Glycosphingolipid composition of epithelial cells isolated along the villus axis of small intestine of a single human individual
Glycosphingolipid composition of epithelial cells isolated along the villus axis of small intestine of a single human individual
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DOI：
10.1093/glycob/cws115
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发表时间：
2012-12-01
期刊：
影响因子：
4.3
通讯作者：
Leffler, Hakon
中科院分区：
文献类型：
--
作者：
Breimer, Michael E.;Hansson, Gunnar C.;Leffler, Hakon
取A(1)Le（A -b+）型血个体6厘米新鲜回肠近端手术标本，用含乙二胺四乙酸缓冲液温和洗涤，从绒毛尖端到隐窝底部逐步分离上皮细胞。从上皮细胞组分和非上皮性肠残渣中制备了酸性和非酸性鞘脂。通过化学和生物（单克隆抗体、霍乱毒素、大肠杆菌）检测试剂的薄层色谱、衍生糖脂质谱和质子核磁共振谱分析，在不进一步分离单个物种的情况下获得鞘脂组成的分子信息。通过这种方法，获得了主要和次要糖的结构、神经酰胺组分及其相对量。上皮细胞和非上皮残基的鞘脂组成有明显差异。鞘磷脂是这两个区室的主要成分。上皮细胞的特征是单糖基神经酰胺、硫化物和血脂(主要是Le（b)六糖基神经酰胺和ALe(b)七糖基神经酰胺）占主导地位。非上皮残基含有约5倍的糖脂，主要是单、二、三和四糖基神经酰胺和神经节苷脂，包括GM1神经节苷脂。与非上皮细胞残基相比，神经酰胺在上皮细胞糖脂中羟基化程度更高（1-2个额外羟基）。结合对同一上皮细胞制剂的糖蛋白的单独详细研究，该人类肠道样品是唯一一种蛋白质和脂质连接糖都被详细表征的上皮细胞制剂。
A 6-cm fresh proximal ileum surgical specimen from a blood group A(1)Le(a-b+) secretor individual was used for stepwise isolation of epithelial cells from villus tip to crypt bottom by gentle washing with ethylenediaminetetraacetic acid-containing buffer. Acid and non-acid sphingolipids were prepared from the epithelial cell fractions and the non-epithelial intestinal residue. Molecular information on the sphingolipid composition was obtained without further isolation of individual species by applying thin-layer chromatography using chemical and biological (monoclonal antibodies, cholera toxin, Escherichia coli) detection reagents, mass spectrometry and proton NMR spectroscopy of derivatized glycolipids. In this way, the structure of major and minor saccharides, ceramide components and their relative amounts were obtained. Epithelial cells and non-epithelial residue were distinctly different in their sphingolipid composition. Sphingomyelin was the major single component in both compartments. Characteristic for epithelial cells was the dominance of monoglycosylceramides, sulphatides and blood group fucolipids (mainly Le(b) hexaglycosylceramides and ALe(b) heptaglycosylceramides). The non-epithelial residue had about five times less glycolipids mainly mono-, di-, tri- and tetra-glycosylceramides and gangliosides, including the GM1 ganglioside. The ceramides were more hydroxylated (1-2 additional hydroxyls) in epithelial cell glycolipids compared with the non-epithelial residue. Combined with a separate detailed study on the glycoproteins of the same epithelial cell preparation, this human intestinal sample is the only epithelial cell preparation where both protein- and lipid-linked saccharides are characterized in detail.