Nonreductive release of O-linked oligosaccharides from mucin glycoproteins for structure/function assignments as neoglycolipids: application in the detection of novel ligands for E-selectin
Nonreductive release of O-linked oligosaccharides from mucin glycoproteins for structure/function assignments as neoglycolipids: application in the detection of novel ligands for E-selectin
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DOI：
10.1093/glycob/7.6.861
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发表时间：
1997-09-01
期刊：
影响因子：
4.3
通讯作者：
Lawson, AM
中科院分区：
文献类型：
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作者：
Chai, WG;Feizi, T;Lawson, AM
新糖脂技术包括几个微程序，涉及生成用于碳水化合物识别研究的脂质连接寡糖探针，以及通过质谱法测定寡糖序列。虽然适用于任何所需的低聚糖，但如果低聚糖是非还原的，则程序将大大简化，因为缀合是通过还原性末端醛还原胺化为磷脂酰乙醇胺来进行的。使用牛颌下粘蛋白作为还原状态下释放O-聚糖的模型，并基于“剥离"反应和降解产生的新糖脂和副产物的产率，选择70%w/v乙胺水溶液在22 ℃下处理48 h，优先于其他条件，三乙胺、氢氧化钠和肼，在这些条件下释放的主要酸性和中性寡糖（二糖至八糖）的完整性通过液体二次离子质谱（LSIMS）分析游离寡糖和TLC-LSIMS分析衍生的新糖脂来确定;与通过常规还原性碱性硼氢化物处理产生的寡糖糖醇的所有成分相比，所述所有成分是有利的。需要乙胺70%w/v在65 ℃下持续6小时的更强的条件以从猪胃粘蛋白释放寡糖;获得二至九糖，其中约三分之一具有完整的核心GalNAc。相对于还原性碱性水解后的产率，这两种糖蛋白的总产率对于酸性和中性寡糖分别为20%和40-50%。在使用乙胺从卵巢囊腺瘤糖蛋白释放的O-聚糖中，硫酸化Le（a/x）序列的三种变体被鉴定为内皮粘附分子E-选择素的配体，其中之一是基于不寻常的主链Gal-3/4GlcNAc-3Gal-3Gal。
The neoglycolipid technology comprises several microprocedures involving the generation of lipid-linked oligosaccharide probes for carbohydrate recognition studies in conjunction with oligosaccharide sequence determination by mass spectrometry. Although applicable to any desired oligosaccharides, procedures are greatly facilitated if the oligosaccharides are nonreduced, as conjugation is by reductive amination of a reducing end aldehyde to a phosphatidylethanolamine. Using bovine submaxillary mucin as a model for release of O-glycans in the reducing state, and based on yields of neoglycolipids and side-products from ''peeling'' reactions and degradation, aqueous ethylamine 70% w/v at 22 degrees C for 48 h has been selected in preference to other conditions, triethylamine, sodium hydroxide, and hydrazine, The integrity of the main acidic and neutral oligosaccharides released under these conditions, di-to octasaccharides, was established by analyses of free oligosaccharides by liquid secondary ion mass spectrometry (LSIMS) and of the derived neoglycolipids by TLC-LSIMS; the repertoire compared favorably with that of the oligosaccharide alditols generated by conventional reductive alkaline borohydride treatment, More forcing conditions of ethylamine 70% w/v at 65 degrees C for 6 h were required to release oligosaccharides from porcine gastric mucin; dito nonasaccharides were obtained of which about one-third had an intact core GalNAc. Relative to yields after reductive alkaline hydrolysis, the overall yields for these two glycoproteins were 20% and 40-50% for acidic and neutral oligosaccharides, respectively, Among O-glycans released from an ovarian cystadenoma glycoprotein using ethylamine, three variants of the sulfated Le(a/x) sequences were identified as ligands for the endothelial adhesion molecule E-selectin, one of which is based on the unusual backbone Gal-3/4GlcNAc-3Gal-3Gal.