The Impact of Glycoengineering on the Endoplasmic Reticulum Quality Control System in Yeasts

Abstract

Yeasts are widely used and established production hosts for biopharmaceuticals. Despite of tremendous advances on creating human-type N-glycosylation, N-glycosylated biopharmaceuticals manufactured with yeasts are missing on the market. The N-linked glycans fulfill several purposes. They are essential for the properties of the final protein product for example modulating half-lives or interactions with cellular components. Still, while the protein is being formed in the endoplasmic reticulum, specific glycan intermediates play crucial roles in the folding of or disposal of proteins which failed to fold. Despite of this intricate interplay between glycan intermediates and the cellular machinery, many of the glycoengineering approaches are based on modifications of the N-glycan processing steps in the endoplasmic reticulum (ER). These N-glycans deviate from the canonical structures required for interactions with the lectins of the ER quality control system. In this review we provide a concise overview on the N-glycan biosynthesis, glycan-dependent protein folding and quality control systems and the wide array glycoengineering approaches. Furthermore, we discuss how the current glycoengineering approaches partially or fully by-pass glycan-dependent protein folding mechanisms or create structures that mimic the glycan epitope required for ER associated protein degradation.

Keywords: endoplasmic reticulum associated protein degradation (ERAD); endoplasmic reticulum quality control (ERQC); glycoengineering; protein N-glycosylation; yeast.

FIGURE 1

(A) Structure of the eukaryotic dolichol-linked oligosaccharide. Linkages and the names of the enzymes catalysing the respective steps are given. Deletion of ALG11 gene prevents formation of the A-branch. Deletion of ALG3 gene prevents formation the B- and C-branch mainly generating a Man₅GlcNAc₂ structure. Glucosylation of the A-branch is impaired in Δalg3 mutants. (B) Canonical N-glycan structures that mediate binding to the lectins calnexin/calreticulin, the ERAD lectin Yos9p, and the ER export receptors Emp46p/Emp47p, respectively. Alternative Yos9p substrates are marked in lighter hues. Blue circles: glucose, green circles: mannose, blue squares: N-acetylglucosamine.

FIGURE 2

N-linked glycosylation pathways leading to complex-type N-glycans. A simplified lipid-linked oligosaccharide (LLO) biosynthesis pathway is depicted in the top of the figure. LLO synthesis is initiated on the cytoplasmic side of the ER membrane by Alg7p. A Man₅GlcNAc₂ structure is assembled on the cytoplasmic side by the consecutive action of the mannosyltransferases Alg13/14p, Alg1p, Alg2p and Alg11p. This structure is flipped into the ER lumen, where the modification of the LLO is continued by Alg3p. The B-branch is completed by the action of Alg9p after which the C-branch is started by Alg12p and completed by Alg9p. The triglucosyl cap is added by Alg6p, Alg8p and Alg10p generating a Glc₃Man₉GlcNAc₂. This structure is transferred onto a protein by the oligosaccharyltransferase complex. Two glucose residues are removed and Glc₁Man₉GlcNAc₂ mediates binding to calnexin. After release of the protein from calnexin and folding of the protein, the terminal mannose of the B-branch is removed (route A). N-glycans of glycoproteins undergo trimming and transfer reactions in the Golgi apparatus. First, α1,2 mannosidase activities (ManI) are trimming the α1,2 mannose residues, after which first GlcNAc residue is added by GnTI. The remaining mannose residues are trimmed off by ManII, which create the substrate for GnTII that adds a second GlcNAc residue. Route B) includes an α1,2 mannosidase activity in the ER that gives rise to the Man₅GlcNAc₂ glycan structure in the ER. This structure is modified in the Golgi apparatus by GnTI and route A and B converge. By deletion of ALG3 gene, LLO biosynthesis is abrogated creating a Man₅GlcNAc₂ that can be glucosylated to varying extent and is transferred onto a protein (route C). After removal of glucose residues and possible interactions with calnexin, an α1,2 mannosidase activity converts this structure into a Man₃GlcNAc₂ structure. Alternatively, the Man₃GlcNAc₂ can be generated biosynthetically by deletion of ALG11 and ALG3 genes and this glycan is transferred onto a protein (route D). The Man₃GlcNAc₂ glycan structure directly serves as a substrate for GnTI and GnTII. The color gradient indicates the presence or absence of canonical glycan features involved in interaction with calnexin/calreticulin and Yos9p, respectively. Blue circles: glucose, green circles: mannose, blue squares: N-acetylglucosamine. Light blue circles indicate reduced glucosylation of the A-branch. Arrows without names: multiple steps; arrows with names: specific enzymes are marked.