key: cord-0760320-etka8z7k
authors: Ghosh, Shyamasree
title: Chapter 2 Sialoglycans and genetically engineered plants
date: 2020-12-31
journal: Sialic Acids and Sialoglycoconjugates in the Biology of Life, Health and Disease
DOI: 10.1016/b978-0-12-816126-5.00002-0
sha: 8b4e7b82e7a3eb242d32d2619ddf4067d9869360
doc_id: 760320
cord_uid: etka8z7k

Abstract Plants express N-glycosylation pathways and produce N-glycosylated proteins but differ from the mammalian-type proteins. Therefore attempts are made to design and engineer plant glycosylation pathways that can produce mammalian-type glycosylated moieties so that large quantities of biopharmaceuticals compatible to the human body can be produced. Most of the studies of plant expression systems for molecular farming have been conducted on Nicotiana sp. and genetic engineering and molecular biology tools have enabled the generation of glycoengineered plant for human use in the production of therapeutic recombinant proteins. We have discussed in this chapter the advances of glycoengineering in plants with special reference to the reconstruction of silaylation pathways in plants and the latest application in the production of antibody and therapeutics in plants.

Thus the synthesis of glycans resembling human glycan structure and its huge complexity remain a major challenge in glycoengineering of plant cells [6] . This is all more challenging due to the complexity and diversity of the glycans in different therapeutic recombinant proteins.

Targeted manipulation of the plant N-glycosylation pathway has enabled the production of human-like oligosaccharides and enabled the generation of functional and effective biopharmaceuticals. In the recent years plant revealing a simple N-glycosylation pathway but lacking the Oglycosylation pathway have been reported to be better and potential glycan expression systems over the conventional ones. Plant systems are being used as effective expression systems of complex sialoglycans and N-glycans and different strategies are being used for the expression of complex therapeutic sialylated glycoforms in plant systems. 

Recently different posttranslational modifications (PTMs) have been reported for peptide maturation and activation, including proteolytic processing, tyrosine sulfation, proline hydroxylation, and hydroxyproline glycosylation [7] in plants. While glycan epitopes of human complex Nglycans are often targets of lectins important for cell-cell communication, the role of plant N-glycans finds importance in protein folding, and other biological functions including salt stress responses, cellulose biosynthesis, microtubule association, and biogenesis of several receptor-like kinases [8] .

N-glycosylation is a major post-translational modification PTM in eukaryotes and is important in maintaining cell viability, where the attached core N-glycans enables proper protein folding of secreted glycoproteins and membrane proteins in the endoplasmic reticulum (ER).

Although the studies of N-glycosylation in human is extensive, the knowledge in plants is restricted due to limited N-linked glycan and mutant phenotypes, limited methods to modify and target N-glycans at specific sites, and limited understanding of protein dynamics within secretory system. Glycoproteins move from the ER to the Golgi apparatus, where the N-glycan moieties undergo further maturation or may exit the ER via an alternative route to vacuoles retaining high-mannose N-glycan structures bypassing the modifications in the Golgi.

In eukaryotes, N-glycans processing is initiated in the ER where the precursor Glc3Man9GlcNAc2 (Man9) is converted to Man8GlcNAc2 (Man8) and processing of Man8 in Golgi leads to the formation of complex Nglycans ( Fig. 1) . N-glycan processing is identical in plants and mammals till the formation of vital intermediate GlcNAc2Man3GlcNAc2 (GnGn). In mammals, GnGn oligosaccharides enables diversification of N-glycosylation but in plants, the GnGn structures are arranged with 1,2-xylose and core 1,3-fucose residues (GnGnXF3). Although in mammals core fucosylation occurs in 1,6-linkage, the fucose residues in plants ( Fig. 3 ) are in 1,3-linkage. Plant cells extend the GnGnXF3 by attaching 1,3-galatose and 1,4-fucose to form Lewis-a epitopes (Lea). Plants reveal formation of paucimannosidic structures due to the removal of terminal GlcNAc residues from GnGnXF3 by endogenous hexosaminidases similar to insects. As compared to human complex N-glycans, N-glycans of the plant systems lack sialic acid but contain core α1,3-fucose (Fuc) and β1,2-xylose (Xyl) modifications, and may contain terminal Lewis-a epitopes (β1, 3-galactose (Gal) and α1,4-Fuc linked to terminal N-acetylglucosamine, GlcNac (Fig. 3) . Human complex N-glycans are often sialylated containing different epitopes, including Lewis x, N-acetyllactosamine (LacNAc), and N,N′-di-N-acetyllactosediamine (LacDiNAc).

Although the plants and mammals reveal differences in the N-glycan structures they share high degree of homology in the secretory pathway.

Glycoengineering aims at the production of recombinant glycoproteins with a defined glycosylation profile, in order to study the impact of glycosylation and for the production of therapeutic agents. The plant expression systems are being designed to generate therapeutically important glycoproteins. Plant systems find importance as they are biologically safe, cost effective, and convenient. However, as plant N-glycosylation pathway differs in many aspects as compared to human N-glycosylation, modification of N-glycosylation pathway in plants is needed to avoid immunological challenges and get humanized authentic N-glycosylated molecules.

Plants reveal highly conserved secretory pathway with folding, assembly, and posttranslational modifications of proteins similar to the mammals. Animal sialyltransferases (STs) consist of four conserved motifs, namely large (L), small (S), very small (VS), and motif III. Although sialic acid has not been detected in plants, three orthologues containing sequences similar to the ST motifs have been identified in Arabidopsis thaliana L. The At3g48820 gene with gene id 824,043 codes for a Golgi resident protein but lacks the ability to transfer sialic acid to asialofetuin or Galβ1,3GalNAc and Galβ1,4GlcNAc oligosaccharide acceptors [10] . Strategies to produce humanized therapeutic glycoproteins in plants involves (i) retaining of the recombinant glycoproteins in ER, where N-glycans undergo modification, (ii) inhibiting the plant endogenous Golgi glycosyltransferase, and (iii) adding new glycosyltransferase from mammals. Different approaches have been used to modify the N-glycosylation pathway in different plant species, using T-DNA insertion mutants [11] , RNA interference (RNAi) [12] [13] [14] , chemical mutagenesis [15] , and targeted nuclease [16] [17] [18] approaches. N. benthamiana finds importance in molecular farming as the transient expression of proteins is fast and yields antibodies [19] by different transient expression systems, including the MagnICON system [20] , the pEAQ vector [21] , and the pTRA vector [22] . Zinc finger nucleases (ZFNs) [23] transcription activator-like effector nucleases (TALENs) [24] have enabled easy knockout of multiple genes. In N. benthamiana, the two XylT genes and two of the five FucT genes were knocked out with TALENs to completely eliminate the β-1,2-xylosyltransferase activity and reduce core α-1,3-fucosyltransferase activity by 60%. CRISPR/ Cas9 system has been used to knockout two β-1,2-xylosyltransferase and four α-1,3-fucosyltransferase genes in N. benthamiana [25] .

Sia and polysialic acid (polySia) play a vital role in biological functions and therapeutic use. Expression system in plants has been designed with multigene vectors enabling the controlled in vivo synthesis of sialylated structures in the human sialylation pathway (Fig. 4 ) that sialylate glycoproteins in α2,6or α2,3-linkage and transient coexpression of human α2,8-polysialyltransferases lead to the production of active and functional polySia structures [26] . 

The conserved secretory pathway between plants and mammals enable the production of IgGs and IgAs efficiently. En block transfer of the Glc3Man9GlcNAc2 precursor onto the growing protein and subsequent trimming in ER and cis/medial-Golgi compartments reveal similarities between mammals and plants up to the synthesis of GnGn structures (Fig. 1) . After this, in mammals, GnGn structures undergo intensive elongation/ modification processes unlike in plants, which add xylose in β1,2-position to the innermost mannose residue and fucose in α1,3-position to the innermost GlcNAc residue of the GnGn core oligosaccharide (Fig. 1) which are absent in mammalian cells. Monoclonal antibodies (mAbs) in plants exhibit a N-glycosylation profile with a single dominant oligosaccharide structure, GnGnXF3.

The humanization of the plant pathways were thoroughly investigated by Palacpac et al. [27] and Bakker et al. [28] . They overexpressed the human β1,4-glacatosyltransferase (GalT) in tobacco plants to elongate the plant-typical GnGnXF3 by β1,4-galactose leading to the formation of galactosylated structures and drastically reduced the degree of xylosylation and fucosylation. Nut production of mAbs (mAbs) suffered from challenges of formation of unexpected glycoforms and incompletely processed and hybrid structures [29, 30] .

Mutants lacking plant-specific β1,2-xylose and core α1,3-fucose achieved by the elimination of endogenous enzymes, β1, 2-xylosyltransferase (XT) and core α1,3-fucosyltransferase (FT3, Fig. 2 ) by knockdown and knockout approaches for the respective genes, and generated mutant plant lines of A. thaliana, Lemna minor, N. benthamian, moss Physcomitrella patens, DXT/FT plants (N. benthamiana glycosylation mutants lack plant-specific core β1,2-xylose and α1,3-fucose residues) were generated and found importance in the production of different mAbs and therapeutics. A schematic diagram of Fc glycoengineering is represented in Fig. 5 .

Fc-N-glycosylation profiles of these mAbs achieved by the elimination of β1,2-xylose and core α1,3-fucose leading to the synthesis of human-type structures containing dominant GnGn with no detectable β1,2-xylose or α1,3-fucose residues revealed unaltered antigen binding and complement-dependent cytotoxicity CDC activity and enhanced antibody-dependent cell-mediated cytotoxicity ADCC, effector functions of antibody. This also enabled the generation of increased galactosylation, sialylation, branching, bisecting GlcNAc, or fucosylation.

GalT when targeted to a late Golgi compartment significantly improved β1, 4-galactosylation in DXT/FT, transgenic plants. mAbs produced in such glycoengineered plants exhibited a single dominant Fc-N-glycan, digalactosylated AA structures which is predominant in serum IgG and mAbs as against HIV produced in these glycoengineered plants which exhibited improved anti-viral activity. GlcNAc bound in b1,4-position to the innermost mannose residue called bisecting GlcNAc is reported to enhance ADCC activity of mAb CAMPATH-1H, glycoengineered Rituxan and Herceptin with increased bisecting structures due to decreased 1,6-fucosylation caused by the blocking of the fucosyltransferase.

Contrasting reports exist that in CHO cells the overexpression of Nacetylglucosaminyltransferase III (GnTIII) done with the hypothesis to increase bisecting GlcNAc, produced typical hybrid structures instead with significantly reduced core-fucose content.

In the DXT/FT mutant lacking plant-specific core modifications, less of bisecting glycoforms were synthesized as compared to Wild type plants. Glycomodified DXT/FT plants produced mammalian-type core α1,6fucosylation by overexpressing core α1,6-fucosyltransferase, generating mAbs with and without fucose with identical N-glycosylation.

Plant based antibody 2G12 batches exhibited glycosylation profiles containing a predominant N-glycan structure, and GnGnXF3, GnGnF6, GnGn, and digalactosylated AA structures with binding similar to FccRI, FccRIIa, and FccRIIb. 2G12 glycoforms lacking core fucose mediated antiviral activity against various lentiviruses including HIV-1.

The most complex step of human N-glycosylation is terminal sialylation and difficult to accomplish in plants as they lack the enzyme cascades. But in planta the sialylation of mAbs has recently become possible [12, 34] by the introduction of enzymes of the mammalian pathway into plants, allowing the biosynthesis of sialic acid its activation, its transport into the Golgi, and finally its transfer onto terminal galactose and mAbs coexpressed with engineered human sialylation pathway carried up to 80% sialylated structures [30] . Six mammalian enzymes were overexpressed in plants [34] .

Advancement has been made in the design and development of plant expression systems for the generation of recombinant N-glycans glycoproteins by glycoengineering. N-glycosylation affects many properties of recombinant glycoproteins produced in planta including efficient plant-made antibodies for passive immunization but with shorter half-life in the blood due to a higher clearance rate [35] [36] [37] [38] . The removal of the core fucose residue from mammalian α-1,6-fucose or the plant α-1,3-fucose from the N-glycan of an antibody has been reported to increase the antibody-dependent cellular cytotoxicity (ADCC) [1, 36, 37] thus proving as an effective biopharmaceutical. 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.

As plant cells lack β1,4-galactosylated and sialylated glycan, which have important biological functions in animal cells [1, 26] , transgenic human β1,4-galactosyltransferase producing tobacco BY2 suspension-cultured cells were developed [1, 27] . Two genes encoding human CMP-Nacetylneuraminic acid synthetase and CMP-sialic acid transporter expressed in tobacco suspension-cultured cell to enable sialic acid biosynthesis in plants can act as bioreactor for mammalian glycoprotein production.

Human butyrylcholinesterase (BChE) is a tetrameric human serum sialylated protein that finds therapeutic importance as a candidate bioscavenger of organophosphorus nerve agents. N. benthamiana has been engineered for the expression of sialylated protein by transient co-expression of BChE cDNA by vectors [41] leading to the generation of rBChE expressing mono-and di-sialylated N-glycans in the intracellular fluid with similarity to the human protein orthologue. β1,4-N-acetylglucosaminyl-transferase IV overexpression in the recombinant engineered plant enabled the generation of branched N-glycans, with tri-sialylated structures with better and effective novel therapeutic role [42] .

Glycoprotein hormone erythropoietin (EPO) finds importance in the maintenance of hematopoiesis and providing tissue protection and recombinant human EPO (rhuEPO) find application in the treatment of anemia. However, rhyEPO at higher doses can cause harmful increase in the RBC masses and reveals limited role in tissue protection. Asialoerythropoietin (asialo-rhuEPO), which is a desialylated form of rhuEPO, has been reported to lack hematopoietic activity, but retain cytoprotective activity. But chemically enzymatic desialylation of rhuEPO suffers from not being cost effective. Although plants are known to synthesize complex N-glycans, they lack enzymes to transfer sialic acid and β1,4-galactose to N-glycan chains, therefore serve as a potential expression for generation of asialoerythropoietin.

Asialo-rhuEPO is being designed to be produced in plants by introducing human β1,4-galactosyltransferase as the penultimate β1,4-linked galactose residues regulating its in vivo biological activity. Co-expression of human β1,4-galactosyltransferase and EPO genes in tobacco plants has been reported to accumulate asialo-rhuEPO confirmed by its specificity to Erythrina cristagalli lectin column, revealing expression of N-glycan structures with terminal β1,4-galactose residues and a functional co-expressed GalT. Asialo-rhuEPO has been reported to interact with the EPO receptor (EPOR) with similar affinity as rhuEPO with desired biological function [43] .

N-glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans but the fact that plants express negligible amounts of free or protein-bound Neu5Ac presents a major disadvantage for their application as biopharmaceutical expression system. Thus to synthesize Neu5Ac-containing N-glycans, plants need to synthesize Neu5Ac and its nucleotide-activated derivative, cytidine monophospho-N-acetylneuraminic acid. Transgenic A. thaliana plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-N-acetylneuraminic acid synthetase, has been designed and developed and their simultaneous expression has led to the generation of significant Neu5Ac amounts in planta, which could be further converted to cytidine monophospho-N-acetylneuraminic acid by the coexpression of CMP-N-acetylneuraminic acid synthetase leading to the production of Neu5Ac-containing glycoproteins in plants [44] .

Neu5Ac could be synthesized in the plant cytosol by the expression of microbial Neu5Ac-synthesizing enzymes including Neu5Ac lyase from Escherichia coli and Neu5Ac synthase (neuB2) from Campylobacter jejuni in two model plants including Bright Yellow 2 (BY2) tobacco cells and Medicago sativa [45] .

Human CMP-N-acetylneuraminic acid (NeuAc) synthase (HCSS) and α2,6-sialyltransferase (HST) enable sialylation of N-linked glycans in mammalian cells. HCSS synthesizes CMP-NeuAc, which HST uses as a donor substrate to transfer NeuAc to the terminal position of N-linked glycans. HCSS and HST genes could be inserted and expressed by the suspension-cultured tobacco BY2 cells to enable sialylation pathway in plants, producing mammalian-type sialoglycoproteins with terminal NeuAc residues in plants [46] .

Two engineered constructs containing either the native signal peptide from human lactoferrin or the signal peptide from sweet potato sporamin fused to human lactoferrin has been reported to produce N-terminal sequences of rhLf purified from tobacco identical to Lf from human milk for both constructs [47] .

The natural insertion of N. benthamiana into the RNA-dependent RNA polymerase 1 gene [1, 49] enables rapid production of high-value hormones, enzymes, and antibodies, and is successful in the production of ZMapp which is a cocktail of neutralizing mAb c13C6 and two chimeric antibodies c2G4 and c4G7, which were applied during the 2014-15 Ebola outbreak [48] , and for the efficient production of vaccines against seasonal flu [49] .

The intravenous immunoglobulin therapeutic application of ZMapp involves direct reaction to the virus and bind as lock and key leading to its deactivation and provides simulated immune response against Ebolaviral proteins Ebolavirus. Genes of the Ebola antibodies needed for the drug are inserted into Agrobacterium, then tobacco plants are injected or infused with the engineered viral vector-encoding Ebola antibodies, and plants produce the antibodies which are later isolated to form the drug known as ZMapp (Figs. 6 and 7, Table 2 ) [48] . In mammals O-glycans on secretory proteins are formed by the attachment of N-acetylgalactosamine (GalNAc) to serine or threonine residues (mucin-type O-glycosylation) which are further modified by the addition of different monosaccharides such as galactose, GlcNAc, sialic acid, forming mucin-type core Oglycan structures that is important in different biological processes [66] (Fig. 8) .

In plants, unlike mammals, proline residues are converted to hydroxyproline (Hyp) by prolyl-4-hydroxylases (P4H) that are linked with arabinose residues. Knockout of P4H genes could eliminate O-glycosylation in P. patens, thereby helping in the modification of recombinantly expressed EPO [1, 6, 67] . Overexpression of human polypeptide GalNAc-transferase 2 (GalNAcT2) in Arabidopsis, tobacco BY2 cells, and N. benthamiana [68] [69] [70] , initiating O-GalNAc formation on different recombinant glycoproteins (including EPO and IgA1 antibodies) [71] , has been reported. This GalNAc residue acts as a substrate for subsequent elongation with β1,3-galactose by overexpressing β1,3galactosyltransferase (C1GalT1) and expression of C1GalT1 and genes for the human sialylation pathway enabled the synthesis of sialylated O-glycans [6, 72] .

Parasitic helminths secrete immunomodulatory with certain N-glycan epitopes including Lewis X and LDN-F glycan motives that find importance in treatment of allergies and autoimmune diseases. Overexpression of glycosyltransferases including FucTs, GalTs, and GalNAcTs in N. benthamiana enabled the reconstruction of Lewis X and LDN-F motives [73] that find importance in the development of anti-helminthic vaccines.

Detection of low-level monosaccharides in the glycoprotein hydrolyzate are accomplished by derivatization prior to high-performance liquid chromatography (HPLC)-fluorescence and liquid chromatography (LC)-sonic spray ionization (SSI)-mass spectrometry (MS) analyses. LC-SSI-MS has been employed to identify the compositional monosaccharides including glucosamine, glucose, mannose, arabinose, xylose, and sialic acid found in the transgenic corn [74] . 

In plant biomanufacturing of human proteins of importance, glycosylation, is one of the most addressed PTMs, as it affects protein homogeneity and functionality. Different engineering expression systems have been designed to control glycosylation and generate engineered N-and O-linked glycans with targeted sugar profiles and their various applications in the generation of human therapeutics .

Despite advances in the study of N-glycosylation pathways in plants, the study is far from complete and not completely known as compared to the human N-glycosylation pathway. The N-glycosylation pathway is not completely known for model plant organism A. thaliana and other different plant species. Although intra-Golgi glycosyltransferases are reported in A. thaliana, their functions remain unknown [79] but is assumed to play a vital role in synthesis of O-glycosylated proteins like arabinogalactan proteins. Studies from A. thaliana and rice have indicated that N-glycans enable growth under stress. However, complete genome sequencing of different plants will enable better understanding of the N-glycan pathway in plants and their efficient modifications and research in this exciting field of biology with human applications in the generation of therapeutics compatible to the human body is increasing across the globe.

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Source Wikipedia: Joachim Müllerchen-Own work Multi-license with GFDL and Creative Commons CC-BY

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Expression of human butyrylcholinesterase with an engineeredglycosylation profile resembling the plasma-derived orthologue

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N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin

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Nglycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulatedthrough co-expression of human β-1,4-galactosyltransferase

Production of human lactoferrin in transgenic tobacco plants

Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp

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