Glyco‐engineering in plants to produce human‐like N‐glycan structures

Abstract

It is now possible to produce complex human proteins, largely correctly folded and N-glycosylated, in plants. Much effort has been invested in engineering expression technologies to develop products with superior characteristics. The results have begun to show success in controlling important posttranslational modifications such as N-glycosylation. With the emerging data increasingly indicating the significance of proper N-glycosylation for the efficacy of a drug, glyco-engineering has become an important issue not only for academia but also for the biopharmaceutical industry. Plants have demonstrated a high degree of tolerance to changes in the N-glycosylation pathway, allowing recombinant proteins to be modified into human-like structures in a specific and controlled manner. Frequently the results are a largely homogeneously glycosylated product, currently unrivalled by that of any other expression platforms. This review provides a comprehensive analysis of recent advances in plant N-glyco-engineering in the context of the expression of therapeutically relevant proteins, highlighting both the challenges and successes in the application of such powerful technologies.

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

“…We intended to generate three glycoforms of SM6 (SM6 wt , SM6 ΔXF , and SM6 sia ) in N. benthamiana by delivering the respective SM6 expression constructs (heavy, light, and joining chain) to WT plants (SM6 wt ), to the glycosylation mutant ΔXT/FT (SM6 ΔXF ; ref. 21) and by coexpressing the genes necessary for in planta sialylation (11) in ΔXT/FT plants (SM6 sia ). IgM expression was monitored by immunoblotting.…”

“…Moreover, heterogeneous and improper N-glycosylation of hybridoma-or cell-culture-produced IgMs might hamper clinical success and impede the investigation of the impact of this important posttranslational modification. These issues can be addressed by using plants as production platform (11). Correctly assembled mammalian proteins like IgGs have been expressed in plants at a high level (12); however, whether plants can also correctly fold and assemble proteins as complex as IgMs is currently unknown.…”

“…Although mammalian cells usually generate proteins with a mixture of glycoforms that are neither identical to the human profile nor optimized for efficacy (13), plants normally produce proteins with a largely homogeneous glycosylation profile. Moreover, plants are amenable to glycoengineering approaches and tolerate the synthesis of human-like N-glycans without obvious negative effects (11). This strategy has been extensively applied for the production of Significance IgM antibodies are increasingly gaining interest as therapeutics; however, knowledge about this antibody class is scarce.…”

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

“…As a result, synthetic homogeneous glycopeptides and glycoproteins emerge as indispensable tools for functional studies and for drug/vaccine discoveries. Many elegant chemical and biochemical strategies have been explored for making homogeneous glycoproteins and mimics, including total chemical synthesis with native chemical ligation (13)(14)(15)(16)(17), chemoselective ligation (18,19), chemoenzymatic synthesis (20 -24), and glycosylation pathway engineering in host expression systems (25)(26)(27)(28). As part of these efforts, we have attempted to develop a chemoenzymatic method for construction of complex N-glycopeptides and glycoproteins that is based on the transglycosylation activity of a class of endo-␤-N-acetylglucosaminidases (the endoglycosidases that hydrolyze N-glycans of glycoproteins) for convergent native ligation of preassembled glycans and GlcNAc-peptide/ protein (20,23,24).…”

Editorial: Biotech reviews on plants, lignocellulose, sequencing, genome engineering and Aspergilli

“…The modification of plant cells to improve and tailor the production of recombinant proteins in different ways is presented in two reviews [1,2]. The first review focuses on strategies to engineer plant cells to change the glycosylation pathway and achieve a human glycosylation pattern [1].…”