Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein -: A highly fucosylated polylactosamine type N-glycan
Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein -: A highly fucosylated polylactosamine type N-glycan
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DOI：
10.1074/jbc.m413092200
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发表时间：
2005-03-25
影响因子：
4.8
通讯作者：
Kawasaki, N
中科院分区：
文献类型：
--
作者：
Terada, M;Khoo, KH;Kawasaki, N
甘露聚糖结合蛋白(MBP)是一种C型血清凝集素，当它与微生物上的甘露糖、N-乙酰氨基葡萄糖、岩藻糖等配体糖结合时，通过凝集素途径激活补体。此外，携带人MBP基因的痘苗病毒在裸鼠体内对人结肠癌SW1116细胞具有很强的生长抑制活性。我们建议将这种活性称为MBP依赖的细胞介导的细胞毒性(MDCC)(Ma，Y.，Uemura，K.，Oka，S.，KozutSumi，Y.，Kawasaki，N.和Kawasaki，T.(1999)proc。娜塔莉。阿卡德。SCI。美国，96,371-375)。本研究对SW1116细胞表面的MBP配体进行了表征。利用植物凝集素和抗Lewis抗体作为MBP与SW1116细胞结合的抑制剂的初步实验表明，岩藻糖在相互作用中起着关键作用。随后，从全细胞裂解产物中制备Pronase糖肽，并通过联氨分解释放寡糖。经吡啶标记后，用MBP亲和层析柱分离MBP配体低聚糖，全甲基化后，结合内切-β-半乳糖苷酶消化和化学脱糖作用，用质谱仪和串联质谱仪测定其序列。MBP配体是大的多天线N-糖链，带有高度岩藻糖化的聚乳糖胺型结构。在非还原末端，Lea结构的Le(B)/Le(A)或串联重复占优势，其中相当大一部分通过内部的Le(X)或N-乙酰乳糖胺单元连接到Trimannosyl核心。所描述的结构是独特的，不同于以前报道的其他肿瘤特异性碳水化合物抗原。结论：MBP需要Le(B)/Le(A)表位的串联重复序列簇才能识别。
Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetyl-glucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Lea structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition.