Analysis of Asparagine-Linked Oligosaccharides by Sequential Lectin-Affinity Chromatography

  • Kazuo Yamamoto
  • Tsutomu Tsuji
  • Toshiaki Osawa
Part of the Methods in Molecular Biology book series (MIMB, volume 14)


Sugar moieties on the cell surface play one of the most important roles in cellular recognition. In order to elucidate the molecular mechanism of these cellular phenomena, assessment of the structure of sugar chains is indispensable. However, it is difficult to elucidate the structures of cell-surface oligosaccharides because of two technical problems. First is the difficulty in fractionating various oligosaccharides heterogeneous in the number, type, and substitution patterns of outer sugar branches. The second problem is that very limited amounts of material can be available, which makes it difficult to perform detailed structural studies. Lectins are proteins with sugar-binding activity. Each lectin binds specifically to a certain sugar sequence in oligosaccharides and glycopeptides. To overcome the problems just described, lectins are very useful tools. Recently, many attempts have been made to fractionate oligosaccharides and glycopeptides on immobilized lectin columns. The use of a series of immobilized lectin columns, whose sugar-binding specificities have been precisely elucidated, enables us to fractionate a very small amount of radioactive oligosaccharides or glycopeptides (ca. 10 ng, depending on the specific activity) into structurally distinct groups.


Sialic Acid Sugar Chain Sugar Sequence Lectin Column Immobilize Lectin 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Kornfeld, R. and Kornfeld, S. (1985) Assembly of asparagine-linked oligosac-charides. Ann. Rev. Biochem. 54, 631–664.PubMedCrossRefGoogle Scholar
  2. 2.
    Tsuji, T., Irimura, T., and Osawa, T. (1981) The carbohydrate moiety of Band 3 glycoprotein of human erythrocyte membrane. J. Biol. Chem. 256, 10,497–10,502.PubMedGoogle Scholar
  3. 3.
    Fukuda, M., Dell, A., Oates, J. E., and Fukuda, M. N. (1984) Structure of branched lactosaminoglycan, the carbohydrate moiety of Band 3 isolated from adult human erythrocytes. J. Biol. Chem. 259, 8260–8273.PubMedGoogle Scholar
  4. 4.
    Merkle, R. K. and Cummings, R. D. (1987) Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell ine BW5147. J. Biol Chem. 262, 8179–8189.PubMedGoogle Scholar
  5. 5.
    Fukuda, M. (1985) Cell surface glycoconjugates as onco-differentiation markers in hematopoietic cells. Biochim. Biophys. Ada. 780, 119–150.Google Scholar
  6. 6.
    Fukuda, M., Kondo, T., and Osawa, T. (1976) Studies on the hydrazino-lysis of glycoproteins. Core structures of oligosaccharides obtained from porcine thyroglobulin and pineapple stem bromelain. J. Biochem. 80, 1223–1232.PubMedGoogle Scholar
  7. 7.
    Takasaki S. and Kobata, A. (1978) Microdetermination of sugar composition by radioisotope labeling. Methods Enzymol. 50, 50–54.PubMedCrossRefGoogle Scholar
  8. 8.
    Tai, T., Yamashita, K., Ogata, M. A., Koide, N., Muramatsu, T., Iwashita, S., Inoue, Y, and Kobata, A. (1975) Structural studies of two ovalbumin glycopeptides in relation to the endo-β-N-acetylglucosaininidase specificity. J. Biol Chem. 250, 8569–8575.PubMedGoogle Scholar
  9. 9.
    Rupley, J. A. (1964) The hydrolysis of chitin by concentrated hydrochloric acid, and the preparation of low-molecular-weight substrates for lysozyme. Biochim. Biophys. Ada. 83, 245–255.Google Scholar
  10. 10.
    Krusius, T., Finne, J., and Rauvala, H. (1978) The poly(glycosyl) chains of glycoproteins. Characterizaion of a novel type of glycoprotein saccharides from human erythrocyte membrane. Eur.J. Biochem. 92, 289–300.PubMedCrossRefGoogle Scholar
  11. 11.
    Yamamoto, K., Tsuji, T., Tarutani, O., and Osawa, T. (1984) Structural changes of carbohydrate chains of human thyroglobulin accompanying malignant transformations of thyroid grands. Eur.J. Biochem. 143, 133–144.PubMedCrossRefGoogle Scholar
  12. 12.
    Tsuji, T., Irimura, T., and Osawa, T. (1980) The carbohydrate moiety of Band-3 glycoprotein of human erythrocyte membranes. Biochem. J. 187, 677–686.PubMedGoogle Scholar
  13. 13.
    Baenziger, J. U. and Fiete, D. (1979) Structural determinants of Ricinus communis ngglutinin and toxin specificity for oligosaccharides. J. Biol. Chem. 254, 9795–9799.PubMedGoogle Scholar
  14. 14.
    Irimura, T., Tsuji, T., Yamamoto, K., Tagami, S., and Osawa, T. (1981) Structure of a complex-type sugar chain of human glycophorin A. Biochemistry 20, 560–566.PubMedCrossRefGoogle Scholar
  15. 15.
    Shibuya, N., Goldstein, I. J., Van Damme, E.J. M., and Peumans, W. J. (1988) Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb J. Biol. Chem. 263, 728–734.PubMedGoogle Scholar
  16. 16.
    Yamamoto, K., Tsuji, T., Matsumoto, I., and Osawa, T. (1981) Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized wheat germ agglutinin Biochemistry 20, 5894–5899.PubMedCrossRefGoogle Scholar
  17. 17.
    Ogata, S., Muramatsu, T., and Kobata, A. (1975) Fractionation of glycopeptides by affinity column chromatography on concanavalin A-Sepharose. J. Biochem. 78, 687–696.PubMedGoogle Scholar
  18. 18.
    Krusius, T., Finne, J., and Rauvala, H. (1976) The structural basis of the different affinities of two types of acidic N-glycosidic glycopeptides from concanavalin A-Sepharose. FEBSLett. 71, 117–120.CrossRefGoogle Scholar
  19. 19.
    Kornfeld, K., Reitman, M. L., and Kornfeld, R. (1981) The carbohydrate-binding specificity of pea and lentil lectins. J. Biol. Chem. 256, 6633–6640PubMedGoogle Scholar
  20. 20.
    Katagiri, Y, Yamamoto, K., Tsuji, T., and Osawa, T. (1984) Structural requirements for the binding of glycopeptides to immobilized vicia faba (fava) lectin. Carbohydr. Res. 129, 257–265.CrossRefGoogle Scholar
  21. 21.
    Yamamoto, K., Tsuji, T., and Osawa, T. (1982) Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil-and pea-lectins. Carbohydr. Res. 110, 283–289.PubMedCrossRefGoogle Scholar
  22. 22.
    Yamashita, K., Hitoi, A., and Kobata, A. (1983) Structural determinants of Phaseolus vulgaris erythroagglutinating lectin for oligosaccharides. J. Biol. Chem. 258, 14,753–14,755.PubMedGoogle Scholar
  23. 23.
    Cummings, R. D. and Kornfeld, S. (1982) Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized Phaseolus vulgaris leukoagglutinating and erythroagglutinating lectins. J. Biol. Chem. 257, 11,230–11,234.PubMedGoogle Scholar
  24. 24.
    Cummings, R. D. and Kornfeld, S. (1984) The distribution of repeating [Galpl,4GlcNAcpl,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell line BW5147 and PHAR2.1. J. BioL Chem. 259, 6253–6260.PubMedGoogle Scholar
  25. 25.
    Yamashita, K., Totani, K., Ohkura, T., Takasaki, S., Goldstein, I. J., and Kobata, A. (1987) Carbohydrate binding properties of complex-type oligosaccharides on immobilized Datura stramonium lectin. J. BioL Chem. 262, 1602–1607.PubMedGoogle Scholar
  26. 26.
    Sato, S., Animashaun, T., and Hughes, R. C. (1991) Carbohydrate-binding specificity of Tetracarpidhcm conophorumlectxn. J. Bid. Chem. 266, 11,485–11,494.Google Scholar
  27. 27.
    Irimura, T. and Nicolson, G. L. (1983) Interaction of pokeweed mitogen with poly(N-acetyllactosamine)-type carbohydrate chains. Carbohydr. Res. 120, 187–195.PubMedCrossRefGoogle Scholar
  28. 28.
    Kawashima, H., Sueyoshi, S., Li, H., Yamamoto, K., and Osawa, T. (1990) Carbohydrate binding specificities of several poly-N-acetyllactosamine-binding lectins. Glycoconjugate J. 7, 323–334.CrossRefGoogle Scholar
  29. 29.
    Wang, W.-C. and Cummings, R. D. (1988) The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked α-2,3 to penultimate galactose residues. J. BioL Chem. 263, 4576–4585.PubMedGoogle Scholar
  30. 30.
    Kawaguchi, T., Matsumoto, I., and Osawa, T. (1974) Studies on hemagglutinins from Maackia amurensis seeds. J. BioL Chem. 249, 2786–2792.PubMedGoogle Scholar
  31. 31.
    Sueyoshi, S., Yamamoto, K., and Osawa, T. (1988) Carbohydrate binding specificity of a beetle (Allomyrina dichotoma) lectin. J. Biochem. 103, 894–899.PubMedGoogle Scholar
  32. 32.
    Yamashita, K., Umetsu, K., Suzuki, T., Iwaki, Y, Endo, T., and Kobata, A. (1988) Carbohydrate binding specificity of immobilized Allomyrina dichotoma lectin II. J. BioL Chem. 263, 17,482–17,489.PubMedGoogle Scholar
  33. 33.
    Shibuya, N., Goldstein, I.J., Broekaen, W. F., Lubaki, M. N., Peeters, B., and Peumans, W. J. (1987) Fractionation of sialylated oligosaccharides, glyco-peptides, and glycoproteins on immobilized elderberry (Sambucus nigra L.) bark lectin. Arch. Biochem. Biophys. 254, 1–8.PubMedCrossRefGoogle Scholar
  34. 34.
    Shibuya, N., Goldstein, I. J., Broekaert, W. F., Lubaki, M. N., Peeters, B., and Peumans, W. J. (1987) The elderberry (Sambucus nigra L.) bark lectin,. recognizes the Neu5Ac(α2-6)Gal/GalNAc sequence. J. BioL Chem. 262, 1596–1601.PubMedGoogle Scholar
  35. 35.
    Osawa, T. and Tsuji, T. (1987) Fractionation and structural assessment of oligosaccharides and glycopeptides by use of immobilized lectins. Ann. Rev. Biochem. 56, 21–42.PubMedCrossRefGoogle Scholar
  36. 36.
    Osawa, T. (1989) Recent progress in the application of plant lectins to glycoprotein chemistry. Pure Appl. Chem. 61, 1283–1292.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1993

Authors and Affiliations

  • Kazuo Yamamoto
    • 1
  • Tsutomu Tsuji
    • 1
  • Toshiaki Osawa
    • 1
  1. 1.Division of Chemical Toxicology and Immunohistochemistry, Faculty of Pharmaceutical SciencesUniversity of TokyoJapan

Personalised recommendations