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USE OF IMMOBILIZED SUGAR CHAINS ON MICROBEADS FOR DETECTION AND ANALYSES OF LECTIN ACTIVITIES

使用微珠上的固定化糖链检测和分析凝集素活性

基本信息

批准号：
06680573
负责人：
KOCHIBE Naohisa
金额：
$ 1.41万
依托单位：
GUNMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

A simple and reliable method for detection of and analyzing lectin activities is developed by using microbeads on which sugar chains had been immobilized. For convenience in handling and adjusting lectin activity, the lectin used as a counterpart was also immobilized to the microbeads.Amino group on the surface of Honensphere (2.5 mum in diameter) was succinylated with succinic anhydride, and then converted to active ester with cardodiimide. Amino group in the glycopeptide and lectin molecules was coupled to the active ester in the presence of N-hydroxysuccinimide under mild alkaline condition to give "sugar-bead" and "lectin-bead", respectively. The glycopeptides were derived from mucin type and Asn-linked type glycoprotems of which sugar structure has bead well known. Each of twelve lectins of known and 2 novel lectins was coupled to the bead.Results of agglutination reaction of sugar-bead and lectin-bead showed good agreement with the reported lectin-sugar structure relationships, indicating that the present method is reliable. When a sugar-bead was sequentially treated with exoglycosidase, the lectin-bead accessible to the sugar-bead changed at every step of treatment, i.e. each lectin-bead reacted to the exposed sugar moiety. This result indicates that the binding properties could be analyzed by using the sugar-bead and its enzymetreated one. Indeed, two novel lectins from fruiting bodies of Basidiomycetes and Ascomycetes bound to a T antigen structure and strictly required a conformation of GalNAc at the reducing terminal.Purification and properties of these lectins will be reported elsewhere.Lectin-beads were applicable to examine cell surface sugar structures. Although it is only qualitative, the method is simple and reliable as compared to flow cytometry or fluorescence microscopy.

建立了一种简便、可靠的糖链固定微珠检测和分析凝集素活性的方法。为了方便处理和调节凝集素的活性，作为对应物的凝集素也被固定在微球上。Honensphere（直径2.5 μ m）表面的氨基被琥珀酸酐琥珀化，然后被cardodi亚胺转化为活性酯。在温和碱性条件下，糖肽和凝集素分子中的氨基在n-羟基琥珀酰亚胺存在下与活性酯偶联，分别生成“糖珠”和“凝集素珠”。糖肽来源于粘蛋白型和asn -连接型糖蛋白，它们的糖结构是已知的。将已知的12种凝集素和2种新的凝集素分别偶联在头上。糖珠和凝集素珠的凝集反应结果与已有的凝集素-糖的结构关系吻合较好，表明本方法是可靠的。当一个糖球被连续地用外糖苷酶处理时，糖球所能接触到的凝集素球在处理的每一步都发生了变化，即每个凝集素球都与暴露的糖部分发生了反应。这一结果表明，可以用糖球及其酶处理后的糖球来分析其结合特性。事实上，来自担子菌和子囊菌子实体的两种新的凝集素结合到T抗原结构上，并且严格要求在还原末端具有GalNAc的构象。这些凝集素的纯化和性质将在其他地方报道。凝集素珠可用于检测细胞表面糖结构。虽然它只是定性的，但与流式细胞术或荧光显微镜相比，该方法简单可靠。