Abstract
We made a comparative study of the structures of the oligosaccharides on the glycoproteins from Caco-2 human colonic adenocarcinoma cells, before and after differentiation. Enterocytic differentiated Caco-2 cells highly express H type 1 blood group antigen on the cell surface as well as activities of brush border membrane hydrolases, such as dipeptidyl peptidase IV and alkaline phosphatase. A strong correlation was observed between the amounts of H type 1 blood group antigen and the degrees of differentiation. Structural analysis with use of lectin affinity high performance liquid chromatography revealed that typical mucin-type sugar chains of the glycoproteins from undifferentiated cells have H type 2 group, linear polylactosamines, and core 1 structure. On the other hand, differentiated cells newly contain H type 1 and Le(b) groups and core 2 structure. Mucins with H type 1 group make contact with brush border membrane enzymes on differentiated cells. Furthermore benzyl 2-acetamide-2-deoxy-alpha-D-galactopyranoside inhibited both expression of H type 1 group on the cell surface and enhancement of brush border membrane enzyme activities even in the presence of a differentiating inducer. These results suggest that the mucin-type sugar chains with H type 1 group have important functions regarding differentiation of Caco-2 cells.
Highlights
Caco-2 cells derived from a human colonic adenocarcinoma differentiate into enterocytes-like cells spontaneously [1] or by induction with sodium butyrate [2]
To clarify which fucose-containing blood group antigens are expressed on the Caco-2 cells before and after differentiation, the obtained proteins were studied by an enzyme immunoassay using lectins, RCA120 and Ulex europaeus agglutinin I (UEA-I), and monoclonal antibodies against blood type A and B, X, Y, Lea, Leb, and H type 1
The reactivities of the glycoproteins from the differentiated cells to the antibodies against H type 1 and Leb were obviously higher than for the undifferentiated cells (Fig. 2, A and B), yet the difference was small between the two kinds of cells regarding reactivities to UEA-I and anti-Y antibody (Fig. 2, C and D)
Summary
Cell Culture—Caco-2 cells obtained from the American Type Culture Collection were cultured (no data shown). Lectin Affinity High Performance Liquid Chromatography of Mucintype Sugar Chains of Glycoproteins—-For determination of the nonreducing termini and the repeating units, mucin-type sugar chains were released from the delipidated proteins by alkaline-borohydride treatment [15] and digested with Escherichia freundii endo--galactosidase (Seikagaku Corporation, Tokyo, Japan) for 3 days at 37 °C by adding 30 milliunits of enzyme every day. Flow Cytometry—Cells were detached by trypsin-EDTA, washed with Tris-buffered saline, and incubated with control mouse IgG, anti-H type 1, or anti-alkaline phosphatase antibody in 0.1% BSA in Trisbuffered saline, washed, and with FITC-conjugated Affinipure F(abЈ) fragment donkey anti-mouse IgG for 30 min on ice. Other cells were incubated with R-PE-conjugated mouse IgG, R-PE-conjugated anti-CD26 antibody, FITC-conjugated streptavidin, or FITC-conjugated UEA-I. The stained preparations were analyzed using confocal microscope, Fluoroview (Olympus, Tokyo, Japan)
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