Glycoconjugate Journal

, Volume 24, Issue 1, pp 33–47 | Cite as

Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris

  • Véronique Blanchard
  • Rupali A. Gadkari
  • Gerrit J. Gerwig
  • Bas R. Leeflang
  • Rajan R. Dighe
  • Johannis P. Kamerling


Human chorionic gonadotropin (hCG) is a heterodimeric, placental glycoprotein hormone involved in the maintenance of the corpus luteum during the first trimester of pregnancy. Biologically active hCG has been successfully expressed in the yeast Pichia pastoris (phCG). In the context of structural studies and therapeutic applications of phCG, detailed information about its glycosylation pattern is a prerequisite. To this end N-glycans were released with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F and fractionated via anion-exchange chromatography (Resource Q) yielding both neutral (80%) and charged, phosphate-containing (20%) high-mannose-type structures. Subfractionations were carried out via normal phase (Lichrosorb-NH2) and high-pH anion-exchange (CarboPac PA-1) chromatography. Structural analyses of the released N-glycans were carried out by using HPLC profiling of fluorescent 2-aminobenzamide derivatives, MALDI-TOF mass spectrometry, and 500-MHz 1H-NMR spectroscopy. Detailed neutral oligosaccharide structures, in the range of Man8GlcNAc2 to Man11GlcNAc2 including molecular isomers, could be established, and structures up to Man15GlcNAc2 were indicated. Phosphate-containing oligosaccharides ranged from Man9PGlcNAc2 to Man13PGlcNAc2. Mannosyl O-glycans were not detected. Profiling studies carried out on different production batches showed that the oligosaccharide structures are similar, but their relative amounts varied with the culturing media.


Glycosylation Pichia pastoris Human chorionic gonadotropin High-mannose-type N-glycans Phosphorylation 



human chorionic gonadotropin


human chorionic gonadotropin expressed in Pichia pastoris


urinary human chorionic gonadotropin

PNGase F

peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F


fast protein liquid chromatography




matrix-assisted laser desorption ionization time-of-flight mass spectrometry


high-performance liquid chromatography


high-pH anion-exchange chromatography


pulsed amperometric detection


total correlation spectroscopy


rotating-frame nuclear Overhauser enhancement spectroscopy


water eliminated Fourier transform


composite pulse devised by M. Levitt


  1. 1.
    Talwar, G.P.: Human chorionic gonadotropin and ovarian and placental steroidogenesis. J. Steroid Biochem. 11, 27–34 (1979)CrossRefPubMedGoogle Scholar
  2. 2.
    Endo, T., Yamashita, K., Tachibana, Y., Tojo, S., Kobata, A.: Structures of the asparagine-linked sugar chains of human chorionic gonadotropin. J. Biochem. (Tokyo) 85, 669–679 (1979)Google Scholar
  3. 3.
    Blithe, D.L.: Carbohydrate composition of the α-subunit of human chorionic gonadotropin (hCGα) and the free α molecules produced in pregnancy: most free α and some combined hCGα molecules are fucosylated. Endocrinology 126, 2788–2799 (1990)PubMedGoogle Scholar
  4. 4.
    Weisshaar, G., Hiyama, J., Renwick, A.G.C.: Site-specific N-glycosylation of human chorionic gonadotropin, Structural analysis of glycopeptides by one- and two-dimensional 1H NMR spectroscopy. Glycobiology 1, 393–404 (1991)PubMedGoogle Scholar
  5. 5.
    Damm, J.B.L., Voshol, H., Hård, K., Kamerling, J.P., van Dedem, G.W.K., Vliegenthart, J.F.G.: The β-subunit of human chorionic gonadotropin contains N-glycosidic trisialo tri- and tri′-antennary carbohydrate chains. Glycoconj. J. 5, 221–233 (1988)CrossRefGoogle Scholar
  6. 6.
    Damm, J.B.L., Kamerling, J.P., van Dedem, G.W.K., Vliegenthart, J.F.G.: A general strategy for the isolation of carbohydrate chains from N-,O-glycoproteins and its application to human chorionic gonadotrophin. Glycoconj. J. 4, 129–144 (1987)CrossRefGoogle Scholar
  7. 7.
    Amano, J., Nishimura, R., Mochizuki, M., Kobata, A.: Comparative study of the mucin-type sugar chains of human chorionic gonadotropin present in the urine of patients with trophoblastic diseases and healthy pregnant women. J. Biol. Chem. 263, 1157–1165 (1988)PubMedGoogle Scholar
  8. 8.
    Liu, C.L., Bowers, L.D.: Mass spectrometry characterisation of the beta-subunit of human chorionic gonadotropin. J. Mass Spectrom. 32, 33–42 (1997)CrossRefPubMedGoogle Scholar
  9. 9.
    Cole, L.A.: Distribution of O-linked sugar units on hCG and its free alpha-subunit. Mol. Cell. Endocrinol. 50, 45–57 (1987)CrossRefPubMedGoogle Scholar
  10. 10.
    Bielinska, M., Boime, I.: The glycoprotein hormone family: structure and function of the carbohydrate chains. In: Montreuil, J., Vliegenthart, J.F.G., Schachter, H. (eds.) Glycoproteins, Vol. 29a, pp. 565–587. New Comprehensive Biochemistry, Elsevier Science BV, Amsterdam, The Netherlands (1995)Google Scholar
  11. 11.
    Kamerling, J.P., Hård, K., Vliegenthart, J.F.G.: Structural analysis of carbohydrate chains of native and recombinant-DNA glycoproteins. In: Crommelin, D.J.A., Schellekens, H. (eds.) Developments in Biotherapy, Vol. 1, From Clone to Clinic, pp. 295–304. Kluwer Academic Publishers, Dordrecht, The Netherlands (1990)Google Scholar
  12. 12.
    Gervais, A., Hammel, Y.-A., Pelloux, S., Lepage, P., Baer, G., Carte, N., Sorokine, O., Strub, J.-M., Koerner, R., Leize, E., Van Dorsselaer, A.: Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency. Glycobiology 13, 179–189 (2003)CrossRefPubMedGoogle Scholar
  13. 13.
    Lustbader, J.W., Birken, S., Pollak, S., Pound, A., Chait, B.T., Mirza, U.A., Ramnarain, S., Canfield, R.E., Brown, J.M.: Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures. J. Biomol. NMR 7, 295–304 (1996)CrossRefPubMedGoogle Scholar
  14. 14.
    Chen, W., Shen, Q.X., Bahl, O.P.: Carbohydrate variant of the recombinant beta-subunit of human choriogonadotropin expressed in baculovirus expression system. J. Biol. Chem. 266, 4081–4087 (1991)PubMedGoogle Scholar
  15. 15.
    Chen, W., Bahl, O.P.: Recombinant carbohydrate variant of human choriogonadotropin beta-subunit (hCGbeta) descarboxyl terminus (115–145). Expression and characterization of carboxyl-terminal deletion mutant of hCG beta in the baculovirus system. J. Biol. Chem. 266, 6246–6251 (1991)PubMedGoogle Scholar
  16. 16.
    Chen, W., Bahl, O.P.: Recombinant carbohydrate and selenomethionyl variants of human choriogonadotropin. J. Biol. Chem. 266, 8192–8197 (1991)PubMedGoogle Scholar
  17. 17.
    Jung, E., Williams, K.L.: The production of recombinant glycoproteins with special reference to simple eukaryotes including Dictyostelium discoideum. Biotechnol. Appl. Biochem. 25, 3–8 (1997)PubMedGoogle Scholar
  18. 18.
    Linskens, M.H.K., Grootenhuis, P.D.J., Blaauw, M., Huisman-de Winkel, B., van Ravenstein, A., van Haastert, P.J.M., Heikoop, J.C.: Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum. FASEB J. 13, 639–645 (1999)PubMedGoogle Scholar
  19. 19.
    Peters, B.P., Krzesicki, R.F., Hartle, R.J., Perini, F., Ruddon, R.W.: A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthetized by human choriocarcinoma cells. J. Biol. Chem. 258, 15123–15130 (1984)Google Scholar
  20. 20.
    Cole, L.A., Kroll, T.D., Ruddon, R.W., Hussa, R.O.: Differential occurrence of free beta and free alpha subunits of human chorionic gonadotropin (hCG) in pregnancy sera. J. Clin. Endocrinol. Metab. 58, 1200–1202 (1984)PubMedGoogle Scholar
  21. 21.
    Lustbader, J., Birken, S., Pollak, S., Levinson, L., Berstine, E., Hsiung, N., Cornfield, R.: Characterization of the expression products of recombinant human choriogonadotropin and subunits. J. Biol. Chem. 262, 14204–14212 (1987)PubMedGoogle Scholar
  22. 22.
    Sen Gupta, C., Dighe, R.R.: Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris. J. Mol. Endocrinol. 22, 273–283 (1999)CrossRefPubMedGoogle Scholar
  23. 23.
    Gadkari, R., Deshpande, R., Dighe, R.R.: Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotrophic yeast, Pichia pastoris. Protein Expr. Purif. 32, 175–184 (2003)CrossRefPubMedGoogle Scholar
  24. 24.
    Grinna, L.S., Tschopp, J.F.: Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris. Yeast 5, 107–115 (1989)CrossRefPubMedGoogle Scholar
  25. 25.
    Trimble, R.B., Atkinson, P.H., Tschopp, J.F., Townsend, R.R., Maley, F.: Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris. J. Biol. Chem. 266, 22807–22817 (1991)PubMedGoogle Scholar
  26. 26.
    Miele, R.G., Nilsen, S.L., Brito, T., Bretthauer, R.K., Castellino, F.J.: Glycosylation properties of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator. Biotechnol. Appl. Biochem. 25, 151–157 (1997)PubMedGoogle Scholar
  27. 27.
    Gemmill, T.R., Trimble, R.B.: Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta 1426, 227–237 (1999)PubMedGoogle Scholar
  28. 28.
    Hirose, M., Kameyama, S., Ohi, H.: Characterization of N-linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris. Yeast 19, 1191–1202 (2002)CrossRefPubMedGoogle Scholar
  29. 29.
    Boraston, A.B., Sandercock, L.E., Warren, R.A.J., Kilburn, D.G.: O-Glycosylation of a recombinant carbohydrate-binding module mutant secreted by Pichia pastoris. J. Mol. Microbiol. Biotechnol. 5, 29–36 (2003)CrossRefPubMedGoogle Scholar
  30. 30.
    Cereghino, J.L., Cregg, J.M.: Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45–66 (2000)CrossRefPubMedGoogle Scholar
  31. 31.
    Letourneur, O., Gervasi, G., Gaïa, S., Pagès, J., Watelet, B., Jolivet, M.: Characterization of Toxoplasma gondii surface antigen I (SAGI) secreted from Pichia pastoris: evidence of hyper O-glycosylation. Biotechnol. Appl. Biochem. 33, 35–45 (2001)CrossRefPubMedGoogle Scholar
  32. 32.
    González, L.J., Cremata, J.A., Guanche, Y., Ramos, Y., Triguero, A., Cabrera, G., Montesino, R., Huerta, V., Pons, T., Boué, O., Farnós, O., Rodríguez, M.: The cattle tick antigen, Bm95, expressed in Pichia pastoris contains short chains of N- and O-glycans. Arch. Biochem. Biophys. 432, 205–211 (2004)CrossRefPubMedGoogle Scholar
  33. 33.
    Kamerling, J.P., Vliegenthart, J.F.G.: Carbohydrates. In: Lawson, A.M. (ed.) Clinical Biochemistry—Principles, Methods, Applications, Vol 1, Mass Spectrometry, pp. 175–263. Walter de Gruyter, Berlin, Germany (1989)Google Scholar
  34. 34.
    Packer, N.H., Lawson, M.A., Jardine, D.R., Redmond, J.W.: A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj. J. 15, 737–747 (1988)CrossRefGoogle Scholar
  35. 35.
    Thieme, T.R., Ballou, C.E.: Nature of the phosphodiester linkage of the phosphomannan from the yeast Kloeckera brevis. Biochemistry 10, 4121–4129 (1971)CrossRefPubMedGoogle Scholar
  36. 36.
    Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., Parekh, R.B.: Nonselective and efficient fluorescent labeling of glycans using 2-aminobenzamide and anthranilic acid. Anal. Biochem. 230, 229–238 (1995)CrossRefPubMedGoogle Scholar
  37. 37.
    Stroop, C.J.M., Weber, W., Gerwig, G.J., Nimtz, M., Kamerling, J.P., Vliegenthart, J.F.G.: Characterization of the carbohydrate chains of the secreted form of the human epidermal growth factor receptor. Glycobiology 10, 901–917 (2000)CrossRefPubMedGoogle Scholar
  38. 38.
    Narui, T., Iwata, S., Takahashi, K., Shibata, S.: Partial hydrolysis of α-D-glucans with acid in the presence of 1,1,3,3-tetramethylurea. Carbohydr. Res. 170, 269–273 (1987)CrossRefGoogle Scholar
  39. 39.
    Guile, G.R., Rudd, P.M., Wing, D.R., Prime, S.B., Dwek, R.A.: A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem. 240, 210–226 (1996)CrossRefPubMedGoogle Scholar
  40. 40.
    Papac, D.I., Briggs, J.B., Chin, E.T., Jones, A.J.S.: A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Glycobiology 8, 445–454 (1998)CrossRefPubMedGoogle Scholar
  41. 41.
    Vliegenthart, J.F.G., Dorland, L., van Halbeek, H.: High-resolution, 1H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Adv. Carbohydr. Chem. Biochem. 41, 209–374 (1983)CrossRefGoogle Scholar
  42. 42.
    Hård, K., van Zadelhoff, G., Moonen, P., Kamerling, J.P., Vliegenthart, J.F.G.: The Asn-linked carbohydrate chains of human Tamm-Horsfall glycoprotein of one male. Novel sulfated and novel N-acetylgalactosamine-containing N-linked carbohydrate chains. Eur. J. Biochem. 209, 895–915 (1992)CrossRefPubMedGoogle Scholar
  43. 43.
    Hernández, L.M., Ballou, L., Alvarado, E., Gillece-Castro, B.L., Burlingame, A.L., Ballou, C.E.: A new Saccharomyces cerevisiae mnn mutant N-linked oligosaccharide structure. J. Biol. Chem. 264, 11849–11856 (1989)PubMedGoogle Scholar
  44. 44.
    Koles, K., van Berkel, P.H.C., Pieper, F.R., Nuijens, J.H., Mannesse, M.L.M., Vliegenthart, J.F.G., Kamerling, J.P.: N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits. Glycobiology 14, 51–64 (2004)CrossRefPubMedGoogle Scholar
  45. 45.
    Michalski, J.-C., Haeuw, J.-F., Wieruszeski, J.-M., Montreuil, J., Strecker, G.: In vitro hydrolysis of oligomannosyl oligosaccharides by the lysosomal α-D-mannosidases. Eur. J. Biochem. 189, 369–379 (1990)CrossRefPubMedGoogle Scholar
  46. 46.
    Tseneklidou-Stoeter, D., Gerwig, G.J., Kamerling, J.P., Spindler, K.-D.: Characterization of N-linked carbohydrate chains of the crayfish, Astacus leptodactylus hemocyanin. Biol. Chem. Hoppe-Seyler 376, 531–537 (1995)PubMedGoogle Scholar
  47. 47.
    Byrd, J.C., Tarentino, A.L., Maley, F., Atkinson, P.H., Trimble, R.B.: Glycoprotein synthesis in yeast; identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing. J. Biol. Chem. 257, 14657–14666 (1982)PubMedGoogle Scholar
  48. 48.
    De Waard, P., Kamerling, J.P., van Halbeek, H., Vliegenthart, J.F.G., Broertjes, J.S.S.: Characterization of N-linked gluco-oligomannose type of carbohydrate chains of glycoproteins from the ovary of the starfish Asterias rubens (L.). Eur. J. Biochem. 168, 679–685 (1987)CrossRefPubMedGoogle Scholar
  49. 49.
    Trimble, R.B., Atkinson, P.H.: Structure of yeast external invertase Man8–14GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy. J. Biol. Chem. 261, 9815–9824 (1986)PubMedGoogle Scholar
  50. 50.
    Cohen, R.E., Ballou, C.E.: Linkage and sequence analysis of mannose-rich glycoprotein core oligosaccharides by proton nuclear magnetic resonance spectroscopy. Biochemistry 19, 4345–4358 (1980)CrossRefPubMedGoogle Scholar
  51. 51.
    Hardy, M.R., Townsend, R.R.: Separation of fucosylated oligosaccharides using high-pH anion-exchange chromatography with pulsed-amperometric detection. Carbohydr. Res. 188, 1–7 (1989)CrossRefPubMedGoogle Scholar
  52. 52.
    Hernández, L.M., Ballou, L., Ballou, C.E.: Separation of yeast asparagine-linked oligosaccharides by high-performance anion-exchange chromatography. Carbohydr. Res. 203, 1–11 (1990)CrossRefPubMedGoogle Scholar
  53. 53.
    Hernández, L.M., Ballou, L., Alvarado, E., Tsai, P., Ballou, C.E.: Structure of the phosphorylated N-linked oligosaccharides from the mnn9 and mnn10 mutants of Saccharomyces cerevisiae. J. Biol. Chem. 264, 13648–13659 (1989)PubMedGoogle Scholar
  54. 54.
    Hernández, L.M., Olivero, I., Alvarado, E., Larriba, G.: Oligosaccharide structures of the major exoglucanase secreted by Saccharomyces cerevisiae. Biochemistry 31, 9823–9831 (1992)CrossRefPubMedGoogle Scholar
  55. 55.
    De Waard, P., Vliegenthart, J.F.G., Kozutsumi, Y., Kawasaki, T., Yamashina, I.: Structural studies on phosphorylated oligosaccharides derived from yeast mannan by 1H(31P) relayed spin-echo difference spectroscopy (RESED). J. Biol. Chem. 264, 12141-12144 (1989)PubMedGoogle Scholar
  56. 56.
    Matzuk, M.M., Hsueh, A.J.W., Lapolt, P., Tsafriri, A., Keene, J.L., Boime, I.: The biological role of the carboxy-terminal extension of human chorionic gonadotropin β-subunit. Endocrinology 126, 376–383 (1990)PubMedCrossRefGoogle Scholar
  57. 57.
    Sairam, M.R., Jiang, L.G.: Comparison of the biological and immunological properties of glycosylation deficient human chorionic gonadotropin variants produced by site directed mutagenesis and chemical deglycosylation. Mol. Cell. Endocrinol. 85, 227–235 (1992)CrossRefPubMedGoogle Scholar
  58. 58.
    Erbel, P.J., Haseley, S.R., Kamerling, J.P., Vliegenthart, J.F.G.: Studies on the relevance of the glycan at Asn-52 of the α-subunit of human chorionic gonadotropin in the αβ dimer. Biochem. J. 364, 485–495 (2002)CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Véronique Blanchard
    • 1
  • Rupali A. Gadkari
    • 2
  • Gerrit J. Gerwig
    • 1
  • Bas R. Leeflang
    • 1
  • Rajan R. Dighe
    • 2
  • Johannis P. Kamerling
    • 1
  1. 1.Bijvoet Center, Department of Bio-Organic ChemistryUtrecht UniversityUtrechtThe Netherlands
  2. 2.Department of Molecular Reproduction, Development and GeneticsIndian Institute of ScienceBangaloreIndia

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