Production of Human Compatible High Mannose-type (Man5GlcNAc2) Sugar Chains in Saccharomyces cerevisiae

Abstract

A yeast mutant capable of producing Man 5 GlcNAc 2 human compatible sugar chains on glycoproteins was constructed. An expression vector for ␣-1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/ retrieval tag was designed and expressed in Saccharomyces cerevisiae. An in vitro ␣-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an ␣-1,2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man 5 GlcNAc 2 . This is the first report of a recombinant S. cerevisiae able to produce Man 5 GlcNAc 2 -oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.Saccharomyces cerevisiae is useful for the production of recombinant proteins of biological interest because of the established expression system, and it can be easily grown in large quantities. Moreover, yeast share the early steps of the mammalian Asn-linked glycosylation pathway. However, the mature Asn-linked oligosaccharides of yeast are mannan glycans and are highly antigenic against mammals. Thus, it would be necessary to eliminate the antigenicity of the sugar chains when recombinant therapeutic glycoproteins are produced in yeast.Several genes concerned with the biosynthesis of yeast sugar chains have been cloned, and the glycosylation pathway of yeast has been clarified. The OCH1 gene encodes an ␣-1,6-mannosyltransferase that initiates ␣-1,6-polymannose outer chain formation on the Asn-linked inner oligosaccharide Man 8 GlcNAc 2 in S. cerevisiae (1). MNN1 has been proposed as the structural gene for the ␣-1,3-mannosyltransferase that elongates the outer chain and the inner core oligosaccharide (2, 3). The ⌬och1 mnn1 double mutant accumulated a single oligosaccharide moiety, Man 8 GlcNAc 2 , a high mannose-type structure (1). This mutant may be useful to produce recombinant therapeutic glycoproteins without any antigenicity toward humans.On the other hand, some glycoproteins of therapeutic value require complex-type sugar chains for their efficacy. Erythropoietin (EPO), 1 a hematopoietic glycoprotein factor produced in the kidney, has three complex-type Asn-linked sugar chains and one mucin-type sugar chain. It is reported that the composition and structure of each sugar chain affected the biological activity, the efficiency of secretion, and had profound effects on the half-life of EPO in the blood circulation (4). It seems that the most active form of the EPO molecule requires tetraantennary Asn-linked sugar chains (5) with full sialylation, to prevent serum clearance by the action of the hepatic asialoglycoprotein binding protein (6, 7). When EPO was expressed in the ⌬och1 mnn1 mutant yeast, the recombinant EPO should have high mannose-type oligosaccharides, which are trapped by the...

Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeastPichiapastoris

“…These data clearly emphasize the importance of choosing the proper combination of (i) a localization sequence and (ii) an ␣-1,2-mannosidase catalytic domain of the proper length. Fungal ␣-1,2-mannosidases with acidic pH optima (e.g., P. citrinium and A. nidulans), when expressed as fusions with the leader library, generally resulted in low (Man) 5 -(GlcNAc) 2 yields (data not shown) consistent with previous findings (11,21).…”

“…This enzyme will trim the (Man) 8 -(GlcNAc) 2 core structure to (Man) 5 -(GlcNAc) 2 and thereby generate the structure that is capable of receiving the GlcNAc that initiates the formation of hybrid N-glycans. A very similar approach was taken in a triple mutant och1 mnn1 mnn4 strain of S. cerevisiae (11). ER localization of a fungal ␣-1,2-mannosidase (from Aspergillus saitoi) was accomplished by adding the tetrapeptide HDEL as an ER retrieval tag to the C terminus of the gene.…”

“…By using the S. cerevisiae GAPDH promoter and a multicopy number plasmid, mannosidase activity was detectable in cell-free extracts; however, only 27% of the N-glycans of an endogenous marker protein (carboxypeptidase Y) were trimmed from (Man) 8 -(GlcNAc) 2 to (Man) 5 -(GlcNAc) 2 in vivo. Although not entirely successful, the pioneering work of Chiba et al (11) demonstrates that N-glycans from S. cerevisiae can be modified substantially by engineering glycosylation pathways. Here the localization of catalytic domains to the secretory pathway of the related yeast P. pastoris using yeast type II membrane protein leader domains is demonstrated.…”

“…Because the activity of the catalytic domain has to be maintained in the environment in which it has been localized, additional aspects such as pH optima must be considered. For example, Chiba et al (11) found that localizing a mannosidase with a pH optimum of 5 in the ER of S. cerevisiae results in only modest intracellular mannosidase activity.…”

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans inPichia pastoris

“…Thus, a triple-knockout strain is needed to obtain a homogeneous Man 8 GlcNAc 2 pattern, and the viability of such a strain is severely impaired. Further introduction of an ER-retained ␣-1,2-mannosidase led to production of only about 20% Man 5 GlcNAc 2 on the yeast glycoprotein carboxypeptidase Y (8). Thus, so far N-glycan homogenization in baker's yeast has been an inefficient process, and the strains obtained might be too weak for general use in glycan engineering and subsequent glycoprotein production.…”