Advertisement

Glycoconjugate Journal

, Volume 9, Issue 6, pp 293–301 | Cite as

Use of large-scale hydrazinolysis in the preparation ofN-linked oligosaccharide libraries: application to brain tissue

  • D. R. Wing
  • T. W. Rademacher
  • M. C. Field
  • R. A. Dwek
  • B. Schmitz
  • G. Thor
  • M. Schachner
Papers

Abstract

In this report, we describe the preparation of a library ofN-linked glycans from whole murine brain obtained by the large-scale hydrazinolysis of an acetone powder of the tissue followed by chromatographic procedures. 84% of the characterized oligosaccharides were found to be anionic, the remainder neutral. The anionic species were successively neutralized by neuraminidase (29%), aq. hydrofluoric acid (30%), and methanolysis (26%), indicating that approximately equal portions were sensitive to desialylation, dephosphorylation and desulfation, respectively. The presence of the sulfated fraction was confirmed by direct35SO4 metabolic labelling. A residual partially characterized fraction was found to be anionic through possession of carboxylic acid groups, unrelated to sialic acid. The purified oligosaccharides, in the absence of their original protein conjugates, were shown to retain those immunological characteristics essential for recognition by a specific monoclonal antibody, LS (412), that is known to recognize a carbohydrate epitope present on a number of neural adhesion molecules and functional in neural cell adhesion. These properties confirm the viability of scaling up the size of the hydrazinolysis procedure and adapting it to whole tissue for the production of glycan libraries and for the probing of structures of interest.

Keywords

N-linked oligosaccharide library L2/HNK-1 epitope anionicity murine brain large scale hydrazinolysis 

Abbreviations

ConA

concanavalin A

ELISA

enzyme-linked immunosorbent assay

Fuc

fucose

Gal

galactose

GalNAc

N-acetylgalactosamine

GlcNAc

N-acetylglucosamine

g.u.

glucose units

HRP

horseradish peroxidase

HVE

high voltage electrophoresis

Man

mannose

MS

mass spectrometry

N-CAM

neural cell adhesion molecule

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Phillips ML, Nudelman E, Gaeta FCA, Perez M, Singhal AK, Hakamori S-I, Paulson JC (1990)Science 250: 1130–2.Google Scholar
  2. 2.
    Walz G, Aruffo A, Kolanus W, Bevilacqua M, Seed B (1990)Science 250: 1132–5.Google Scholar
  3. 3.
    Brandley BK, Swiedler SJ, Robbins PW (1990)Cell 63: 861–3.Google Scholar
  4. 4.
    Takasaki S, Mizuochi T, Kobata A (1982)Methods Enzymol 83: 263–8.Google Scholar
  5. 5.
    Margolis RK, Margolis RU (1989) InNeurobiology of Glycoconjugates (Margolis RU, Margolis RK, eds) pp. 85–126. New York: Plenum.Google Scholar
  6. 6.
    Schachner M, Antonicek H, Fahrig T, Faissner A, Fischer G, Kunemund V, Martini R, Meyer A, Persohn E, Pollerberg E, Probstmeier R, Sadoul K, Sadoul R, Seilheimer B, Thor G (1990) InMorphoregulatory Molecules (Edelman GM, Cunningham BA, Thiery JP, eds) pp. 443–68. New York: Wiley.Google Scholar
  7. 7.
    Fahrig T, Schmitz B, Weber D, Kucherer-Ehret A, Faissner A, Schachner M (1990)Eur J Neurosci 2, 153–61.Google Scholar
  8. 8.
    Kunemund V, Jungalwala FB, Fischer G, Chou DKH, Keilhauer G, Schachner M (1988)J Cell Biol 106: 213–23.Google Scholar
  9. 9.
    Long C (1961) InBiochemist's Handbook, p. 640. London: Spon.Google Scholar
  10. 10.
    Ashford D, Dwek RA, Welply JK, Amatayakul S, Homans SW, Lis H, Taylor GN, Sharon N, Rademacher TW (1987)Eur J Biochem 166: 311–20.Google Scholar
  11. 11.
    Ferguson MAJ, Homans SW, Dwek RA, Rademacher TW (1988)Science 239: 753–9.Google Scholar
  12. 12.
    Kruse J, Mailhammer R, Wernecke H, Faissner A, Sommer I, Goridis C, Schachner M (1984)Nature 311: 153–5.Google Scholar
  13. 13.
    Noronha A, Ilyas A, Antonicek H, Schachner M, Quarles RH (1986)Brain Res 385: 237–44.Google Scholar
  14. 14.
    Faissner A, Kruse J, Goridis C, Bock E, Schachner M (1984)EMBO J 3: 733–7.Google Scholar
  15. 15.
    Field MC, Wing DR, Dwek RA, Rademacher TW, Schmitz B, Bollensen E, Schachner M (1992)J Neurochem 58: 993–1000.Google Scholar
  16. 16.
    Chou DKH, Ilyas AA, Evans JE, Costello C, Quarles RH, Jungalwala FB (1986)J Biol Chem 261: 11717–25.Google Scholar
  17. 17.
    Parekh RB, Tse AGD, Dwek RA, Williams AF, Rademacher TW (1987)EMBO J 6: 1233–44.Google Scholar
  18. 18.
    Freeze HH, Yeh R, Miller AL, Kornfeld S (1983)J Biol Chem 258: 14874–9.Google Scholar
  19. 19.
    Green ED, Baenziger JU (1988)J Biol Chem 263: 25–35.Google Scholar
  20. 20.
    Field MC (1989) D. Phil. Thesis, University of Oxford.Google Scholar
  21. 21.
    Sorkin BC, Hoffman S, Edelman GM, Cunningham BA (1984)Science 225: 1476–8.Google Scholar
  22. 22.
    Sundblad G, Kajiji S, Quaranta V, Freeze HH, Varki A (1988)J Biol Chem 263: 8897–903.Google Scholar
  23. 23.
    Amatayakul-Chantler S, Ferguson MAJ, Dwek RA, Rademacher TW, Parekh RB, Crandall IE, Newell PC (1991)J Cell Sci 99: 485–95.Google Scholar
  24. 24.
    Sundblad G, Holojda S, Roux L, Varki A, Freeze HH (1988)J Biol Chem 263: 8890–6.Google Scholar
  25. 25.
    Gowda DC, Margolis RU, Margolis RK (1989)Biochemistry 28: 4468–74.Google Scholar

Copyright information

© Chapman & Hall 1992

Authors and Affiliations

  • D. R. Wing
    • 1
  • T. W. Rademacher
    • 1
  • M. C. Field
    • 1
  • R. A. Dwek
    • 1
  • B. Schmitz
    • 2
  • G. Thor
    • 2
  • M. Schachner
    • 2
  1. 1.Department of BiochemistryGlycobiology InstituteOxfordUK
  2. 2.Department of NeurobiologyETH-Hönggerberg, HPMZürichSwitzerland

Personalised recommendations