RESUMO
We have shown previously that human colon cancer CX-1 cells contain lipid- and protein-bound sialosyl Lewis(a) structures that support the adhesion of these cells to E-selectin. Treatment of cancer cells with O-sialoglycoprotease did not decrease either the binding of anti-sialosyl Le(a) antibodies or binding to E-selectin-expressing CHO cells. This suggested that cleavage of sialomucins uncovered cryptic sialosyl Le(a) gangliosides that support such interactions. In the present study, inhibitors of glycolipid and O-glycan biosynthesis, d,l-threo-PPPP and GalNAc-alpha-O-benzyl, respectively, were used to study whether the binding of anti-sialosyl Le(a) antibody and adhesion of CX-1 cells to E-selectin can be mediated by sialosyl Le(a) gangliosides. Treatment of cancer cells with each of the inhibitors decreased the expression of the respective glycoconjugates as shown by TLC-binding assay and immunoblotting with anti-sialosyl Le(a) antibody. However, only slight differences in binding of antisialosyl Le(a) antibody to the surfaces of control and inhibitor-treated CX-1 cells were found by flow cytometry, as well no differences were observed in binding of control and inhibitor-treated CX-1 cells to E-selectin-expressing CHO cells, supporting the earlier hypothesis on the involvement of gangliosides in binding of anti-sialosyl Lewis(a) in the partial absence of mucin O-glycans. This hypothesis was further proven by electron microscopy data. Both native CX-1 and d,l-threo-PPPP-treated cells were labelled with anti-sialosyl Lewis(a) antibody mostly at a distance 70-90 nm from cell surface, suggesting interaction with protein-bound carbohydrate structures only. In contrast, the cancer cells treated with GalNAc-alpha-O-benzyl showed most of the staining around 20 nm distance from the plasmalemma, implying that the antibody interacts with lipid-bound sialosyl Lewis(a) instead. The electron microscopy data in conjunction with other results described in this report strongly support the hypothesis that sialosyl Lea gangliosides are not involved in the adhesion of CX-1 cells to E-selectin when mucins are present on the cell surface, but they may be involved in binding to E-selectin in their absence.