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

血清甘露聚糖结合蛋白（MBP）是一种c型哺乳动物凝集素，对甘露糖、n-乙酰氨基葡萄糖和焦糖具有特异性，在体内对人类结直肠癌细胞系具有有效的生长抑制活性。在这项研究中，我们表征了在人结直肠癌细胞系SW 1116上表达的mbp配体低聚糖，这些低聚糖被认为可以触发MBP.1的抗肿瘤活性。fitc标记的MBP与SW1116细胞的结合被配体糖、聚焦特异性凝集素、抗lewis a和抗lewis B单克隆抗体特异性抑制，表明MBP与i型碳水化合物结合。通过MBP亲和柱从SW 1116细胞裂解物的pronase-digest的高分子量糖肽部分分离到MBP配体糖肽。将MBP配体糖肽进行肼解和吡啶层合后，再通过MBP亲和柱分离得到pa衍生的MBP配体低聚糖。(1)碳水化合物分析表明，mbp配体低聚糖含有高分子量的n -聚糖，具有高的半乳糖、n -乙酰氨基葡萄糖和焦糖含量。(2)Endo-β-半乳糖苷酶对MBP配体低聚糖的酶切导致其与MBP柱的结合活性明显降低，其分子大小减小，表明存在聚n -乙酰乳胺结构。选择性去除MBP配体寡糖的焦点残基导致与MBP和AAL亲和柱的结合活性几乎完全丧失。(3) mbp配体低聚糖的MALDI-MSIMS和纳米ESI-MS/MS分析表明，mbp配体低聚糖存在高度聚焦的结构。mbp配体寡糖表现为高分子量聚n -乙酰乳胺型，具有高聚焦含量。

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.