Novel Carbohydrate Ligands for a Serum Lectin Expressed on Colon Cancer Cells and Associated with Anti-tumor Activity

血清甘露聚糖结合蛋白（MBP）是一种对甘露糖、N-乙酰葡糖胺和岩藻糖残基具有特异性的宿主防御C型凝集素，对人结肠癌细胞具有生长抑制活性。在我们之前的研究中，从人结肠癌细胞SW 1116中分离的MBP-配体寡糖（MLO）被表征为具有高度岩藻糖基化的聚乳糖胺型结构的大的多触角N-聚糖，在其非还原末端具有Le^b-Le^a或莱亚结构的串联重复。在这项研究中，首先，我们试图确定的糖蛋白，携带独特的MLO。通过AAL柱和MBP柱从细胞裂解液中分离出MBP配体糖蛋白（MLGP），并通过LC-MS/MS分析鉴定出两种主要的MLGP，分别为CD 26（二肽基肽酶IV（DPPPIV））和CD 98重链（CD 98 hc）.这些蛋白质的糖苷酶二聚体表明，CD 26含有以高亲和力结合MBP的复合型N-聚糖，而CD 98 hc仅包含不结合MBP的高甘露糖型N-聚糖。然后，我们使用抗人CD 26 mAb亲和柱从SW 1116细胞裂解物中纯化CD 26。从纯化的CD 26中释放的PNGase F N-聚糖的MALDI-MS分析证明存在岩藻糖基化N-乙酰乳糖胺（LacNAc）的一系列串联重复序列。因此，CD 26被鉴定为主要的MLO携带蛋白。通过比较从MBP结合和非结合CD 26糖肽释放的N-聚糖，显示Le^a表位的串联重复结构（长于四聚体）对于配体与MBP的高亲和力结合至关重要。对CD 26糖肽的N-糖链连接位点的分析表明，该独特的配体优先表达在一些潜在的N-糖基化位点，但偏好不是那么严格。

The serum mannan-binding protein (MBP) is a host defense C-type lectin specific for mannose, N-acetylglucosamine and fucose residues, and has growth inhibitory activity to human colon cancer cells. In our previous study, the MBP-ligand oligosaccharides (MLO) isolated from a human colon cancer cell, SW1116, were characterized as large, multi-antennary N-glycans with highly fucosylated polylactosamine-type structures having Le^b-Le^a or tandem repeats of the Lea structure at their non-reducing ends. In this study, first we attempted to identify the glycoproteins, which carried the unique MLO.1. We isolated the MBP-ligand glycoproteins (MLGPs) from the cell lysates by AAL and MBP-columns, and the two major MLGPs were identified as CD26 (Dipeptidylpeptidase IV (DPPPIV)) and CD98 heavy chain (CD98hc) by LC-MS/MS analysis.2. The glycosidase digestions of these proteins indicated that the CD26 contained the complex-type N-glycans that bound MBP with high affinity, while CD98hc comprised only of high-mannose type N-glycans that did not bind MBP. Then, we purified CD26 from SW1116 cell lysates using an anti-human CD26 mAb affinity column.3. The MALDI-MS analysis of the PNGase F released N-glycans from the purified CD26 demonstrated the presence of a series of tandem repeats of fucosylated N-acetyllactosamine (LacNAc). Thus CD26 was identified as a major MLO carrying protein.4. By the comparison of the N-glycans released from the MBP-binding and-non-binding CD26 glycopeptides, the tandem repeat structure, longer than tetramer, of the Le^a epitope was shown to be critically important for the high affinity binding of the ligands to MBP.5. The analysis of the N-glycan attachment sites of the CD26 glycopeptides demonstrated that the unique ligand was expressed preferentially at some potential N-glycosylation sites, but the preference was not so strict.