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Structural analysis of oligosaccharides ligands to a serum lectin inducing an anti-tumor activity

诱导抗肿瘤活性的血清凝集素寡糖配体的结构分析

基本信息

批准号：
14572054
负责人：
KAWASAKI Nobuko
金额：
$ 2.5万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572054/
关键词：
serum lectin mannan-binding protein mannose-binding protein human colon cancer cell anti-tumor activity host defence factor oligosaccharide ligand polylactosamine structure ヒト結腸ガン細胞赤血球Band3糖タンパク質

项目摘要

Serum Mannan-binding protein (MBP), a C-type mammalian lectin specific for mannose, N-acetylglucosamine and fucose, has a potent growth inhibitory activity to a human colorectal carcinoma cell line in vivo. In this study, we have characterized the MBP-ligand oligosaccharides expressed on the human colorectal carcinoma cell line, SW 1116, which are suggested to trigger anti-tumor activity of MBP.1.FITC-labeled MBP binding to SW1116 cells was inhibited specifically by the ligand sugars, a fucose-specific lectin, and anti-Lewis A and anti-Lewis B mAbs, suggesting that MBP binds to type-I carbohydrates.2.MBP-ligand glycopeptides were isolated by a MBP affinity column from the high molecular weight glycopeptides fraction of the pronase-digest of SW 1116 cell lysates. After hydrazinolysis followed by pyridylamination of MBP-ligand glycopeptides, PA-derivatized MBP-ligand oligosaccharides were isolated again by the MBP affinity column.(1)Carbohydrate analysis indicated that MBP-ligand oligosaccharides contained high molecular size N-glycans with high galactose, N-acetylglucosamine and fucose contents.(2)Endo-β-galactosidase digestion of the MBP-ligand oligosaccharides resulted in a marked reduction of the binding activity to the MBP column together with the decrease of their molecular sizes, indicating the presence of poly N-acetyllactosamine structures. The selective removal of fucose residues from MBP-ligand oligosaccharides resulted in almost complete loss of the binding activity to the MBP and the AAL affinity columns.(3)MALDI-MSIMS and nano ESI-MS/MS analyses of the MBP-ligand oligosaccharides indicated the presence of highly fucosylated structure.MBP-ligand oligosaccharides appear to be high molecular weight poly N-acetyllactosamine-type with high fucose content.

血清甘露聚糖结合蛋白（MBP）是一种对甘露糖、N-乙酰葡萄糖胺和岩藻糖具有特异性的C型哺乳动物凝集素，对人大肠癌细胞株具有较强的体内生长抑制活性。在本研究中，我们对人结肠癌细胞系SW 1116上表达的MBP配体寡糖进行了表征，这些寡糖被认为是触发MBP抗肿瘤活性的物质：1. FITC标记的MBP与SW 1116细胞的结合被配体糖、岩藻糖特异性凝集素、抗Lewis A和抗Lewis B mAb特异性抑制，2.通过MBP亲和柱从SW 1116细胞裂解物的链霉蛋白酶消化物的高分子量糖肽级分中分离MBP-配体糖肽。MBP-配体糖肽肼解后，然后吡啶胺化，PA-衍生的MBP-配体寡糖再次分离的MBP亲和柱。（1）糖分析表明，MBP-配体寡糖含有高分子量的N-聚糖，具有高半乳糖、N-乙酰葡萄糖胺和岩藻糖含量。（2）内切-β-半乳糖苷酶消化MBP-配体寡糖后，MBP-配体寡糖与MBP柱的结合活性显著降低，分子大小减小，表明存在聚N-乙酰乳糖胺结构。从MBP-配体寡糖中选择性去除岩藻糖残基导致MBP和AAL亲和柱的结合活性几乎完全丧失。（3）MALDI-MSIMS和NanoESI-MS/MS分析表明，MBP配体寡糖具有高度岩藻糖基化的结构，属于高分子量聚N-乙酰乳糖胺型，岩藻糖含量高。