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

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-mannosetype 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. Phosphatecontaining 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.

Modification of paper properties using carbohydrate-binding module 3 from the Clostridium thermocellum CipA scaffolding protein produced in Pichia pastoris: elucidation of the glycosylation effect

“…Wan et al (2011) previously reported that only the Asn68 of CBM3wt was actually glycosylated when this CBM was produced in P. pastoris in fusion with the enhanced green fluorescent protein (EGFP). Therefore, considering that the N-glycans added by P. pastoris to other recombinant glycoproteins have been shown to generally contain two N-acetylglucosamines (0.4 kDa) plus 8-14 mannoses (1.3-2.3 kDa) (Montesino et al 1999;Blanchard et al 2007), N-glycosylation is estimated to account for around 10 % of the molecular weight of the recombinant CBM3wt produced in this yeast.…”

“…Glycosylated CBM3wt three-dimensional (3D) model building Previous work by Wan et al (2011) indicated that only the Asn68 of CBM3wt was N-glycosylated by P. pastoris, which is known to attach to recombinant proteins N-glycans containing predominantly 8-14 mannoses (Montesino et al 1999;Blanchard et al 2007). Based on this information, the 3D structure of the CBM3 from Clostridium thermocellum CipA deposited in the Protein Data Bank by Tormo et al (1996) (PDB ID: 1NBC) was uploaded in GlyProt (Bohne-Lang and von der Lieth 2005), which was used to build a hybrid CBM3wt structure containing a high mannose-type N-glycan with 9 mannoses at the position Asn68.…”

Profiling of Endo H‐released serum N‐glycans using CE‐LIF and MALDI‐TOF‐MS – Application to rheumatoid arthritis

“…Many methods are used to analyze the structure of glycoprotein glycans: lectin staining [7], 2D-HPLC profiling [8], high-pH anino-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) [9], MS [10], NMR [11]. The advantage of CE-LIF over many other methods is the rapid nature of the analysis; conditioning takes only a few minutes and the analysis itself takes less than 20 min.…”

Invasion of Trypanosoma cruzi into host cells is impaired by N-propionylmannosamine and other N-acylmannosamines

“…Samples were then digested with 1 U peptide-N 4 -(N-acetyl-β-glucosaminyl)asparagine amidase F (Roche Applied Science, IN) [23][24][25] for 16 h at 37°C. N-Glycans were isolated using reversed-phase C18 cartridges (Grace Davison Discovery Sciences, Deerfield, IL) and desalted on graphitized carbon columns (Grace Davison Discovery Sciences, Deerfield, IL) [26].…”