Skip to main content
SpringerLink
Log in

A general strategy for the isolation of carbohydrate chains fromN-,O-glycoproteins and its application to human chorionic gonadotrophin

  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

For the structural analysis of the carbohydrate chains ofN-,O-glycoproteins a straightforward strategy was developed based on the cleavage of theN-linked chains with immobilized peptide-N 4-(N-acetyl-β-glucosaminyl) asparagine amidase-F (PN-Gase-F) fromFlavobacterium meningosepticum, followed by alkaline borohydride treatment of the remainingO-glycoprotein material. This methodology was applied to the isolation of the Asn- and Ser-linked carbohydrate chains of human chorionic gonadotrophin. The structures of the isolated oligosaccharides were verified by 500-MHz1H-NMR spectroscopy. The Asn-linked sugar chains were shown to be: NeuAcα2-3Galβ1-4GlcNAcβ1-2Manα1-6[NeuAcα2-3Galβ1-4GlcNAcβ1-2Manα1-3]Man β1-4GlcNAcβ1-4[Fucα1-6]0-1GlcNAc and Manα1-6[NeuAcα2-3Galβ1-4GlcNAcβ1-2Man α1-3]Manβ1-4GlcNAcβ1-4GlcNAc. Also some minor constituents occurred. The structures of the Ser-linked oligosaccharides were established in the form of their oligosaccharide-alditols as: NeuAcα2-3Galβ1-3[NeuAcα2-6]GalNAc, NeuAcα2-3Galβ 1-3GalNAc and NeuAcα2-3Galβ1-3[NeuAcα2-3Galβ1-4GlcNAcβ1-6]GalNAc.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

hCG:

human chorionic gonadotrophin

hCG-α:

α-subunit

hCG-β:

β-subunit

ElA:

enzyme immunoassay

PNGase-F:

peptide-N 4-(N-acetyl-β-glucosaminyl)asparagine amidase-F (EC 3.5.1.52)

SDS:

sodium dodecyl sulphate

GalNAc:

N-acetylgalactosamine

GlcNAc:

N-acetylglucosamine

NeuAc:

N-acetylneuraminic acid

Man:

mannose

Gal:

galactose

Fuc:

fucose

References

  1. Nilsson B (1978) Ph.D. Thesis, University of Lund, Sweden.

  2. Ogata Sl, Lloyd KO (1982) Anal Biochem 119:351–59.

    PubMed  Google Scholar 

  3. Spiro RG (1976) Methods Carbohydr Chem 7:185–90.

    Google Scholar 

  4. Takasaki S, Mizuochi T, Kobata A (1982) Methods Enzymol 83:263–68.

    PubMed  Google Scholar 

  5. Zinn AB, Plantner JJ, Carlson DM (1977) in The Glycoconjugates, Vol. 1, eds. Horowitz Ml, Pigman W. Academic Press, New York, p 69–85.

    Google Scholar 

  6. Berger EG, Buddecke E, Kamerling JP, Kobata A, Paulson JC, Vliegenthart JFG (1982) Experientia 38:1129–62.

    PubMed  Google Scholar 

  7. Korrel SAM, Clemetson KJ, van Halbeek H, Kamerling JP, Sixma JJ, Vliegenthart JFG (1984) Eur J Biochem 140:571–76.

    PubMed  Google Scholar 

  8. Egge H, Peter-Katalinic J, Paz-Parente J, Strecker G, Montreuil J, Fournet B (1983) FEBS Lett 156:357–62.

    PubMed  Google Scholar 

  9. Michalski J-C, Peter-Katalinic J, Egge H, Paz-Parente J, Montreuil J, Strecker G (1984) Carbohydr Res 134:177–89.

    Google Scholar 

  10. Mutsaers JHGM, van Halbeek H, Kamerling JP, Vliegenthart JFG (1985) Eur J Biochem 147:569–74.

    PubMed  Google Scholar 

  11. Bendiak B, Cumming DA (1985) Carbohydr Res 144:1–12.

    PubMed  Google Scholar 

  12. Kobata A (1979) Anal Biochem 100:1–14.

    PubMed  Google Scholar 

  13. Plummer TH Jr., Elder JH, Alexander S, Phelan AW, Tarentino AL (1984) J Biol Chem 259:10700–4.

    PubMed  Google Scholar 

  14. Tarentino AL, Gómez CM, Plummer TH Jr. (1985) Biochemistry 24:4665–71.

    PubMed  Google Scholar 

  15. Green ED, van Halbeek H, Boime l, Baenziger JU (1985) J Biol Chem 260:15623–30.

    PubMed  Google Scholar 

  16. Chu FK (1986) J Biol Chem 261:172–77.

    PubMed  Google Scholar 

  17. Steube K, Gross V, Hösel W, Tran-Thi T-A, Decker K, Heinrich PC (1986) Glycoconjugate J 3:247–54.

    Google Scholar 

  18. Lee K-O, Gesundheit N, Chen H-C, Weintraub BD (1986) Biochem Biophys Res Commun 138:230–37.

    PubMed  Google Scholar 

  19. Swedlow JR, Matteri RL, Papkoff H (1986) Proc Soc Exp Biol Med 181:432–37.

    PubMed  Google Scholar 

  20. Takahashi N, Nishibe H (1978) J Biochem (Tokyo) 84:1467–73.

    Google Scholar 

  21. Risley JM, van Etten RL (1985) J Biol Chem 260:15488–94.

    PubMed  Google Scholar 

  22. Sugiyama K, Ishihara H, Tejima S, Takahashi N (1983) Biochem Biophys Res Commun 112:155–60.

    PubMed  Google Scholar 

  23. Kamerling JP, Vliegenthart JFG (1982) Cell Biol Monogr 10:95–125.

    Google Scholar 

  24. Weaghe TJ, Darvill AG, McNeil M, Albersheim P (1983) Carbohydr Res 123:281–304.

    Google Scholar 

  25. Vliegenthart JFG, Dorland L, van Halbeek H (1983) Adv Carbohydr Chem Biochem 41:209–374.

    Google Scholar 

  26. Ernst RR (1966) Adv Magn Res 2:1–135.

    Google Scholar 

  27. Haasnoot CAG (1983) J Magn Reseon 52:153–58.

    Google Scholar 

  28. Laemmli UK (1970) Nature 227:680–85.

    PubMed  Google Scholar 

  29. Van Pelt J, Damm JBL, Kamerling JP, Vliegenthart JFG (1987) Carbohydr Res, in press.

  30. Endo Y, Yamashita K, Tachibana Y, Tojo S, Kobata A (1979) J Biochem (Tokyo) 85:669–79.

    Google Scholar 

  31. Gleeson PA, Feeney J, Hughes RC (1985) Biochemistry 24:493–503.

    PubMed  Google Scholar 

  32. Baussant T, Strecker G, Wieruszeski J-M, Montreuil J, Michalski J-C (1986) Eur J Biochem 159:381–85.

    PubMed  Google Scholar 

  33. Cahour A, Debeire P, Hartmann L, Montreuil J, van Halbeek H, Vliegenthart JFG (1984) FEBS Lett 170:343–49.

    PubMed  Google Scholar 

  34. Mutsaers JHGM, Kamerling JP, Devos R, Guisez Y, Fiers W, Vliegenthart JFG (1986) Eur J Biochem 156:651–54.

    PubMed  Google Scholar 

  35. Kessler MJ, Reddy MS, Shah RH, Bahl OP (1979) J Biol Chem 254:7901–8.

    PubMed  Google Scholar 

  36. Van Halbeek H, Dorland L, Vliegenthart JFG, Fiat A-M, Jollés P (1981) FEBS Lett 133:45–50.

    PubMed  Google Scholar 

  37. Mizuochi T, Kobata A (1980) Biochem Biophys Res Commun 97:772–78.

    PubMed  Google Scholar 

  38. Muzuochi T, Nishimura R, Taniguchi T, Utsunomiya T, Mochizuki M, Derappe C, Kobata A (1985) Jpn J Cancer Res 76:752–59.

    PubMed  Google Scholar 

  39. Nilsson B, Rosen SW, Weintraub BD, Zopf DA (1986) Endocrinology 119:2737–43.

    PubMed  Google Scholar 

  40. Kessler MJ, Mise T, Ghai RD, Bahl OP (1979) J Biol Chem 254:7909–14.

    PubMed  Google Scholar 

  41. Cole LA, Birken S, Perini F (1985) Biochem Biophys Res Commun 126:333–39.

    PubMed  Google Scholar 

  42. Carver JP, Brisson J-R (1984) in Biology of Carbohydrates, Vol 2, eds. Ginsburg V, Robbins PW, Wiley, New York, p 289–331.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80.075, NL-3508, Utrecht, The Netherlands

    Jan B L Damm, Johannis P Kamerling & Johannes F G Vliegenthart

  2. Diosynth B.V., P.O. Box 20, NL-5340 BH, Oss, The Netherlands

    Gijs W K van Dedem

Authors
  1. Jan B L Damm
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Johannis P Kamerling
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gijs W K van Dedem
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Johannes F G Vliegenthart
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Damm, J.B.L., Kamerling, J.P., van Dedem, G.W.K. et al. A general strategy for the isolation of carbohydrate chains fromN-,O-glycoproteins and its application to human chorionic gonadotrophin. Glycoconjugate J 4, 129–144 (1987). https://doi.org/10.1007/BF01049451

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01049451

Key words

Search

Navigation