Characterization of Oligosaccharide Ligands Expressed on SW1116 Cells Recognized by Mannan-binding Protein-A HIGHLY FUCOSYLATED POLYLACTOSAMINE TYPE N-GLYCAN
Resource
Journal of Biological Chemistry 280 (12): 10897-10913
Journal
Journal of Biological Chemistry
Pages
10897-10913
Date Issued
2005
Date
2005
Author(s)
Terada, Motoki
Khoo, Kay-Hooi
Inoue, Risa
Chen, Chun-I
Yamada, Kanako
Sakaguchi, Hiromi
Kadowaki, Naoko
Ma, Bruce Yong
Oka, Shogo
Kawasaki, Toshisuke
Kawasaki, Nobuko
Abstract
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-acetylglucosamine, 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-β-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/Lea 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 Leb/Lea epitope for recognition. ? 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
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Other Subjects
Antigens; Cells; Mass spectrometry; Molecular structure; Sugars; Toxicity; Tumors; Ligands; Mannan-binding proteins (MBP); MBP-dependent cell-mediated cytotoxicity (MDCC); Permethylation; Proteins; epitope; fucose; glycopeptide; ligand; mannan binding lectin; mannose; n acetylglucosamine; oligosaccharide; plant lectin; pronase; amino acid sequence; article; cancer inhibition; cell lysate; chemical analysis; colorectal carcinoma; controlled study; cytotoxicity; human; human cell; mass spectrometry; methylation; priority journal; protein analysis; protein binding; protein expression; protein interaction; tandem mass spectrometry; tandem repeat; Amino Sugars; Cell Line, Tumor; Fucose; Humans; Ligands; Mannose-Binding Lectin; Molecular Weight; Oligosaccharides; Polysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Repeat Sequences; Mus musculus; Vaccinia; Vaccinia virus
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