Glycosylation of human α1-antitrypsin inSaccharomyces cerevisiae and methylotrophic yeasts

Abstract

Human α1‐antitrypsin (α1‐AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α1‐AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α1‐AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α1‐AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α1‐AT. The human α1‐AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α1‐AT in the methylotrophic yeasts appeared to be relatively shorter than those of α1‐AT in S. cerevisiae. The α1‐AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α1‐AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.

Functional Characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 Genes as Members of the Yeast OCH1 Mannosyltransferase Family Involved in Protein Glycosylation

“…Furthermore, the less extensive hyperglycosylation of glycoproteins from H. polymorpha than from S. cerevisiae has been suggested to be another factor that favors the production of mammalian cell-originated proteins in this yeast (12,13). Our recent study on the structural characterization of the oligosaccharides assembled on glycoproteins secreted by H. polymorpha showed that most N-linked glycans synthesized in H. polymorpha have core-type structures (Man 8 -12GlcNAc 2 ) and that they are mainly branched by ␣-1,2-linkages without hyper-immunogenic terminal ␣-1,3-linked mannose residues.…”

Display of malaria transmission-blocking antigens on chimeric duck hepatitis B virus-derived virus-like particles produced in Hansenula polymorpha

“…polymorpha to high cell densities reaching dimensions of 100 g DCW per L culture broth [77]. Additional advantages of the methylotrophic yeast for the production of recombinant proteins are its relatively high optimal growth temperature of 37°C which allows a better temperature management in large-scale fermentations and the tendency to reduced N -linked hyperglycosylation of recombinant proteins compared to Saccharomyces cerevisiae [74,78] combined with the lack of the terminal, hyperallergenic α-1,3-linked mannose [79].…”

Glucosidase and Mannosidase Inhibitors Mediate Increased Secretion of Mutant α1 Antitrypsin Z

“…These data indicate that ␣1-ATZ secreted in the presence of CST, KIF, or DMJ is functionally active although, in each case, it is apparently slightly lower in activity than wild type ␣1-AT secreted by HepG2 cells. Similar results have been described for wild type human ␣1-AT secreted with high-mannose type carbohydrate side chains by Saccharomyces cerevisiae and methylotrophic yeasts (27,28).…”