| Budget Amount *help |
¥3,920,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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.
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