Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1985 Apr;77(4):840–846. doi: 10.1104/pp.77.4.840

Biosynthesis of Mannose-Containing Lipid-Linked Oligosaccharides by Solubilized Enzyme Preparation from Cultured Soybean Cells 1

Hidetaka Hori 1, Gur P Kaushal 1, Alan D Elbein 1
PMCID: PMC1064616  PMID: 16664147

Abstract

Glycosyl transferases that participate in the assembly of the lipid-linked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P-40 (NP-40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at −10°C for 1 month. The solubilized enzyme synthesized [14C]Man3GlcNAc2-pyrophosphoryl-polyprenol and [14C]Man5GlcNAc2-pyrophosphoryl-polyprenol when incubated with GDP-[14C]mannose plus a partially purified acceptor lipid isolated from calf liver. The formation of these lipid-linked oligosaccharides did not require the addition of dolichyl-phosphate or metal ions. In fact, the addition of 5 to 10 millimolar ethylenediaminetetraacetate stimulated the incorporation of mannose into lipid-linked oligosaccharides 2- to 3-fold. Since little or no dolichyl-phosphoryl-mannose is formed in the presence of ethylenediaminetetraacetate, the results suggest that the mannosyl residues added to form Man3GlcNAc2-lipid and Man5GlcNAc2-lipid come directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. On the other hand, the formation of significant amounts of Man6GlcNAc2-lipid, Man7GlcNAc2-lipid, and Man8GlcNAc2-lipid occurred when the above incubations were supplemented with dolichyl-phosphate and metal ions. Based on various time course studies and supplementation studies with various additions, it appears likely that the first five mannose residues to form Man5GlcNAc2-lipid come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichyl-phosphoryl-mannose.

Full text

PDF
840

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arakawa H., Mookerjea S. Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A. Eur J Biochem. 1984 Apr 16;140(2):297–302. doi: 10.1111/j.1432-1033.1984.tb08101.x. [DOI] [PubMed] [Google Scholar]
  2. Bailey D. S., Dürr M., Burke J., Maclachlan G. The assembly of lipid-linked oligosaccharides in plant and animal membranes. J Supramol Struct. 1979;11(2):123–138. doi: 10.1002/jss.400110203. [DOI] [PubMed] [Google Scholar]
  3. Chambers J., Forsee W. T., Elbein A. D. Enzymatic transfer of mannose from mannosyl-phosphoryl-polyprenol to lipid-linked oligosaccharides by pig aorta. J Biol Chem. 1977 Apr 25;252(8):2498–2506. [PubMed] [Google Scholar]
  4. Chapman A., Fujimoto K., Kornfeld S. The primary glycosylation defect in class E Thy-1-negative mutant mouse lymphoma cells is an inability to synthesize dolichol-P-mannose. J Biol Chem. 1980 May 25;255(10):4441–4446. [PubMed] [Google Scholar]
  5. Ericson M. C., Gafford J. T., Elbein A. D. Tunicamycin inhibits GlcNAc-lipid formation in plants. J Biol Chem. 1977 Nov 10;252(21):7431–7433. [PubMed] [Google Scholar]
  6. Forsee W. T., Elbein A. D. Biosynthesis of lipid-linked oligosaccharides and glycoprotein in aorta: stimulation by acceptor lipids isolated from liver. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2574–2578. doi: 10.1073/pnas.73.8.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Forsee W. T., Elbein A. D. Biosynthesis of lipid-linked oligosaccharides in cotton fibers. Stimulation by lipids from pig liver. J Biol Chem. 1977 Apr 10;252(7):2444–2446. [PubMed] [Google Scholar]
  8. Forsee W. T., Elbein A. D. Glycoprotein biosynthesis in plants. Demonstration of lipid-linked oligosaccharides of mannose and N-acetylglucosamine. J Biol Chem. 1975 Dec 25;250(24):9283–9293. [PubMed] [Google Scholar]
  9. Heifetz A., Elbein A. D. Solubilization and properties of mannose and N-acetylglucosamine transferases involved in formation of polyprenyl-sugar intermediates. J Biol Chem. 1977 May 10;252(9):3057–3063. [PubMed] [Google Scholar]
  10. Hori H., Elbein A. D. Characterization of the oligosaccharides from lipid-linked oligosaccharides of mung bean seedlings. Plant Physiol. 1982 Jul;70(1):12–20. doi: 10.1104/pp.70.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hori H., Elbein A. D. Processing of N-linked oligosaccharides in soybean cultured cells. Arch Biochem Biophys. 1983 Feb 1;220(2):415–425. doi: 10.1016/0003-9861(83)90431-9. [DOI] [PubMed] [Google Scholar]
  12. Hori H., James D. W., Jr, Elbein A. D. Isolation and characterization of the Glc3Man9GlcNAc2 from lipid-linked oligosaccharides of plants. Arch Biochem Biophys. 1982 Apr 15;215(1):12–21. doi: 10.1016/0003-9861(82)90273-9. [DOI] [PubMed] [Google Scholar]
  13. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  14. Jensen J. W., Schutzbach J. S. The biosynthesis of oligosaccharide-lipids. Partial purification and characterization of mannosyltransferase II. J Biol Chem. 1981 Dec 25;256(24):12899–12904. [PubMed] [Google Scholar]
  15. Jensen J. W., Springfield J. D., Schutzbach J. S. The biosynthesis of oligosaccharide-lipids. Isolation of an oligosaccharide-P-P-lipid acceptor. J Biol Chem. 1980 Dec 10;255(23):11268–11272. [PubMed] [Google Scholar]
  16. Kang M. S., Spencer J. P., Elbein A. D. Amphomycin inhibition of mannose and GlcNAc incorporation into lipid-linked saccharides. J Biol Chem. 1978 Dec 25;253(24):8860–8866. [PubMed] [Google Scholar]
  17. Kang M. S., Spencer J. P., Elbein A. D. Amphomycin inhibits the incorporation of mannose and GlcNAc into lipid-linked saccharides by aorta extracts. Biochem Biophys Res Commun. 1978 May 30;82(2):568–574. doi: 10.1016/0006-291x(78)90912-9. [DOI] [PubMed] [Google Scholar]
  18. Lehle L., Tanner W. Polyprenol-linked sugars and glycoprotein synthesis in plants. Biochem Soc Trans. 1983 Oct;11(5):568–574. doi: 10.1042/bst0110568. [DOI] [PubMed] [Google Scholar]
  19. Lehle L., Tanner W. The specific site of tunicamycin inhibition in the formation of dolichol-bound N-acetylglucosamine derivatives. FEBS Lett. 1976 Nov 15;72(1):167–170. doi: 10.1016/0014-5793(76)80922-2. [DOI] [PubMed] [Google Scholar]
  20. Schutzbach J. S., Springfield J. D., Jensen J. W. The biosynthesis of oligosaccharide-lipids. Formation of an alpha-1,2-mannosyl-mannose linkage. J Biol Chem. 1980 May 10;255(9):4170–4175. [PubMed] [Google Scholar]
  21. Spencer J. P., Elbein A. D. Transfer of mannose from GDP-mannose to lipid-linked oligosaccharide by soluble mannosyl transferase. Proc Natl Acad Sci U S A. 1980 May;77(5):2524–2527. doi: 10.1073/pnas.77.5.2524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Staneloni R. J., Leloir L. F. The biosynthetic pathway of the asparagine-linked oligosaccharides of glycoproteins. CRC Crit Rev Biochem. 1982 Apr;12(4):289–326. doi: 10.1080/10409238209104422. [DOI] [PubMed] [Google Scholar]
  23. Staneloni R. J., Tolmasky M. E., Petriella C., Ugalde R. A., Leloir L. F. Presence in a plant of a compound similar to the dolichyl diphosphate oligosaccharide of animal tissue. Biochem J. 1980 Oct 1;191(1):257–260. doi: 10.1042/bj1910257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tkacz J. S., Lampen O. Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun. 1975 Jul 8;65(1):248–257. doi: 10.1016/s0006-291x(75)80086-6. [DOI] [PubMed] [Google Scholar]
  25. WELKER N. E., CAMPBELL L. L. EFFECT OF CARBON SOURCES ON FORMATION OF ALPHA-AMYLASE BY BACILLUS STEAROTHERMOPHILUS. J Bacteriol. 1963 Oct;86:681–686. doi: 10.1128/jb.86.4.681-686.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Waechter C. J., Lennarz W. J. The role of polyprenol-linked sugars in glycoprotein synthesis. Annu Rev Biochem. 1976;45:95–112. doi: 10.1146/annurev.bi.45.070176.000523. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES