated with the % of CD19+ cells expressing KN343, for each of the PBMC samples. In addition, the same analytical workflow was applied to various lineages of murine T lymphocytes including the naive, and activated Th1, Th2, Th17 and iTreg subsets derived thereof. Unlike murine B lymphocytes, sulfated N-glycans could be readily detected for all these different T lymphocyte subsets but at variable level. Moreover, di-sialyl, α-Gal and Neu5Ac capping were found to be more prominently expressed on both N- and O-glycans of murine naive T, Th1 and Th2 cells, respectively. Both the derived glyco-signatures specific to each lymphocyte subset and the multifaceted N-sulfoglycomic approaches developed in this thesis work should prove to be highly valuable for future follow-up studies on the glycobiology of human and murine lymphocytes.

Chapter 1: Introduction………………………………………………………….….....1
1.1: N- and O-linked glycosylation……………………………………………….…..1
1.2: A diverse range of sulfated glycans and lymphocyte homing……...…….….......7
1.2.1: Sulfated GAGs…………………………..…………………..……..……......7
1.2.2: Sulfated LacdiNAc……………………………..………..……………….....8
1.2.3: Sulfo-sialyl Lewis X, L-selectin and lymphocyte homing…………..….......9
1.2.4: Sulfo-α-2,6-sialyl LacNAc and CD22……………………..……..….…….12
1.3: GlcNAc 6-O-Sulfotranferases…………………………………………….……14
1.4: α-2,6/2,3-Sialyltransferases……………………………………………....….....16
1.5: Current applications for analytical sulfoglycomics………………………….…18
1.5.1: Liquid chromatography (LC) and analytical columns………………….….19
1.5.2: Matrix assisted laser desorption ionization (MALDI)-MS and electrospray ionization (ESI)-MS……………………………………………………………...….…21
1.5.3: MS/MS analysis of glycans………………………………………….....….23
1.6: The limitation of sulfoglycomics by current mass spectrometry-based methods………………………………………………………………………………...26
1.7: Current unsolved questions of sulfoglycomics in immune system…………….27
1.8: Specific Aims………………………………………………………………......28
Chapter 2: Materials and Methods…………………………………………………31
2.1: Cells and Clinical Samples…………………………………………………….31
2.2: N- and O-glycan preparation………………………………………………..…..32
2.3: Permethylation of oligosaccharides and enrichment of permethylated sulfated glycans by Oasis MAX cartridge…………………………………………………..…..34
2.4: Clean up of permethylated glycans by ZipTipC18………………………………35
2.5: MALDI/TOF-MS and MS/MS…………………………………………………35
2.6: NanoLC-MS and MS/MS analysis…………………………………….……….36
2.7: Glypick software…………………………….………………………………….39
Chapter 3: Results…………………………………………………………………….40
3.1: Human pre-B and B-CLL cells and N-Sulfoglycomic workflow………..……..40
3.1.1: Overall N-glycosylation profiles of NALM-6 and MEC-1 Cells………....40
3.1.2: MALDI-MS/MS analysis of permethylated N-glycans……………….…..43
3.1.3: NanoLC-MS/MS analysis of permethylated sulfated N-glycans………….44
3.1.4: NanoLC-MS/MS analysis of native N-glycans on PGC…………………..46
3.2: Human lymphocytes and PBMC derived from donors and CLL patients…………………………………………………………...…………………….55
3.2.1: Evidence of KN343 in human CD19+, CD4+ cells and PBMC...…………55
3.2.2: Quantifying the relative amount of total sulfated and non-sulfated N-glycans..……………………………………………………………………………..58
3.2.3: Overall strategic workflow and practical considerations………………….60
3.3: Murine T lymphocyte……………………………….……………………….....71
3.3.1: NeuGc remodeling in N-glycans of murine T lymphocytes upon activation…………………………………………………………………………….....71
3.3.2: Sulfated N-glycans of murine T lymphocyte subsets………………….…..72
3.3.3: O-glycans of mutine T lymphocyte subsets…………………………….….73
Chapter 4: Discussion and perspectives………………………………………….….87
4.1: Analytical workflow…………………………………………………………....88
4.2: Biological relevance of identified glycomic features……………………….….90
4.2.1: Sulfation is more relevant in humans than murine lymphocytes……...…..90
4.2.2: Disialylation in murine T cells is absent after differentiation…………..…92
4.2.3: α-Gal and Neu5Ac capping are the glycan signatures in murine Th1 and Th2, respectively……………………………………………………………………….....…93
4.2.4: Overall view of N-glycosylation in mouse CD4+ lymphocytes…………...95
4.3: Future work on sulfoglycomics of immune system……………………...….96
Reference………………………………………………………………………….…..98

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