Expression Patterns of α2,3-Sialyltransferases and α1,3-Fucosyltransferases Determine the Mode of Sialyl Lewis X Inhibition by Disaccharide Decoys

Abstract

A variety of human adenocarcinomas express sialylated, fucosylated Lewis blood group antigens on cell surface and secreted mucins. Binding of these antigens to P-selectin on platelets is thought to facilitate formation of platelet-tumor emboli in the circulation, which in turn allows sequestration of the tumor cells in the microvasculature. Here we report a pharmacologic approach for blocking these interactions through metabolic inhibition of sialylation. Peracetylated forms of Gal␤1,4GlcNAc␤-O-naphthalenemethanol and GlcNAc-␤1,3Gal␤-O-naphthalenemethanol were taken up by LS180 human colon carcinoma cells, O-deacetylated, and utilized as biosynthetic intermediates, resulting in heterogeneous oligosaccharides. The primed oligosaccharides included sialylated, sulfated, and fucosylated products based on mass spectrometry. Assembly of free oligosaccharides on the glycosides decoyed glycosylation of cellular glycoproteins, as assessed by altered binding of lectins and carbohydrate-specific antibodies. Expression of ␣2,3-sialylated oligosaccharides on the cell surface was diminished specifically, whereas ␣2,6-sialylation and fucosylation were not. In U937 lymphoma cells, the glycosides decreased fucosylation without affecting sialylation. The differential inhibitory activities correlated inversely with fucosyltransferase and sialyltransferase activity based on enzyme assays and microarray analysis. Regardless of the mechanism, the disaccharides blocked the cells from forming selectin ligands and inhibited adhesion to immobilized selectins, suggesting that the glycosides might prove useful for interfering with tumor cell adhesion and metastasis.

Deoxygenated Disaccharide Analogs as Specific Inhibitors of β1–4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

“…6, b and d), whereas multiple neutral and charged (sialylated and sulfated) oligosaccharides were assembled on 1 (Fig. 6, a and c) (7,9). The lack of any glycosylated products was confirmed in nonradioactive experiments using mass spectrometry (data not shown).…”

“…lation and sialylation should not occur, since the relevant transferases require Gal␤1-4GlcNAc-R core as a substrate (9). As predicted, treating U937 cells with 2 in the presence of ]fucose did not yield any radiolabeled oligosaccharides generated on the glycoside (Fig.…”

“…Binding of monoclonal antibody CSLEX-1 and P-selectin Ig chimera to cells fixed with 1% paraformaldehyde was measured by an enzyme-linked immunosorbent assay as described (9,13). Background binding was measured after treatment with sialidase, which showed that nonspecific binding was less than 5%.…”

“…Cells were radiolabeled in growth medium containing 1 mM glucose. Samples of radiolabeled oligosaccharide products were analyzed by reverse phase HPLC as described (9). Nonradioactive samples were analyzed by mass spectrometry on a Thermo Finnigan LCQ classic mass spectrometer (San Jose, CA).…”

“…In contrast, the parent compound 1 inhibits cell surface sLe X expression by a mechanism that involves priming of oligosaccharides and diversion of downstream enzymes involved in sLe X biosynthesis on tumor cells, such as ␣2-3sialyltransferases and ␣1-4- fucosyltransferase (9). The deacetylated form of the 4Ј-deoxy analog can bind to ␤4Gal-T1 in vitro, and assuming that this is the primary target in vivo, binding leads to effective inhibition of the enzyme for natural glycoprotein substrates.…”