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
Article

Abstract

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.

Keywords

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

Abbreviations

hCG

human chorionic gonadotropin

phCG

human chorionic gonadotropin expressed in Pichia pastoris

uhCG

urinary human chorionic gonadotropin

PNGase F

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

FPLC

fast protein liquid chromatography

2AB

2-aminobenzamide

MALDI-TOF-MS

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

HPLC

high-performance liquid chromatography

HPAEC

high-pH anion-exchange chromatography

PAD

pulsed amperometric detection

TOCSY

total correlation spectroscopy

ROESY

rotating-frame nuclear Overhauser enhancement spectroscopy

WEFT

water eliminated Fourier transform

MLEV

composite pulse devised by M. Levitt

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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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