chains, because these glycans also utilize GlcNAc-PP-dolichol (Hart and Lennarz, 1978). Glycolipid biosynthesis may also be inhibited, although the mechanism underlying this has not been established (Yusuf et al., 1983; Guarnaccia et al., 1987).

The glycosidase inhibitors castanosper-mine, deoxynojirimycin, deoxymannojirimy-cin, and swainsonine are considerably less toxic than tunicamycin. Castanospermine inhibits glucosidase I, and deoxynojirimycin inhibits both glucosidases I and II (Fig. 12.3.1; Pan et al., 1983; Saunier et al., 1982). In some studies, the metabolic block produced by these two drugs is not complete, yielding a mixture of normally processed and underprocessed structures. More recently, an endo-a-D-mannosi-dase has been described that cleaves the tetrasaccharide Glc3-Man1 from Glc3Man9-GlcNAc2, effectively bypassing the block produced by the glucosidase inhibitors (Lubas and Spiro, 1987). Although the question has not been examined fully, the level of this endo-a-D-mannosidase may vary between different cell lines.

Deoxymannojirimycin and swainsonine are inhibitors of mannosidases I and II, respectively (Fig. 12.3.1; Fuhrmann et al., 1984; Tul-siani et al., 1982). By inhibiting mannosidase I, deoxymannojirimycin prevents addition of the a1-2 N-acetylglucosamine (GlcNAc) residue required to produce structure D, Figure 12.3.1, which is a necessary step for the action of mannosidase II. Thus, deoxymannojirimy-cin prevents addition of any GlcNAc residues, blocking galactosylation and sialylation as well. Swainsonine blocks mannosidase II. However, it does not prevent the single GlcNAc residue (structure D, Fig. 12.3.1) from being galactosylated and sialylated, resulting in hybrid structures (Tulsiani and Touster, 1983).

Some of the glycosidase inhibitors are also active against lysosomal enzymes, although the significance of this effect in tissue culture cells has been little explored. Deoxynojirimycin can inhibit synthesis of the lipid-linked precursor in cell-free extracts (Romero et al., 1985); this also has not been thoroughly investigated with intact cells. No information is available on the metabolism or half-lives of these inhibitors in cultured cells.

Critical Parameters and Troubleshooting

The two most critical variables are duration of exposure and inhibitor concentration. The treatment times suggested in this unit (24 hr)

will permit a cell to replace most of its endogenous glycoproteins with glycoproteins synthesized in the presence of inhibitor. For proteins that turn over slower or faster than average, longer or shorter incubation times may be appropriate. If pulse-chase studies with radiolabeled sugar or amino acid precursors (unit 12.2) are being carried out, it is necessary to incubate cells in inhibitor for only 1 to 2 hr before adding label.

N-linked glycosylation inhibitors may be toxic to the cells, as indicated by decreased cell viability and/or reduced protein synthesis. The concentration of tunicamycin that can be used is generally limited by its toxicity, which may be considerable in some cell lines (more so with tumor cells than nontransformed cells). Other glycosidase inhibitors are usually not toxic in the concentration ranges suggested in Table 12.3.1 (the highest concentrations listed represent those used in most published studies).

It is important to realize that in a given cell line, not all glycoproteins will be affected to the same extent by a given inhibitor. Moreover, it is possible that not all sites on the same glyco-protein will be affected equally. These caveats should be kept in mind when interpreting the results from studies employing these inhibitors.

Failure to observe an effect from an inhibitor may indicate that concentration of inhibitor was too low and/or length of exposure was too short. Cell lines differ in their susceptibility to these compounds.

Anticipated Results

These experiments will demonstrate the chosen inhibitor's toxicity, as assessed by protein synthesis, and its ability to alter oligosac-charide biosynthesis, as assessed by total [3H]mannose incorporation (for tunicamycin) or endo H release of the oligosaccharides (for the other inhibitors listed in Table 12.3.1).

Time Considerations

Setting up the initial round of cell cultures for determining the optimal inhibitor concentration takes 2 hr, after which they are incubated 24 hr. On the second day, the dilution series of medium containing inhibitor is set up, the cells are transferred to these media (<1 hr), and the plates incubated 24 hr. On the third day, the incubation of the cells with the radiolabeled precursor is set up (~4 hr); the actual incubation takes 4 to 16 hours. Following incubation, the cells are assayed for radiolabel incorporation by TCA precipitation (2 to 4 hr).

The main part of the experiment follows a similar time course if the inhibitor used is tunicamycin. If a different inhibitor is used, analysis of endo H susceptibility by Sephacryl S-200 chromatography will take ~1 day, including the overnight incubation.

Literature Cited

Elbein, A.D. 1987. Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu. Rev. Biochem. 56:497-534.

Fuhrmann, U., Bause, E., Legler, G., and Ploegh, H. 1984. Novel mannosidase inhibitor blocking conversion of high mannose to complex oligosaccharides. Nature 307:755-758.

Guarnaccia, S.P., Shaper, J.H., and Schnaar, R.L. 1987. Tunicamycin inhibits ganglioside biosynthesis in neuronal cells. Proc. Natl. Acad. Sci. U.S.A. 80:1551-1555.

Hart, G.W. and Lennarz, W.J. 1978. Effects of tunicamycin on the biosynthesis of glycosaminogly-cans by embryonic chick cornea. J. Biol. Chem. 253:5795-5801.

Hubbard, S.C. and Ivatt, R.J. 1981. Synthesis and processing of asparagine-linked oligosaccha-rides. Annu. Rev. Biochem. 50:555-583.

Kobata, A. and Takasaki, S. 1992. Structure and biosynthesis of cell surface carbohydrates. In Cell Surface Carbohydrates and Cell Development (M. Fukuda, ed.) pp. 1-24. CRC Press, Boca Raton, Fla.

Lubas, W.A. and Spiro, R.G. 1987. Golgi endo-a-D-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme. J. Biol. Chem. 262:3775-3781.

McDowell, W. and Schwarz, R.T. 1988. Dissecting glycoprotein biosynthesis by the use of specific inhibitors. Biochimie 70:1535-1549.

Pan, Y.T., Hori, H., Saul, R., Sanford, B.A., Moly-neux, R.J., and Elbein, A.D., 1983. Castanosper-mine inhibits the processing of the oligosaccha-ride portion of the influenza viral hemagglutinin. Biochemistry 22:3975-3984.

Romero, P., Friedlander, P., and Herscovics, A. 1985. Deoxynojirimycin inhibits the formation of Glc3Man9GlcNAc2-PP-dolichol in intestinal epithelial cells in cultures. FEBS Lett. 183:2932.

Saunier, B., Kilker, R.D., Tkacz, J.S., Quaroni, A., and Herscovics, A. 1982. Inhibition of N-linked complex oligosaccharide formation by 1-deoxy-nojirimycin, an inhibitor of processing glucosi-dases. J. Biol. Chem. 257:14155-14161.

Tkacz, J.S. and Lampen, J.O. 1975. Tunicamycin inhibition of polyisoprenyl N-acetylglucos-aminyl pyrophosphate formation in calf liver microsomes. Biochem. Biophys. Res. Commun. 65:248-55.

Tulsiani, D.R.P., Harris, T.M., and Touster, O. 1982. Swainsonine inhibits the biosynthesis of complex glycoproteins by inhibition of Golgi man-nosidase II. J. Biol. Chem. 257:7936-7939.

Tulsiani, D.R.P. and Touster, O. 1983. Swainsonine causes the production of hybrid glycoproteins by human skin fibroblasts and rat liver Golgi preparations. J. Biol. Chem. 258:7578-7585.

Yusuf, H.K.M., Pohlentz, G., Schwarzmann, G., and Sandhoff, K. 1983. Ganglioside biosynthesis in Golgi apparatus of rat liver. Eur. J. Biochem. 134:47-54.

Key References

Useful general review.

McDowell, W. and Schwarz, R.T. 1988. See above.

Useful general review.

Good review of early work on processing pathway.

Kobata, A. and Takasaki, S. 1992. See above.

Good review of recent work on some of the complexities in oligosaccharide processing.

Contributed by Leland D. Powell University of California San Diego La Jolla, California




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