Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> |
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Authors: | Véronique Blanchard Rupali A Gadkari Gerrit J Gerwig Bas R Leeflang Rajan R Dighe Johannis P Kamerling |
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Institution: | (1) Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands;(2) Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India |
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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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Keywords: | Glycosylation Pichia pastoris Human chorionic gonadotropin High-mannose-type N-glycans Phosphorylation |
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