Size distribution and general structural features of N‐linked oligosaccharides from the methylotrophic yeast, Pichia pastoris

Abstract

The secreted glycoproteins of Pichia pastoris contain more than 35% of their N-linked oligosaccharides as structures smaller than Man14GlcNAc2 (Man = mannose; GlcNAc = N-acetylglucosamine). On heterologous invertase produced in P. pastoris, approximately 85% of the oligosaccharides are in the size range Man8-14GlcNAc2. The structures appear to contain alpha-linked mannose. In addition, one-third of the structures contain net negative charge and can be radio-labelled in vivo with 32P. The largest oligosaccharides isolated from P. pastoris are significantly shorter than the hypermannosylated structures typical of S. cerevisiae, indicating that the factors which influence the processing of N-linked oligosaccharides in P. pastoris are different from those which influence processing in S. cerevisiae. The smaller N-linked oligosaccharides synthesized by P. pastoris resemble high-mannose oligosaccharides synthesized by animal cells, and this finding increases the utility of P. pastoris as a host for the production of heterologous glycoproteins.

Genome sequence of the recombinant protein production host Pichia pastoris

“…In terms of N-glycosylation, yeasts such as P. pastoris modify proteins with a range of heterogenous highmannose glycans 28 , which introduce a large amount of heterogeneity in the protein (reducing downstream processing efficiency and complicating product characterization) and induce fast clearance from the bloodstream. The highly immunogenic terminal alpha-1, 3-mannosyl glycotopes that are abundantly produced by S. cerevisiae are not detected on Pichia-produced glycoproteins 29 .…”

Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris

“…In particular, N-linked carbohydrate moieties significantly influence the biological activity of gonadotropins (Thotakura & Blithe 1995, Ulloa-Aguirre et al 1995, Arey et al 1997, as they are required for activation of intracellular signalling pathways in addition to being determinants of in vivo bioactivity through their influence on the metabolic clearance rate (MCR) of the hormones (Ulloa-Aguirre et al 1995, Arey et al 1997. As the glycosylation moieties synthesized by P. pastoris differ from those of mammalian cells, being of the high-mannose type characteristic of yeast (Grinna & Tschopp 1989), an important future goal will be to determine the glycosylation patterns and MCR of Pichia-derived roFSH. Comparison of the mean concentrations of the secreted roFSH and subunits with those of the roFSH heterodimer indicates that only approximately 9% and 4% of the subunits, 26 and 32% of the subunits, secreted by strains GSpAOM M and GSpAO(M ) 2 M respectively, are present in the culture media as dimers.…”

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

“…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.…”