用於質譜測序和功能分析的不對稱N-聚醣的合成

細胞表面的N型聚醣具有獨特的特徵，可被不同的聚醣結合蛋白（GBP）和病原體結合及辨識。人類中大多數Ｎ型聚醣是不對稱和存在結構異構物，但是由於缺乏具結構異構物之純Ｎ型聚醣，人們對其生物學功能的了解還不夠。在這項研究中，我們先利用化學方法製備用於可被岩藻糖和唾液酸轉移酶接受的通用核心結構，再以此兩種酵素接上岩藻糖或唾液酸形成最後的不對稱Ｎ型聚醣，此方法提供不對稱N型聚醣合成的改進策略。利用此方法合成出產的26個在不同觸角上帶有唾液酸殘基的純Ｎ型聚醣接著以點片機製成醣晶片，用於分析禽流感病毒（H5N2）的血凝素（HA）的結合特異性。我們發現不同分支的N-乙酰氨基葡萄糖（GlcNAc）連接的Neu5Ac-Gal異構物表現出不同的結合力，不同分支上具末端半乳糖的異構物對結合力的影響較小。總體而言，醣晶片分析顯示了不同異構物間對HA的結合力差別。為了進一步開發質譜法以解析N型聚醣異構物，我們對三天線和四天線N-聚醣進行了CID MS2 / MS3裂解，裂解過程產生了B和Y離子。片段化過程中產生的結構指紋有助於開發利用質譜儀解析N型聚醣異構物的新方法。

關鍵字

流感；酶促的；糖阵列；多样化；合成；聚糖

並列摘要

N-glycans on the cell surface provide distinct signatures that are recognized by different glycan-binding proteins (GBPs) and pathogens. Most glycans in humans are asymmetric and isomeric, yet their biological functions are not well understood due to their lack of availability. In this study, we have developed an improved strategy for asymmetric N-glycan assembly and diversification using the designed common core substrates prepared chemically for selective enzymatic fucosylation and sialylation. The resulted 26 well-defined glycans that carry the sialic acid residue on different antennae were used in a microarray format as a representative application to profile the binding specificity of hemagglutinin (HA) from the avian influenza virus (H5N2). We found that the Neu5Ac-Gal epitope linked to the N-acetylglucosamine (GlcNAc) of different branches exhibited distinct binding affinity and the terminal galactose on different branches had minor influence in the binding. Overall, the microarray analysis showed branch-biased and context-based recognition patterns. To elucidate the structures of N-glycans under mass spectrometry, we carried out CID MS2/ MS3 fragmentation of tri-antennary and tetra-antennary N-glycans and the outcome of the fragmentation process produced B and Y ions. The structural fingerprints generated in the fragmentation could be useful for the development of new glycan sequencing methods.