Characterization of N‐linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris

Abstract

We studied the structures of four N-linked oligosaccharide chains of the recombinant human antithrombin (rAT) expressed in the yeast Pichia pastoris. rAT was fully glycosylated at Asn 96 and Asn 155, whereas the glycosylation on Asn 135 and Asn 192 was partial. The glycosylation level on Asn 135 was only 12% and this reduction is assumed to be one of the reasons for a higher heparin-binding affinity of rAT than plasma-derived human antithrombin (pAT). In order to determine the sizes and electrostatic charges of the N-linked oligosaccharides, rAT was treated with PNGase F, and the reduced ends were labelled by pyridylamination followed by analysis using anion exchange and amide adsorption columns. The N-linked oligosaccharides were 78% neutral and 22% phosphomannosylated. The neutral oligosaccharides were thought to be Man 9 -12 GlcNAc 2 as their major components. The phosphomannosylated oligosaccharides were then subjected to mild acid hydrolysis and/or digestion with alkaline phosphatase, and their charge shifts were analysed by the affinity to an anion exchange column. Among phosphomannosylated oligosaccharides, monophosphate diester type was predominant, whereas negatively charged diphosphate diester and monophosphate monoester types were minor components. The mannose residues at the non-reducing end(s) of Man 9 -12 GlcNAc 2 were phosphomannosylated or phosphorylated and these are the major components. Because rAT is less negatively charged than pAT, which has disialyl biantennary N-glycans, it might be less repulsive to pentasaccharide-bearing anticoagulantly active heparan sulphate proteoglycan molecules exposed on the surface of the damaged vascular vessels.

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

“…Treatment of subfraction Q1-1 with mild acid led to a shift of the position of peak A to that of peak C (Fig. 9c), indicating that A corresponds to structures with an acid-labile monocharged diesterified phosphate group (Man-P-Man-→ P-Man-) [28]. Peak B seems to be a mixture of structures with terminal or diesterified phosphate groups, as deduced from the observation that peak B totally disappears only after both alkaline phosphatase and mild acid incubations.…”

“…Mild acid hydrolysis followed by an alkaline phosphatase digestion resulted in the complete disappearance of the charged components' peaks. Taking into account their retention times, it is speculated that peak A reflects glycans with one terminal Man-P-Man motif, and peaks B + C glycans with two terminal Man-P-Man motifs [28].…”

“…However, P. pastoris does not produce the typical oligomannose-, hybrid-and complex-type glycosylation patterns as generally found in mammalian cell systems. Several studies on other recombinant glycoproteins expressed in P. pastoris have shown that relatively small (compared with glycoproteins from other yeast species) (phosphorylated) high-mannose-type structures are present, generally containing 8 to 14 Man residues [24][25][26][27][28]. O-Mannosylation of proteins expressed in P. pastoris has also been reported (1-5 mannose residues) [29][30][31][32]; however, it occurs less frequently than N-glycosylation.…”

Molecular cloning and characterization of a Pichia pastoris ortholog of the yeast Golgi GDP-mannose transporter gene

“…Several improved host-vector systems for the intracellular or secretory production of foreign gene products in P. pastoris are now commercially available. The secretory production is coupled with glycosylation with high-mannose yeast-type carbohydrates in most cases, during the vesicular transport from the endoplasmic reticulum (ER) to Golgi (Hirose et al, 2002;Yoda and Noda, 2001). …”

Substitution of <i>Pichia pastoris</i>-Derived Recombinant Proteins with Mannose Containing O- and N-Linked Glycans Decreases Specificity of Diagnostic Tests

“…P. pastoris is generally regarded to produce smaller high-mannose N-glycans. In both S. cerevisiae and P. pastoris, charged groups can be linked to N-glycans in the form of mannose phosphate diesters [7, 8, 9]. Yeast can also substitute serines and threonines with O-linked glycans.…”