Abstract
Capillary endothelial cells can be induced to form capillary-like structures in vitro by plating on fibronectin-coated dishes (Ingber, D. E., and Folkman, J. (1989) J. Cell Biol. 109, 317-330), thereby mimicking angiogenesis. To assess the role of glycoproteins bearing asparagine-linked oligosaccharides in this process, we tested the effect of oligosaccharide processing inhibitors on the formation of capillary tubes. Deoxymannojirimycin, a compound that prevents synthesis of hybrid and complex-type oligosaccharides, inhibited the formation of capillary tubes. In contrast, swainsonine, an inhibitor that blocks synthesis of complex- but not hybrid-type oligosaccharides, did not inhibit tube formation. Lectin affinity chromatography of 2-[3H] mannose-labeled glycopeptides from endothelial cells induced to form tubes did not reveal a striking difference in the spectrum of oligosaccharides compared to uninduced cells. Since endothelial cells formed tubes normally in the presence of swainsonine, we analyzed glycopeptides from swainsonine-treated induced and uninduced cells. Cells induced to form tubes were enriched in monosialylated hybrid-type oligosaccharides sensitive to alpha-fucosidase, beta-galactosidase, and beta-N-acetylhexosaminidase, suggestive of sialyl Lewis-X determinants. We used an enzyme-linked immunoassay to measure sialyl Lewis-X epitopes on capillary endothelial cells and found that both induced and uninduced cells expressed sialyl Lewis-X epitopes. Deoxymannojirimycin and, to a lesser extent, swainsonine reduced the level of sialyl Lewis-X epitopes in cells induced to form capillary tubes, but neither compound affected the level of epitopes in cell monolayers. We conclude that synthesis of at least hybrid-type oligosaccharides is required for capillary tube formation in vitro and that an increase in monosialylated, fucosylated glycans on asparagine-linked oligosaccharides occurs during this process.
Highlights
Lary tube formation, the hybrid- and high mannose-type oli- Sephadex (Fig. 5,Panels C and D). the same percent gosaccharides synthesized in its presence must be sufficient of counts/min (23%)from total cell-associated glycopeptides for capillary differentiation
The major peak (0.9% of the counts/min), which co-migrated with the Man7GlcNAc standard (Fig. 5, Panel F ),was resistant to a-fucosidase digestion but was digested completely by @galactosidase (Table 111).The detectable peak (Panel F, first open arrow) was partially sensitive to a-fucosidase and P-galactosidase (Table 111).This a-fucosidaselp-galactosidase-sensitivoeligosaccharide represented less than 0.02% of the counts/min recovered fromcellassociated glycopeptides
Individual peaks separated by HPLC were subjected to afucosidase and P-galactosidase.Of the capillary tube oligosaccharides that co-fractionated with the Man5.7GlcNAc oligosaccharide standards, 0.5% of the counts/min recovered from total cellular glycopeptides were sensitive to a-fucosidase
Summary
1-DeoxymunmjirimycinInhibits Capillary Tube Formation in a Reversible Manner-BCE cells induced to form capillary tubes in vitro undergo dramatic changes incell architecture. BCE cells, either induced to form tubes or (Fig. 2, Panel D).Swainsonine blocks synthesis of complex- controls in which cells were platedin growth medium on but not hybrid-type oligosaccharides by inhibiting Golgi a- gelatin-coated dishes (monolayers), were labeled with 2-[3H]. Lary tube formation, the hybrid- and high mannose-type oli- Sephadex (Fig. 5,Panels C and D). the same percent gosaccharides synthesized in its presence must be sufficient of counts/min (23%)from total cell-associated glycopeptides for capillary differentiation. I n Fig. 5, the QAE-Sephadex fractionations of ConA-Sepha- from BCE tubes (Panel E ) , whichmigratedslightlylarger rose purified oligosaccharides fromtube (Panel A) anmdono- than theMan6GlcNAc standard, was resistant to a-fucosidase layer (PanelB ) BCE cells are shown In both cases, the bulk digestion but completely sensitive to@-galactosidaseand . The third peak (Fig. 5 , Panel E, second open arrow)contained
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