N-glycosylation Engineering of Biopharmaceutical Expression Systems

Abstract

N-glycosylation, the enzymatic coupling of oligosaccharides to specific asparagine residues of nascent polypeptide chains, is one of the most widespread post-translational modifications. Following transfer of an N-glycan precursor in the ER, this structure is further modified by a number of glycosidases and glyco-syltransferases in the ER and the Golgi complex. The processing reactions occurring in the ER are highly conserved between lower and higher eukaryotes. In contrast, the reactions that take place in the Golgi complex are species- and cell type-specific. Due to its non-template driven nature, glycoproteins typically occur as a mixture of glycoforms. Since N-glycans influence circulation half-life, tissue distribution, and biological activity each glycoform has its own pharmacokinetic, pharmacodynamic and efficacy profile. Moreover, modification of glycoproteins with non-human oligosaccharides can result in undesired immunogenicity. Therefore, engineering of the N-glycosylation pathway of most currently used heterologous protein expression systems (bacteria, mammalian cells, insect cells, yeasts and plants) is actively pursued by several academic and industrial laboratories. These research efforts are in the first place directed at humanizing the N-glycosylation pathway and eliminating immunogenic glycotopes. Moreover, one wants to establish new structure-function relationships of different glycoforms, which helps to decreasing the complexity of the N-glycan repertoire towards one defined N-glycan structure. In this review, we discuss the most important recent milestones in the glycoengineering field.

Engineering of complex protein sialylation in plants

“…Although such cells produce properly sialylated glycoproteins, they have several limitations, like glycan heterogeneity and the incapability to produce distinct glycoforms on demand, that hamper further investigation on the biological impact of sialylation and the development of optimized therapeutic proteins. Substantial efforts have been devoted to genetic engineering of mammalian cells and other organisms to expand their glycosylation capacity and to improve or alter glycosylation, including sialylation (9,10). Despite impressive recent achievements, designed sialylation strategies are largely elusive.…”

A Combined System for Engineering Glycosylation Efficiency and Glycan Structure in Saccharomyces cerevisiae

“…The growing demand for production capacity for therapeutic glycoproteins is driving extensive efforts to generate alternative expression systems for glycoproteins, including plants (6), yeast, fungi (7), and bacteria (8). The most challenging issues of these alternative expression systems are to prevent the introduction of non-human-like, potentially immunogenic sugar moieties and to reproduce the human N-glycosylation patterns or even tailor them for production of highly active therapeutic glycoproteins (9). N-linked glycosylation is initiated at the cytosolic side of the endoplasmic reticulum (ER) membrane, where in a stepwise manner Man 5 GlcNAc 2 is assembled on dolichol pyrophosphate (PP-Dol) (Fig.…”

Glycoengineering of yeasts from the perspective of glycosylation efficiency

“…The glycoengineering of yeasts has been extensively reviewed, with a strong focus on the steps taking place in the Golgi apparatus [7,8]. The architecture of Golgi-localized glycosyltransferases and mannosidases is highly conserved between species and consists of an N-terminal transmembrane domain (TMD) with an extension into the cytoplasm of different length, a stem domain and a C-terminal catalytic domain.…”