, 180:96 | Cite as

The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins

  • Azeddine Driouich
  • Pascale Gonnet
  • Mouna Makkie
  • Anne-Catherine Laine
  • Loïc Faye


Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) secrete a number of acid hydrolases and other proteins that have both highmannose and complex asparagine-linked glycans. We used affinity chromatography with concanavalin A and an antiserum specific for complex glycans in conjunction with in vivo-labeling studies to show that all of the secreted proteins carry glycans. The presence of complex glycans on secretory proteins indicates that they are passing through the Golgi complex on the way to the extracellular compartment. The sodium ionophore, monensin, did not block the transport of proteins to the extracellular medium, even though monensin efficiently inhibited the Golgi-mediated processing of complex glycans. The inhibition of N-glycosylation by tunicamycin reduced by 76% to 84% the accumulation of newly synthesized (i.e. radioactively labeled) protein that was secreted by the sycamore cells, while cytoplasmic protein biosynthesis was not affected by this antibiotic. However, in the presence of glycoprotein-processing inhibitors, such as castanospermine and deoxymannojirimycin, the formation of complex glycans was prevented but glycoprotein secretion was unchanged. These results support the conclusion that N-linked glycan processing is not necessary for sorting, but glycosylation is required for accumulation of secreted proteins in the extracellular compartment.

Key words

Acer (glycoprotein) Glycoprotein Secretion (glycoprotein) 



concanavalin A


endoplasmic reticulum


relative molecular mass


sodium dodecylsulfate-polyacrylamide gel electrophoresis






  1. Alibert, G., Carrasco, A., Boudet, A.M. (1982) Topographic aspects of biosynthesis, extracellular secretion, and intracellular storage of proteins in plant cells. Biochim. Biophys. Acta721, 22–29CrossRefGoogle Scholar
  2. Boller, T., Kende, H. (1979) Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol.63, 1123–1132PubMedGoogle Scholar
  3. Bonner, W.M., Laskey, R.M. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem.46, 83–88CrossRefPubMedGoogle Scholar
  4. Bowles, D.J., Marcus, S.E., Pappin, D.J.C., Findlay, J.B.C., Eliopoulos, E., Maycox, P.R., Burgess, J. (1986) Posttranslalational processing of concanavalin A precursors in jackbean cotyledons. J. Cell. Biol.102, 1284–1294CrossRefPubMedGoogle Scholar
  5. Chrispeels, M.J., Vitale, A. (1985) Abnormal processing of the modified oligosaccharide side chains of phytohemagglutinin in the presence of swainsonine and deoxynojirimycin. Plant Physiol.78, 704–709PubMedGoogle Scholar
  6. Chrispeels, M.J., Faye, L., Sturm, A., Johnson, K.D. (1987) Are oligosaccharide sidechains necessary for glycoprotein transport or targeting? In: Plant membranes: structure, function, biogenesis, pp. 275–287, UCLA Symp. Mol. Cell. Biol., N. S., vol. 63, Leaver, C., Sze, H., eds., Liss, New YorkGoogle Scholar
  7. Craig, S., Goodchild, D.J. (1984) Golgi-mediated vicilin accumulation in pea cotyledon cells is redirected by monensin and by nigericin. Protoplasma122, 91–97CrossRefGoogle Scholar
  8. Elbein, A.D. (1987) Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu. Rev. Biochem.56, 497–537CrossRefPubMedGoogle Scholar
  9. Elbein, A.D. (1988) Glycoprotein processing and glycoprotein processing inhibitors. Plant Physiol.87, 291–295PubMedCrossRefGoogle Scholar
  10. Faye, L., Chrispeels, M.J. (1985) Characterization of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases. Anal. Biochem.149, 218–224CrossRefPubMedGoogle Scholar
  11. Faye, L., Chrispeels, M.J. (1987) Transport and processing of the glycosylated precursor of concanavalin A in jack-bean. Planta170, 217–224CrossRefGoogle Scholar
  12. Faye, L., Chrispeels, M.J. (1988) Common antigenic determinants in the glycoproteins of plants, molluscs and insects. Glycoconjugate J.5, 245–256CrossRefGoogle Scholar
  13. Faye, L., Chrispeels, M.J. (1989) Apparent inhibition of β-fructosidase secretion by tunicamycin may be explained by breakdown of the unglycosylated protein during secretion. Plant Physiol.89, 845–851PubMedGoogle Scholar
  14. Faye, L., Johnson, K.D., Chrispeels, M.J. (1986) Oligosaccharide side chains of glycoproteins that remain in the highmannose form are not accessible to glycosidases. Plant Physiol.81, 206–211PubMedGoogle Scholar
  15. Faye, L., Johnson, K.D., Sturm, A., Chrispeels, M.J. (1989) Structure, biosynthesis, and function of asparagine-linked glycans of plant glycoproteins. Physiol. Plant.75, 309–314CrossRefGoogle Scholar
  16. Gibson, R., Kornfeld, S., Schlesinger, S. (1981) The effect of oligosaccharide chains of different sizes on the maturation and physical properties of vesicular stomatitis virus. J. Biol. Chem.256, 456–462PubMedGoogle Scholar
  17. Heupke, H.J., Robinson, D.G. (1985) Intracellular transport of α-amylase in barley aleurone cells: evidence for the participation of the Golgi apparatus. Eur. J. Cell Biol.39, 265–272Google Scholar
  18. Hori, H., Elbein, A.D. (1981) Tunicamycin inhibits protein glycosylation in suspension cultured soybean cells. Plant Physiol.67, 882–886PubMedGoogle Scholar
  19. Johnson-Flanagan, A.M., Owens, J.N. (1985) Peroxidase activity in relation to suberisation and respiration in white spruce (Picea glauca [Moench] Voss) seedlings roots. Plant Physiol.79, 103–107PubMedGoogle Scholar
  20. Jones, R.L., Bush, D.S., Sticher, L., Simon, P., Jacobsen, J.V. (1987) Intracellular transport and secretion of barley aleurone α-amylase. In: Plant membranes: structure, function, biogenesis, pp. 325–340, Leaver, C., Sze, H., eds. Liss, New YorkGoogle Scholar
  21. Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685CrossRefPubMedGoogle Scholar
  22. Laine, A.-C., Faye, L. (1988) Significant immunological crossreactivity of plant glycoproteins. Electrophoresis9, 841–844CrossRefPubMedGoogle Scholar
  23. Lamport, D.T.A. (1964) Cell suspension cultures of higher plants: isolation and growth energetics. Exp. Cell. Res.33, 195–206PubMedCrossRefGoogle Scholar
  24. Lescure, A.M. (1966) Etude quantitative de la croissance d'une culture d'Acer pseudoplatanus L. Physiol. Vég.4, 365–378Google Scholar
  25. Mäder, M.C., Walter, C. (1986) De-novo synthesis and release of peroxidase in cell suspension cultures ofNicotiana tabacum. Planta189, 273–277CrossRefGoogle Scholar
  26. Melroy, D., Jones, R.L. (1986) The effect of monensin on intracellular transport and secretion of α-amylase isoenzymes in barley aleurone. Planta167, 252–259CrossRefGoogle Scholar
  27. Miernyk, J.A. (1987) Extracellular secretion of acid hydrolases by maize endosperm cells grown in liquid medium. J. Plant Physiol.129, 19–32Google Scholar
  28. Mollenhauer, H.H., Morré, D.J., Backer, C.E. (1988) Swelling of Golgi apparatus cisternae in monensin-treated tissues is modulated by fixation conditions. Protoplasma145, 66–69CrossRefGoogle Scholar
  29. Montreuil, J. (1984) Spatial conformation of glycans and glycoproteins. Biol. Cell.51, 115–132PubMedGoogle Scholar
  30. Olden, K., Parent, J.B., White, S.L. (1982) Carbohydrate moieties of glycoproteins. A re-evaluation of their function. Biochim. Biophys. Acta650, 209–232PubMedGoogle Scholar
  31. Pfeffer, S.R., Rothman, J.E. (1987) Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu. Rev. Biochem.56, 829–852CrossRefPubMedGoogle Scholar
  32. Prives, J.M., Olden, K. (1980) Carbohydrate requirement for expression and stability of acetylcholine receptor on the embryonic muscle cells in culture. Proc. Natl. Acad. Sci. USA77, 5263–5267PubMedGoogle Scholar
  33. Ravi, K., Hu, C., Reddi, P.S., Van Huystee, R.B. (1986) Effect of tunicamycin on peroxidase release by cultured peanut suspension cells. J. Exp. Bot.37, 1708–1715Google Scholar
  34. Sly, W.S., Fischer, H.D. (1982) The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J. Cell Biochem.18, 67–85CrossRefPubMedGoogle Scholar
  35. Sturm, A., Johnson, K.D., Szumilo, T., Elbein, A.D., Chrispeels, M.J. (1987) Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides. Plant Physiol.85, 741–745PubMedGoogle Scholar
  36. Tartakoff, A.M. (1983) Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell32, 1026–1028CrossRefPubMedGoogle Scholar
  37. Tsaftari, A.S., Sorenson, J.C., Scandalios, J.c (1980) Glycosylation of catalase inhibitor is necessary for activity. Biochem. Biophys. Res. Commun.92, 889–895CrossRefGoogle Scholar
  38. Tulsiani, D.R.P., Harris, T.M., Touster, O. (1982) Swainsonine inhibits the biosynthesis of complex glycoproteins by inhibition of Golgi mannosidase II. J. Biol. Chem.257, 7936–7939PubMedGoogle Scholar
  39. Wink, M. (1984) Composition of the spent cell culture medium. I. Time course of ethanol formation and the excretion of hydrolytic enzymes into the medium of suspension-cultured cells ofLupinus polyphyllus. Naturwissenschaften71, 635–637CrossRefGoogle Scholar
  40. Winkler, J.R., Segal, H.C. (1984) Swainsonine inhibits degradation by isolated rat liver lysosomes. J. Biol. Chem.259, 15369–15372PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Azeddine Driouich
    • 1
  • Pascale Gonnet
    • 1
  • Mouna Makkie
    • 1
  • Anne-Catherine Laine
    • 1
  • Loïc Faye
    • 1
  1. 1.C.N.R.S.-UA 203Université de Rouen, Faculté des SciencesMont Saint AignanFrance

Personalised recommendations