Recombinant therapeutic proteins produced in plants: towards engineering of human-type O-and N-glycosylation

Abstract

Background and Purpose: Recombinant DNA technology has allowed expression of different heterologous proteins in many host systems, ranging from prokaryotic to eukaryotic organisms. Therapeutic properties of recombinant proteins are very often affected by the composition and heterogeneity of their glycans. Conventional expression systems for recombinant pharmaceuticals typically do not address this problem and result with products that contain a mixture of glycoforms that are neither identical to human glycans nor optimized for enhanced biological activity. Over the last decade plants have been developed as production platforms for recombinant proteins with pharmaceutical or industrial applications. Namely, plant expression systems contain very small differences in the post-translational modifications, mainly glycosylation, which can partly be overpowered by glycoengineering, whose goal is production of recombinant proteins with highly homogenous glycosylation that closely resembles the native system. This review attempts to present current accomplishments in the production of plant-derived glycoconjugates with humanized N-and O-glycans. Materials and Methods: Main goal of N-glycoengineering is to reduce or eliminate plant-specific N-glycans, and at the same time to introduce mammalian-specific N-glycans through the several approaches. The easiest way is to change intracellular targeting of plant-made recombinant proteins and to ensure their retention in the ER; next approach is to eliminate the addition of plant-specific glycans; while the final step is engineering the plant glycosylation pathway to introduce mammalian glycotransferases into plants with generation of biantennary and multi-antennary structures on complex N-glycans. Due to significant differences in O-glycosylation between humans and plants, different approaches to engineering of O-glycosylation have been taken. Besides having their typical O-glycoslyation on Hyp-residues, plants in general miss the machinery for production of mammalian-type O-glycosylation. Attempts have been made to mimic mammalian O-glycosylation in plants, specifically the mucin-type addition of GalNAc residues. Result: Efficient generation of bisected tetraantennary complex N-glycans without typical plant glycoepitopes on human erythropoietin (hEPO) and human transferrin (hTF) was obtained in Nicotiana benthamiana plants, thus demonstrating generation of recombinant proteins with human-type N-glycosylation at great uniformity. As for the O-glycosylation, attempts to produce mucin-type O-GalNAc and disialylated core 1 Olinked glycan structures on hEPO in N. benthamiana transgenic plants proved to be successful. Moreover, although small amounts of Hyp residues were found on recombinant EPO, no plant-specific O-glycans were de-IntroductIon R ecombinant DNA technology has allowed expression of different heterologous proteins in many host systems, ranging from prokaryotic to eukaryotic organisms. Recombinant microbial systems (mainly bacteria Escherichia coli) are in use ...

Transient co-expression with three O-glycosylation enzymes allows production of GalNAc-O-glycosylated Granulocyte-Colony Stimulating Factor in N. benthamiana

“…Accumulation has been a major focal point for the production of recombinant proteins in plants [ 4 , 38 , 57 , 58 ]. Although it may be assumed that post-translational modifications, such as glycosylation which might have an impact on protein stability or activity [ 6 , 11 ], would be produced by plants and other eukaryotic expression platforms, most studies concerning expression of glycoproteins do not address this issue [ 9 , 11 , 16 , 32 , 59 – 63 ]. In this study, we address the feasibility of introducing the mammalian-specific mucin-type O -glycosylation to the pharmaceutical glycoprotein human G-CSF, in N. benthamiana plants via transient expression.…”

“…Terminal Lewis A epitopes are rarely observed in human proteins, but are widely distributed in plant glycoproteins. Maturation of human N -glycans will present a multi-antennary structure with two or more terminal branches, further extended with galactose and sialic (neuraminic) acid [ 4 , 11 ]. Major efforts to humanize N -glycosylation patterns in plants have focused on retaining the recombinant protein in the ER to avoid further modification of the core N -glycan with α(1,3)-fucose, β(1,2)-xylose and Lewis A, abolishing expression of glycosyltransferases responsible for the attachment of α(1,3)-fucose and β(1,2)-xylose, and further humanization has included the expression of human β(1,4)-galactosyltransferase and heterologous enzymes required to generate sialylation, which are present in human cells but absent in plants [ 4 , 11 – 14 ].…”

“…In plants, protein O -glycosylation is characterized by the presence of O -glycans attached to hydroxyprolines (Hyp) and Ser residues. Typically, Hyp residues in plants are decorated with arabinogalactan polysaccharides or arabinose oligosaccharides, and Ser residues with galactose [ 6 , 11 , 15 , 16 ]. While human mucin-type O -glycosylation is characterized by the attachment of N -acetylgalactosamine (GalNAc) to Ser or Thr residues, which can be further elongated with other sugars to form specific and highly complex glycans.…”

“…While human mucin-type O -glycosylation is characterized by the attachment of N -acetylgalactosamine (GalNAc) to Ser or Thr residues, which can be further elongated with other sugars to form specific and highly complex glycans. Although mucin-type O -glycosylation is widely present in human proteins, plants lack the enzymatic machinery to perform this specific glycosylation [ 11 , 15 , 16 ].…”