Engineering of human-type O-glycosylation in<i><i>Nicotiana benthamiana</i></i>plants

Strasser, Richard

doi:10.4161/bioe.22857

Cited by 26 publications

(19 citation statements)

References 51 publications

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“…Compared to N -glycans, engineering of mucin-type O -glycosylation is highly challenging as a mammalian-like mucin-type O -glycan biosynthesis pathway is absent from plants ( Strasser, 2013 ). The de novo synthesis of defined O -glycan structures in plants requires the coordinated expression of several different mammalian proteins in the secretory pathway of plants.…”

Section: Discussionmentioning

confidence: 99%

Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants

et al. 2016

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The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans.

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Section: Discussionmentioning

confidence: 99%

Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants

et al. 2016

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“…Thus, yeast cells, insect cell lines and mammalian cell cultures have been utilized for eukaryotic protein production (4,5), although the implementation of each of these expression systems has its shortcomings (6,7). Interestingly, these conventional expression systems typically do not address the problem of glycan heterogeneity albeit they result with products that contain a mixture of glycoforms that are neither identical to human glycans nor optimized for enhanced biological activity (8).…”

Section: Conclusion: Plants and Methods Of Plant Molecular Farming Ofmentioning

confidence: 99%

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

2016

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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 ...

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“…The Food and Drug Administration (FDA) has approved first plant-made pharmaceutical protein for human parenteral administration including taliglucerase alfa [38], also named as Elelyso, produced by Protalix Biotherapeutics for the application as a replacement therapy for Gaucher disease, which is advantageous due to the structure of the exposed terminal mannose residues on α-1,3-fucoseand β-1,2-xylose-containing N-glycan structures generated in plant cell vacuoles [1] that are required for the efficient uptake of the enzyme into macrophages. N. benthamiana has been extensively researched for the production of mucin-type O-glycans [39] and N-glycans [40] of recombinant proteins.…”

Section: Applicationsmentioning

confidence: 99%

Sialoglycans and genetically engineered plants

Ghosh¹

2020

Sialic Acids and Sialoglycoconjugates in the Biology of Life, Health and Disease

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Engineering of human-type O-glycosylation inNicotiana benthamianaplants

Cited by 26 publications

References 51 publications

Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants

Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants

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

Sialoglycans and genetically engineered plants

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