Abstract
Plants are increasingly being used for the production of recombinant proteins. One reason is that plants are highly amenable to glycan engineering processes and allow the production of therapeutic proteins with increased efficacies due to optimized glycosylation profiles. Removal and insertion of glycosylation reactions by knock-out/knock-down approaches and introduction of glycosylation enzymes have paved the way for the humanization of the plant glycosylation pathway. The insertion of heterologous enzymes at exactly the right stage of the existing glycosylation pathway has turned out to be of utmost importance. To enable such precise targeting chimeric enzymes have been constructed. In this short review we will exemplify the importance of correct targeting of glycosyltransferases, we will give an overview of the targeting mechanism of glycosyltransferases, describe chimeric enzymes used in plant N-glycosylation engineering and illustrate how plant glycoengineering builds on the tools offered by synthetic biology to construct such chimeric enzymes.
INTRODUCTION
The medicinal use of proteins and blood products has a long history. Already in the 15th century ailing pope Innocent VIII was reportedly infused with blood from three healthy boys to give him back vigor and strength, however, with a fatal outcome for all four of them (). The first successful blood transfusion was made in 1665 between dogs () and it took over 150 years for the first successful transfusion between humans (). Proteins purified from animal or human tissues (growth hormones, insulin, clotting factors, or other blood components) have been used for medicinal purposes since the beginning of the 20th century (e.g., ; ) and with the advent of recombinant protein production possibilities, many of those proteins are now produced recombinantly. The market for pharmaceutical proteins is assumed to currently amount to roughly 150–200 billion US$, and develops strongly with growth rates of ∼10% and more (; ; ). Special drivers of this growth are antibodies and antibody related products, but also other types of proteins are selling well, like insulin, vaccines, erythropoietin, etc. ().
A current major concern in producing biopharmaceuticals is a special type of post-translation modification (PTM), namely N-glycosylation. This PTM is found on a large proportion of pharmaceutically relevant proteins () and can influence protein characteristics like folding and assembly, solubility and charge, serum half-life, functionality, etc. (e.g., ; ; ). As different cell types attach different glycans, the characteristics of the N-glycosylated protein can be strongly affected by the expression host – a fact that should be considered carefully when choosing the production system. For example, bacteria generally do not glycosylate proteins and yeasts attach larger glycan structures than mammals. Insect cells decorate proteins with paucimannosidic N-glycans which are normally not present in humans. Plants produce complex type N-glycans similar to humans, however, certain non-mammalian epitopes are attached and more complex human-type glycosylation cannot be produced (for reviews on typcial N-glycosylation patterns and glycoengineering of different expression hosts see, e.g., ; ). Another concern is glycan microheterogeneity, i.e., attachment of different N-glycans to the same N-glycosylation site, as homogeneously glycosylated products are required by the regulatory authorities. Thus, research has focused on modifying the glycosylation characteristics of a variety of expression systems to allow homogeneous, human-type N-glycosylation (; ; ; ; ; ; ; ) and resulted in the production of proteins carrying modified glycans and often showing improved in vivo functions.
Plants have proven their capability regarding production speed, ease of scale up and to meet quality standards demanded by regulatory agencies for clinical applications (; ). Also governmental agencies like the have recognized the advantages of this technology for the quick manufacturing of vaccines, difficult to produce biopharmaceuticals, etc. This has led to massive investments in research, production facilities complying with current quality standards (; ; ) and the first products on the market. Glucocerebrosidase, an enzyme to treat Gaucher’s disease, has been approved by the FDA in 2013 as the first plant-produced, parenterally applied biopharmaceutical (; ). Additionally, several plant-made pharmaceuticals have received approval for clinical trials and other plant-produced products are already marketed as research/diagnostic reagent, medical device, cosmetic product etc. (recently reviewed by ; ; ). Many of these proteins are glycosylated.
In this review we will discuss the approaches taken to engineer the N-glycosylation pathway in Nicotiana benthamiana and put a strong focus on recently developed and applied semi-synthetic strategies using chimeric glycosyltransferases.
PLANT GLYCOSYLATION
In plants as in other eukaryotes, the endoplasmic reticulum (ER) and the Golgi apparatus play the central role in protein glycosylation and contain the majority of glycan modifying enzymes (reviewed by, e.g., ). While the ER and its glycan processing repertoire are largely conserved between phyla (and kingdoms), morphology and function of the Golgi differ to some extent (; ). For example, a main function of the plant but not the mammalian Golgi is to provide large amounts of polysaccharides, a fundamental component of the cell wall (). Early N-glycosylation steps that take place in ER and cis-Golgi are virtually identical in higher eukaryotes, while further processing differs (recently reviewd by, e.g., ; ). This is mainly due to a drastically reduced repertoire of glycosylation enzymes in plants, where a small number of Golgi-located N-glycan processing enzymes gives rise to typically two different glycan structures (). By comparison, over 2000 different N-glycans have been described on mammalian proteins which arise from several 100 enzymes in the secretory pathway (; ; ). Notwithstanding these differences, the Golgi of higher eukaryotes shares a remarkably high degree of homology, especially with respect to organization, proteome, and N-glycosylation capabilities.
Plant proteins typically carry two major N-glycans, complex GnGnXF and paucimannosidic MMXF (, ; for glycan nomenclature see http://www.proglycan.com/upload/nomen_2007.pdf). These two glycans contain core α1,3-fucose and β1,2-xylose which are plant-specific glyco-epitopes. They are not produced by mammalian cells and up to 50% of humans have been shown to carry substantial amounts of antibodies directed against these epitopes in their blood (). The abundantly present paucimannosidic structures (MMXF, truncated glycans lacking terminal GlcNAc residues; ; ; ) are also a plant peculiarity, otherwise only found in insect cells (, ). In some cases plant proteins carry so-called Lewis A epitopes, terminally β1,3-galactosylated and α1,4-fucosylated structures (; ). The abundance of this epitope differs strongly between species (; ) and organs (), but seems low in Arabidopsis and Nicotiana (; , ; ). Noteworthy is also the absence of core α1,6-fucosylation in plants, a glycan residue present on the vast majority of proteins produced in mammalian cells. Removal of this residue from immunoglobulin G (IgG) glycans increases functional activities of antibodies due to a higher affinity to the antibody-dependent cell-mediated cytotoxicity (ADCC) inducing IgG receptor FcγRIIIa (; ).
The limited glycosylation capacity of plants has turned out to be an advantage for the generation of proteins that need homogeneous glycosylation. For example, IgG antibodies produced in plants carry usually 1–2 different glycan structures (mainly GnGnXF) while the same antibodies produced in Chinese hamster ovary (CHO) cells bear 5–7 structures (, ). For some applications, like testing of functional activities, and according to the demands from regulatory agencies, homogeneous glycosylation is required.
Plants display a remarkable tolerance toward the manipulation of their intrinsic glycan biosynthetic pathways. Elimination of complex glycans, knock-out of plant-specific xylosyl- and fucosyltransferases (XT and FT) or reduction/overproduction of the Lewis A epitope did not lead to any obvious phenotype in Arabidopsis thaliana under standard growth conditions (; , ). Also Lemna minor and N. benthamiana, one of the major plant-based protein production platforms, tolerate a variety of glycoengineering steps without obvious phenotypes or impact on development (; ; ). Only few cases of sensitive reactions to glycosylation changes have been described (). This general tolerance for glycoengineering was a prerequisite for humanizing the plant N-glycosylation pathway. A combination of knock-out/knock-down and knock-in approaches together with transient expression techniques has allowed the removal of potentially immunogenic residues, and the addition of new, human-type glyco-structures. Modular, semi-synthetic constructs assembled on multi-gene vectors enable the efficient manipulation of the glycosylation pathway. These glycoengineering strategies are addressed below.
PLANT GLYCOENGINEERING
Engineering of plant glycans toward human structures requires two main types of modification: (i) plant-specific reactions have to be eliminated and (ii) reactions taking place in humans but not in plants have to be introduced. Reducing the unwanted plant-specific modifications, i.e., β1,2-xylosylation and core α1,3-fucosylation, has initially been achieved by targeting the recombinant protein to the ER or co-overexpressing glycosylation enzymes competing for the same substrate (e.g., ; ; ; ; ; see below). However, as these approaches interfere with the execution of endogenous glycosylation processes and cause the attachment of oligomannosidic or incompletely processed and aberrant glycans they are only of limited use. RNAi approaches targeting the transcript of the unwanted glycosyltransferases or complete knock-outs by T-DNA insertion have proven more successful (; , ; ; ; ; ). Importantly, such plants produce human-type GnGn glycans, which serve as an acceptor substrate for further mammalian modifications and were thus important milestones in the engineering of the plant N-glycosylation pathway toward the production of human-type structures.
Consequently, work over the past decade on the controlled expression of mammalian glycosyltransferases has established plant-based systems that synthesize a series of defined human-type glycan structures (; recently reviewed by ). Recent studies demonstrate how even entire glycosylation-associated biosynthetic pathways can be introduced. Plants do not have the machinery to synthesize the sugar nucleotide precursor CMP-sialic acid (CMP-N-acetylneuraminic acid) necessary for sialylation. The simultaneous overexpression of six mammalian genes enabled the in planta generation of activated sialic acid, the transfer of the activated sugar nucleotide to the Golgi, the production of terminally galactosylated glycans and the transfer of sialic acid to these terminal galactoses (see Figures 1A–D; , ).
FIGURE 1
In planta sialylation of glycans thereby highlights some of the reasons why simple overexpression of a mammalian glycosyltransferase in plants has not always proven successful in generating human-type glycans: acceptor as well as donor substrates need to be present. For example, when expressed the human α2,6-sialyltransferase in Arabidopsis, activity of the enzyme could only be shown after applying donor and acceptor substrates in trans, as plants lack both. The achievements by , and others pointed out one more challenge, namely how delicate the glycosylation system is – coexpression of the human β1,4-galactosyltransferase (β1,4-GalT) had led to the production of galactosylated, but also of unusual, hybrid-type glycans. The latter was due to activity of the galactosyltransferase at a suboptimal stage of the glycosylation pathway and interference with the endogenous glycosylation reactions (described in detail below). Similar findings were reported upon overexpression of N-acetylglucosaminyltransferase (GnT) III (; ; ; ; detailed description see below). These examples show that the final glycosylation profile of a co-expression approach depends on various factors, including the availability of (i) acceptor glycan and (ii) donor substrate as well as (iii) the correct subcellular targeting of the recombinant glycosyltransferase in order to avoid interference with the endogenous glycosylation machinery.
TARGETING MECHANISM OF GLYCOSYLTRANSFERASES
The glycosylation reactions within the Golgi are carried out in a sequential, stepwise manner, and one reaction can be the prerequisite for another one – or inhibit it. Therefore, the ordered sequential arrangement of enzymatic activities, i.e., the correct subcellular localization of the involved enzymes is of utmost importance. This tight regulation has consequences for the expression and targeting of heterologous glycosylation enzymes, as they need to fit precisely into the existing pathway. Fine-tuning the subcellular localization of heterologously expressed glycosylation enzymes requires vast knowledge of the underlying targeting mechanisms.
All known Golgi-resident N-glycosyltransferases are type II transmembrane proteins (reviewed by, e.g., ). Their N-terminus is exposed to the cytoplasm, followed by a transmembrane domain, a stem, and the catalytic domain (see Figure 2). The cytoplasmic part, transmembrane domain, and stem are referred to as CTS region and are responsible for targeting the enzyme to the correct compartment (), and even sub-compartment. This was shown by different Golgi-localized glycosyltransferases not present within the same sub-compartment (; , ).
FIGURE 2
Research activities to elucidate the targeting mechanism(s) of type II transmembrane proteins revealed a basic conservation of processes between plants and mammals (). However, the question on how the fine-tuning of targeting works has not been answered definitively, but several factors have been identified. For example, the cytoplasmic tail influences ER export of the enzyme () and can change the final destination of a protein (). In mammalian cells it has been shown to relocate the catalytic domain from one to another Golgi subcompartment (). This might be due to formation of homo- or heterodimers of the enzymes or interaction with other proteins (). Also the length of the transmembrane domain might influence targeting (), as the membrane thickness of the Golgi changes from cis to trans. Finally, the composition of the lipid bilayer surrounding the enzyme might influence targeting, too (reviewed by ). This makes the CTS regions of glycosyltransferases key regulators for precise sub-Golgi targeting. Theoretically, CTS domains from any eukaryotic organism may serve as a suitable targeting domain. Recent genome sequencing projects provided an abundance of such sequences that may be used for targeted sub-Golgi localization (; ). Nevertheless, as the molecular mechanisms that lie behind this fine-tuning of targeting are not fully understood, it is not entirely predictable how CTS sequences actually perform when fused to the catalytic domain of another glycosyltransferase and expressed in a foreign cell. Thus, experimental testing is required. Also the prediction of the exact size of the individual glycosyltransferase-domains (cytosolic tail, transmembrane domain, stem, catalytic domain) is difficult and even though bioinformatics prediction technology has improved in recent years, different algorithms can lead to different results. Therefore, the reliability of the identification of the separate domains should be considered carefully.
CHIMERIC GLYCOSYLTRANSFERASES USED FOR IN PLANTA GLYCOENGINEERING
First steps to shift the plant N-glycosylation pattern from plant-specific N-glycans (i.e., GnGnXF, MMXF, etc.; see Figure 3A) toward the production of human-like structures were based on full-length mammalian glycosyltransferases. Expressing a human β1,4-GalT in tobacco plants () and tobacco BY2 cells () in fact resulted in galactosylated glycans, however, other oligosaccharides were produced as well. In BY2 cells, unusual and incompletely processed glycans lacking xylose and fucose (e.g., Man5A) were abundant (see Figure 3B), indicating interference of the heterologously expressed GalT with endogenous glycan processing enzymes like mannosidase II, GnTII and plant-specific β1,2-xylosyltransferase and α1,3-fucosyltransferase (XT and FT). In tobacco plants, xylosylated, and fucosylated GnGnXF remained the main oligosaccharide (see Figure 3B) and only minor amounts of galactosylated oligosaccharides like AAXF or GnAXF were found. This points to activity of the GalT at a later stage in tobacco plants, after completion of the endogenous glycosylation reactions. At this later stage, interference with the endogenous glycosylation reactions did not take place and therefore the amount of β1,2-xylose and α1,3-fucose – residues unwanted on proteins needed for human applications – remained basically unchanged. In order to transfer the down-regulation effect observed in BY2 cells to tobacco plants and reduce/eliminate the two plant-specific glycan residues (β1,2-xylose and α1,3-fucose), a chimeric version of the human GalT was constructed that contained the CTS region of the A. thaliana β1,2-xylosyltransferase (). The intention was to generate a chimeric enzyme that acts in the medial Golgi, simultaneously or prior to the endogenously present β1,2-xylosyltransferase and core α1,3-fucosyltransferase (XT and FT). As β1,4-galactosylated proteins are no longer substrates for XT and FT (; ), the expression in tobacco plants led to the intended, drastic decrease in plant-specific glycans. However, the early activity of the β1,4-GalT also led to the inhibition of other enzymes – like mannosidase II and GnTII – and thus to the generation of substantial amounts of unusual, incompletely processed glycans (oligomannosidic glycans, Man5A, etc.; see Figure 3C). Altogether, the glycan profile was similar to the profile of BY2 cells expressing the full-length human β1,4-GalT (). This indicates that the chimeric construct in tobacco plants and the full-length, human GalT in BY2 cells show activity at a comparable stage of the glycosylation pathway.
FIGURE 3
Targeting the human β1,4-GalT to an even earlier compartment (ER/cis-Golgi) by fusion with the CTS region of the A. thaliana GnTI further increased the amount of oligomannosidic glycans (; see Figure 3D). Galactosylated oligosaccharides were hardly found, indicating increased interference with the glycosylation machinery and the secretory pathway.
With the advent of XT/FT knock-down or knock-out lines (; ; ; ; ) a more elegant way to prevent plant-specific glycosylation had been established and the aim of co-expressing human GalT in plants shifted from “interfering with endogenous reactions” to “generating homogeneously galactosylated, human-type glycans.” This was achieved by a rationally designed construct targeting the GalT to a late Golgi compartment. Fusions of the catalytic GalT domain to the CTS region of α2,6-sialyltransferase, an enzyme acting in the final steps of the mammalian glycosylation pathway, indeed resulted in the generation of human-type, mono- and di-galactosylated glycans in XT/FT knock-down plants (; see Figure 3E). This was an important step for the in planta generation of proteins carrying fully human glycans.
This semi-synthetic approach was applied to GnTs to further explore the consequences of generating hybrid constructs carrying foreign CTS regions. One of the GnTs, β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIII), catalyzes the formation of so-called bisected glycans (; ), a modification frequently found on human proteins but not present in plants (). Importantly, bisected – as well as the previously mentioned β1,4-galactosylated glycans – cannot be modified with plant-specific xylose or fucose residues (). In order to produce such bisected glycans and thus prevent the addition of plant-specific glyco-epitopes, fully human GnTIII and a hybrid construct (the catalytic domain was fused to the CTS region of A. thaliana α-mannosidase II) were expressed in tobacco plants and BY-2 tobacco cells. The chimeric constructs led to a stronger decrease in plant-specific glyco-epitopes most probably due to targeting to an early/medial Golgi subcompartment (; ; ). However, targeting of GnTIII to an early compartment not only inhibited unwanted reactions but also led to the generation of non-standard, mainly hybrid-type glycans. Transiently expressing a series of GnTIII-constructs containing different CTS regions (A. thaliana Golgi mannosidase II, A. thaliana core α1,3-fucosyltransferase, A. thaliana β1,2-xyloslytransferase, and rat α2,6-sialyltransferase) in XT/FT knock-down N. benthamiana plants identified late targeting sequences as preferential for the production of naturally occurring, bisected N-glycans ().
In a similar approach, enzymes responsible for branched glycans (i.e., tri- and tetraantennary glycans; human α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase IVa (GnTIV) and human α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase V (GnTV)) were tested with different CTS regions (endogenous CTS region, rat α2,6-sialyltransferase, A. thaliana core α1,3-fucosyltransferase, A. thaliana β1,2-xyloslytransferase; ; , ,). The fusions with the medial Golgi-targeting CTS region of, e.g., FT allowed the generation of a high degree of branched glycans on co-expressed reporter proteins, whereas the endogenous CTS and the late Golgi CTS from α2,6-sialyltransferase did not lead to substantial amounts of branched glycans.
Collectively these results demonstrate that it is not sufficient to “simply” introduce a foreign glycosylation enzyme into a plant to obtain a desired glycan structure. Instead, the successful production of proteins with human-type N-glycosylation in plants harbors a large number of challenges and requires knowledge of glycosylation pathways, enzyme specificities and related topics, like subcellular protein transport. Semi-synthetic approaches serve as useful tools to approach these challenges.
FURTHER CHALLENGES
In recent years, a variety of expression hosts were glyco-engineered (recently reviewed by, e.g., ; ) and the first products with enhanced properties in animal studies have reached the clinic (). Plants, with their similar-to-human yet more simple N-glycosylation machinery and their amenability to glyco-engineering have been on the forefront of this development. Despite substantial achievements, the advantages of this system have been used only in a few in vivo studies (; ; , ; ). So far only one plant-produced product has reached the market, i.e., glucocerebrosidase to treat Gaucher’s disease. This protein carries terminal mannose residues, a glycosylation form that confers enhanced efficacies (). Another plant-produced glyco-optimized protein drug has recently been used to treat patients: ZMapp, an experimental mAb cocktail against Ebola virus was given to several individuals during the ongoing Ebola epidemic (critically reviewed by ). These antibodies had not yet gone through clinical studies but due to the dire predictions of Ebola virus infection and lack of other treatment options its application had been approved by regulatory authorities in several countries.
With the appearance of efficient, transient expression methods, the rapid, scalable and cost-effective production of high-value recombinant proteins became possible (). However, to realize the full potential of plant biotechnology, advanced, stably glyco-engineered plant strains in combination with semi-synthetic approaches will be needed. Versatile, modular expression vectors like MoClo () and GoldenBraid () allow efficient shuffling of domains and will certainly speed up the generation of constructs. Quick assembly of multi-gene vectors also simplifies the remodeling of glycosylation pathways as recently demonstrated (). Moreover, new technologies for genome editing, like CRISPR and TALENs () allow efficient elimination of genes and facilitate metabolic engineering and reprogramming of biosynthetic processes. These developments in combination with computer modeling and simulation approaches that predict protein–glycan interactions will accelerate the development of drugs with optimized and even new functions. In sum, the currently available gene expression systems and the new tools offered by synthetic biology create an ideal environment for establishing a plant-based biomanufacturing platform that can compete with or even surpass current industry standards.
Statements
Acknowledgments
We thank Jürgen Retzer for assistance with figure preparation. This work was supported by grants from the Austrian Research Promotion Agency (FFG) Laura Bassi Centres of Expertise (Number 822757, to Herta Steinkellner) and from the Austrian Science Fund (FWF; Grant Number L575-B13, to Herta Steinkellner).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Summary
Keywords
glycoengineering, plant, glycosyltransferase, CTS, sub-Golgi targeting
Citation
Loos A and Steinkellner H (2014) Plant glyco-biotechnology on the way to synthetic biology. Front. Plant Sci. 5:523. doi: 10.3389/fpls.2014.00523
Received
10 July 2014
Accepted
16 September 2014
Published
08 October 2014
Volume
5 - 2014
Edited by
Els J. M. Van Damme, Ghent University, Belgium
Reviewed by
Beronda L. Montgomery, Michigan State University, USA; Bjoern Usadel, RWTH Aachen University, Germany; Thomas De Meyer, Vlaams Instituut voor Biotechnologie, Belgium
Copyright
© 2014 Loos and Steinkellner.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Herta Steinkellner, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria e-mail: herta.steinkellner@boku.ac.at
This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science.
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