Involvement of cell surface glycans in adhesion of human colon carcinoma cells to liver tissue in a frozen section assay: role of endo-beta-galactosidase-sensitive structures.
Adhesion of human colon carcinoma variant cell lines expressing different levels of the cell surface sialyl Lewis X (sLeX) antigen to frozen sections of mouse liver was examined. KM12-HX cells that bound the monoclonal antibody (mAb) FH6 (anti-sLeX) and thus expressed a high level of sLeX demonstrated a greater degree of adhesion to liver sections than their low-binding counterparts, KM12-LX cells. The adhesion of KM12-HX cells to liver sections was partially blocked by mAb FH6, but not by another anti-sLeX mAb, KM93. The adhesion was Ca2+ dependent but was not inhibited by anti-E-selectin. Endo-beta-galactosidase treatment significantly reduced adhesion and resulted in the loss of cell surface binding sites for mAb FH6. O-linked oligosaccharides from KM12-HX cells incubated in the presence of p-nitrophenyl-N-acetylgalactosaminide were fractionated by a combination of gel filtration, anion exchange chromatography, and normal phase high-performance liquid chromatography. The structure of a mAb FH6-reactive and endo-beta-galactosidase-sensitive glycan was estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in a post source decay mode and by glycosidase digestions to be NeuAc alpha2-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc-NAc beta1-3Gal beta1-4(+/-Fuc alpha1-3)GlcNAc beta1-6(NeuAc alpha2-3Gal beta1-3)GalNAc-pNP. Mild detergent lysates of mouse liver surface-labeled with sulfo-NHS biotin were incubated with glutaraldehyde-fixed monolayers of KM12-HX cells, and bound components were isolated after EDTA treatment. A Mr 49,000 component that bound only to KM12-HX cells and not to KM12-LX cells was identified.
Ota
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M
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
Takamura
N
N
Irimura
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002448:Cell Adhesion / Q000502:physiology; D002846:Chromatography, Affinity; D003110:Colonic Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D006224:Cricetinae; D005434:Flow Cytometry; D005629:Frozen Sections; D006026:Glycoside Hydrolases; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D011506:Proteins / Q000276:immunology / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D009844:Oligosaccharides; D011506:Proteins; D000080506:Sialyl Lewis X Antigen; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase