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

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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-N 4-(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 Man9 PGlcNAc2 to Man13 PGlcNAc2. 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.

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Abbreviations

hCG:

human chorionic gonadotropin

phCG:

human chorionic gonadotropin expressed in Pichia pastoris

uhCG:

urinary human chorionic gonadotropin

PNGase F:

peptide-N 4-(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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Acknowledgment

This research has been financially supported by the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO).

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Authors and Affiliations

  1. Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, Padualaan 8, NL-3584 CH, Utrecht, The Netherlands

    Véronique Blanchard, Gerrit J. Gerwig, Bas R. Leeflang & Johannis P. Kamerling

  2. Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India

    Rupali A. Gadkari & Rajan R. Dighe

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  1. Véronique Blanchard
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  2. Rupali A. Gadkari
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  6. Johannis P. Kamerling
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Correspondence to Johannis P. Kamerling.

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Blanchard, V., Gadkari, R.A., Gerwig, G.J. et al. Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris . Glycoconj J 24, 33–47 (2007). https://doi.org/10.1007/s10719-006-9010-3

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