Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris.
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-beta-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-NH(2)) 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(1)H-NMR spectroscopy. Detailed neutral oligosaccharide structures, in the range of Man(8)GlcNAc(2) to Man(11)GlcNAc(2) including molecular isomers, could be established, and structures up to Man(15)GlcNAc(2) were indicated. Phosphate-containing oligosaccharides ranged from Man(9)PGlcNAc(2) to Man(13)PGlcNAc(2). 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.
Blanchard
Véronique
V
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.
Gadkari
Rupali A
RA
Gerwig
Gerrit J
GJ
Leeflang
Bas R
BR
Dighe
Rajan R
RR
Kamerling
Johannis P
JP
D000117:Acetylglucosamine / Q000032:analysis / Q000737:chemistry; D006063:Chorionic Gonadotropin / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006063:Chorionic Gonadotropin; D009844:Oligosaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000117:Acetylglucosamine