Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein. A highly fucosylated polylactosamine type N-glycan.
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-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 Le(a) 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.
Terada
Motoki
M
Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Shogoin, Kyoto 606-8501, Japan.
Khoo
Kay-Hooi
KH
Inoue
Risa
R
Chen
Chun-I
CI
Yamada
Kanako
K
Sakaguchi
Hiromi
H
Kadowaki
Naoko
N
Ma
Bruce Yong
BY
Oka
Shogo
S
Kawasaki
Toshisuke
T
Kawasaki
Nobuko
N
D000606:Amino Sugars / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D005643:Fucose / Q000737:chemistry; D006801:Humans; D008024:Ligands; D037601:Mannose-Binding Lectin / Q000378:metabolism*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020080:Tandem Repeat Sequences
D000606:Amino Sugars; D008024:Ligands; D037601:Mannose-Binding Lectin; D009844:Oligosaccharides; D011134:Polysaccharides; C066929:polylactosamine; D005643:Fucose