Mol. Cells, Vol. 25, No. 4, pp. 494-503
Molecules
and
Cells
Minireview
©KSMCB 2008
Glyco-engineering of Biotherapeutic Proteins in Plants
Kisung Ko*, Mi-Hyun Ahn, Mira Song, Young-Kug Choo, Hyun Soon Kim1, Kinarm Ko2, and
Hyouk Joung1
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea;
1
Plant Cell Biotechnology Lab., Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-335, Korea;
2
Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, Germany.
(Received September 28, 2007; Accepted January 24, 2008)
Plants have emerged as an attractive system for the production of therapeutic proteins, as plants are generally considered to have several advantages, including the lack of
animal pathogens, low cost and the large-scale production
of safe and biologically active valuable recombinant proteins, the ease of scale-up, efficient harvesting, and storage.
One of the principal advantages of plants over microorganism-based production systems is their posttranslational
modification ability, which is required for glycosylation,
phosphorylation, and methylation, including proteolytic
processing and non-enzymatic modifications for biological
activity and longevity, as well as pharmacokinetics (Gomord and Faye, 2004). Among these modifications, glycosylation has been shown to modulate vital biological
characteristics, including stability, immunogenicity/allogenicity, and ligand-receptor protein interactions (Faye et
al., 2005). Many valuable therapeutic glycoproteins expressed in plants are glycosylated. However, the biosynthesis of N-linked glycans in plants differs from that of
mammalian cells (Rayon et al., 1999). In plants, β(1,2)xylose and α(1,3)-fucose residues have been shown to be
linked to the core Man3GlcNAc2 of glycans, whereas
they are not detected on mammalian glycans, where sialic
acid residues exist instead (Gomord et al., 2005; Shin et
al., 2006). These plant-specific glycan residues that lack
of sialic acid may cause concerns regarding the safety and
efficacy of such plant-expressed glycoproteins (Bakker et
al., 2001a; 2001b). Thus, the commercial production of
biotherapeutic glycoproteins of human origin in plants is
currently hampered due to differences in the N-glycosylation patterns between plants and humans. Glycoengineering with the combined knock-out/knock-in approach of glycosylation-related enzyme genes has been
recognized for the avoidance of plant-specific glycan residues and the introduction of human glycosylation machinery. In this context, we discuss recent approaches and perspectives regarding glyco-engineering in plants.
* To whom correspondence should be addressed.
Tel: 82-63-850-6088; Fax: 82-63-857-8837
Email: [email protected]
Structure of plant N-linked glycans Plant glycoproteins
harbor two types of N-linked glycan: oligomannose and
biantennary complex type N-glycans (Ma et al., 2003) (Fig.
Many therapeutic glycoproteins have been successfully
generated in plants. Plants have advantages regarding
practical and economic concerns, and safety of protein
production over other existing systems. However, plants
are not ideal expression systems for the production of
biopharmaceutical proteins, due to the fact that they
are incapable of the authentic human N-glycosylation
process. The majority of therapeutic proteins are
glycoproteins which harbor N-glycans, which are often
essential for their stability, folding, and biological
activity. Thus, several glyco-engineering strategies have
emerged for the tailor-making of N-glycosylation in
plants, including glycoprotein subcellular targeting, the
inhibition of plant specific glycosyltranferases, or the
addition of human specific glycosyltransferases. This
article focuses on plant N-glycosylation structure,
glycosylation variation in plant cell, plant expression
system of glycoproteins, and impact of glycosylation on
immunological function. Furthermore, plant glycoengineering techniques currently being developed to
overcome the limitations of plant expression systems in
the production of therapeutic glycoproteins will be discussed in this review.
Keywords: Glycoprotein; Glycosylation; Molecular Biopharming; Monoclonal Antibody; Recombinant Protein;
Transgenic Plant.
Introduction
Kisung Ko et al.
495
Fig. 1. N-glycosylation process in the endoplasmic reticulum (ER) and Golgi apparatus of plant and human cells. The oligosaccharides
attached to specific asparagine residues are processed sequentially with the removal of glucoses and mannoses in ER and with the addition of species-specific glycan residues in Golgi apparatus (Cabanes-Macheteau et al., 1999; Helenius and Aebi, 2001; Wright and Morrison, 1997). Abbreviations: Glu I, glucosidase I; Glu II, glucosidase II; ER Man, ER mannosidase; GNT I, N-acetylglucosaminyltransferase I; GNT II, N-acetylglucosaminyltransferase II; β(1,2)XyT, β(1,2)xylosyltransferase α(1,3)FucT, α(1,3)fucosyltransferase;
β(1,3)GalT, β(1,3)galactosyltransferase; α(1,4)FucT, α(1,4)fucosyltransferase; β(1,4)GalT, β(1,4)galactosyltransferase.
1). The oligomannose type of N-glycans harbors the structure of mannose (Man)5-9 N-aceltylglucosamine (GlcNAc)2,
which exists in both plant and mammalian glycoproteins
(Kim et al., 2007). The biantennary complex type of Nglycans is acquired from the maturation processes of glycosylation in the endoplasmic reticulum (ER) and the Golgi
complex. In contrast with the oligomannose type, the structure of the biantennary complex type of N-glycans differs
between plants and mammals. The plant complex type of
N-glycan has β(1,2)-xylose and α(1,3)-fucose, and β(1,3)galactose and α(1,3)-fucose to the terminal N-GlcNAc,
whereas the mammalian N-glycan harbors α(1,6)-fucose on
the core glycan structure and β(1,4)-galactose attached with
sialic acids, respectively (Fig. 1). N-glycosylation begins
on the cytosolic surface of the ER membrane via the
biosynthesis of N-linked core oligosaccharides (Fig. 1). The
initial synthesis occurs via the addition of two GlcNAc and
five mannose sugars to the lipid carrier (Freeze and Aebi,
2005).
The oligosaccharide attached to the dolichnol lipid
carrier is flipped to the luminal side of the ER membrane,
and the three glucose molecules and four mannose molecules are added to the terminal mannose chains (Faye et al.,
2005). In ER, once transferring to specific asparagine
(Asn) residues of N-glycosylation sequences (Asn-X-Ser/
Thr, X = any amino acid except proline and aspartic acid) of
polypeptide chain, the glucose and mannose are trimmed
by ER glucosidase I and II and mannosidases. Subsequent
steps occur within the Golgi complex, in which consequent
processes of mannose removal are carried out by Golgi
mannosidases. The further maturation of glycosylation
involves the addition of GlcNAc, β(1,3)-galactose, and
α(1,3)-fucose by GlcNAc transferase I and II, galactosyltransferase, and fucosyltransferase, respectively.
N-glycosylation variation in plants The profiles of Nglycan structures vary depending on the protein structures,
plant subcellular compartments (Gomord et al., 2005),
plant developmental stage, and plant growth conditions
(Elbers et al., 2001) (Fig. 2). Many glycoproteins are cotranslated with N-glycosylation in the ER, transferred into
the Golgi complex, and secreted into subcellular compartments such as the vacuoles and extracellular space (Fig.
2A). In plants, the glycosylation maturation levels vary at
the locations of each leaf, and thus the matured glycoproteins harbor structurally diverse N-linked glycan moieties,
high-mannose, complex, and paucimannosidic types (Fig.
2B). The high mannose type of glycan structures are principally detected in glycoproteins retained in the ER (Fig.
2A). Complex-type glycans mature after the trimming of
the mannoses in the Golgi complex. Following the maturation of glycosylation in the ER and the Golgi complex, the
further glycosylation modification of complex-type Nglycans including β(1,3)Gal[α(1,4)Fuc], referred to as the
Lewis a (Lea)-containing N-glycans, can be processed during glycoprotein transport and/or subcellular compartment
496
Plant Glycosylation
A
B
Fig. 2. Profile heterogeneity of N-glycan structures in plant tissues and subcellular compartment levels (Gomord et al., 2004; Rudd et al.,
2004). A. The profile of N-glycan structures on glycoproteins in different subcellular compartments in plant cells (Pagny et al., 2000;
Sriraman et al., 2004). N-glycan profile varies in the plant subcellular compartments, in which glycoproteins are localized. In ER, the
glycoproteins harbor primarily the oligomannose type of N-glycans, whereas they principally harbor (Lea)-containing N-glycans in the
extracellular space. In the vacuoles, the N-glycan structures of the glycoproteins are abundantly paucimanosidic N-glycans. B. The relative percentage of variable N-glycan structures along with plant leaf position (Elbers et al., 2001). The total N-glycans of plant-derived
antibodies isolated from young leaves (30%) evidenced a high percentage of oligomannose type as compared to old leaves (10%).
localization (Fitchette-Laine et al., 1997). The Lea-containing N-glycans are abundantly detected on extracellular
glycoproteins, but are only rarely observed on the vacuolar
glycoproteins. It has been determined that vacuolar glycoproteins harbor paucimannosidic-type N-glycans containing
fucose and/or xylose residues, but lack terminal galactose
residues (Fig. 2A). The paucimannosidic-type N-glycans
are resultant from the post-Golgi removal of terminal Nacetyl-glucosamine residues or the degradation of larger
complex type glycans including Lea-containing N-glycans,
probably as the result of exoglycosidase activity during
transference to or following arrival in the vacuole (Fitchette
et al., 1999). As mentioned above, a variety of N-glycans
are modified by glycosyltransferases and glycosidases during glycoprotein transport from the ER to their final destination. The accessibility of modifying enzymes to oligosaccharide side-chains on glycoproteins differs from the
protein structure, consequently generating the diverse
glycoform profile, depending on the relevant proteins (Faye
et al., 1986b; Vitale and Chirspeels, 1984). For instance,
monoclonal antibody generated in plants harbor complex
type N-glycans which lack of xylose and/or fucose (Elbers
et al., 2001; Ko et al., 2005). This can be explained by the
steric hindrance of xylosyltransferase, fucosyltransferase,
and GlcNAcsidases, due to the buried nature of the Nglycan as in natural IgG (Deisenhofer, 1981). It has been
frequently observed that, during their passing through the
secretory pathway, certain oligosaccharide side-chains on
the glycoproteins are processed, while others are not (Faye
et al., 1986a). The leaf age also affects the glycoform profile
(Fig. 2). Young leaves harbor less oligomannose-type glycans than are generally observed in the older leaves. Senescence appears to affect the glycosylation profile of endogenous proteins. The relative quantity of N-glycans without
Kisung Ko et al.
terminal GlcNAc increases with the age of the leaf, thereby
indicating that gradual N-glycan processing occurs with the
maturation of the leaf. By way of contrast, plant growth
conditions including temperature and light conditions appear not to significantly influence N-glycosylation (Elbers
et al., 2001). Taken together, when plant is used as a glycoprotein production system, it is important to consider how
to increase homozygosity of glycosylation of therapeutic
proteins expressed in plant.
Expression of recombinant glycoproteins in plant Plants
provide many potential advantages for the large-scale production of valuable recombinant proteins, as compared to
other production systems, including those involving animals and microorganisms (Werner et al., 2007). Among
these advantages, the most notable selling points of plantbased expression systems are the low cost of mass production, rapid scale-up, simple distribution by seeds, ease of
handling, and the lack of human pathogenic contaminants.
The most valuable proteins expressed in plants are pharmaceutical proteins, including human biopharmaceuticals,
antibodies, and recombinant subunit vaccines (Ma et al.,
2003), which are largely glycoproteins. Since the first recombinant biopharmaceutical protein was expressed in
transgenic plants two decades ago, many other heterologous proteins including antibodies (Hiatt et al., 1989) and
vaccines (Mason et al., 1992) have been cloned and expressed. The antibodies bind specifically to foreign antigens and recruit immune cells to protect the human body
from pathogens and abnormal cells. They have become
highly recognizable biopharmaceutical proteins, and are
utilized as immunotherapeutic and diagnostic agents. The
global market for therapeutic antibodies was more than US
$11 billion in 2004 (AS Insights, 2005). The market is expected to increase to at least triple its current size by the
end of the decade. The variety of therapeutic antibodies
used thus far has been produced principally in hybridoma
cell technology or other mammalian cell culture systems.
The cell culturing systems are quite expensive, and can
only be conducted in a limited capacity. This limitation
may hamper the fulfillment of the dramatically increasing
future demand. Thus, economically feasible alternative
production systems, such as plant and yeast-based systems,
have been developed by many companies and universities.
Several different forms of recombinant antibodies have
been expressed in plants (Ko et al., 2005; Ma et al., 2003),
including full-size, large single-chain (Mayfield et al.,
2003), camelid heavy-chain (Jobling et al., 2003), Fab
fragments (Peeters et al., 2001), and single-chain antibodies
(scFv) (Conrad and Fiedler, 1998). Antibody-based immunotherapy primarily necessitates two antibody bioactivities,
specific binding activity to antigens and interactions between the Fc portion and the Fc receptors of immune cells,
which are described later. Depending on the required immunotherapeutic mechanisms, both or only the binding
497
activity is required. For example, full-size or large singlechain antibody are essential forms for the induction of antibody-dependent cell mediated cytotoxicity (ADCC), which
requires both bioactivities, whereas smaller recombinant
antibody derivatives without Fc regions, which evidence
antigen-binding activity only, including Fab fragments or
scFvs, are sufficient for the achievement of therapeutic
effectiveness. The smaller size of the antibodies is preferable to anti-tumor immunotherapy for the killing of large
volumes of tumors. Full-size antibodies are applied for
immunotherapy, which particularly requires immune cell
recruiting relevant to the interaction between the Fc antibody and Fc receptors on the immune cells.
Several full-size antibodies expressed in plants have
been determined to be effective in the prevention of infectious diseases, including Streptococcus mutans and herpes
simplex virus, by topical application (Ma et al., 1998;
Zeitlin et al., 1998). Furthermore, we have reported that
recombinant full-size monoclonal antibodies were expressed successfully in plants, and evidenced an efficacy
similar to that of the mammalian-derived counterparts in
passive immunization (Brodzik et al., 2006; Ko et al.,
2003; 2005). Unlike topically applicable antibodies, these
recent reports indicated that plants can be utilized in the production of full-size antibodies for systemically applicable
immunotherapy against infectious diseases and cancers in
in vivo animal models. In order to generate such potent
immunotherapeutics, the expression system should allow
for an economically satisfactory production level and
should allow the apparatus to properly fold and assemble
proteins with primarily disulfide bond formation and authentic glycosylation. Plants are eukaryotes with posttranslational abilities similar to those of mammalian cells.
The therapeutic proteins can be readily targeted and localized into the subcellular compartments of plant cells: the
intercellular space, chloroplast, and ER. When secreted into
the intercellular space, the glycoproteins are attached with
fully matured glycosylation, as described above. By way of
contrast, when localized in the ER, the proteins are attached with a high degree of mannose glycosylation. In
general, ER localization augments overall production levels
as the result of a high level of accumulation in the ER environment, which is characterized by high pH, low degradation, and stabilization. It is generally recognized that
chloroplast localization or chloroplast transformation can
be employed as a high-capacity production system, with a
yield of 7−50% of total cellular proteins. So far, the most
promising approach for protein expression in transgenic
plants, except for chloroplast transformation, is to target
and localize proteins into the ER.
N-glycan influencing immunogenicity and biological
activity of glycoproteins Despite the economically efficient
production of biologically active recombinant therapeutic
glycoproteins using a plant system, their potential immuno-
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Plant Glycosylation
Fig. 3. Antibody Fc glycosylation can be tailored to therapeutic applications. Complex N-linked nonfucosylated type glycans improve
ADCC and complement-dependent cytotoxicity (CDC) (Okazaki et al., 2004). Sialic acid plays an important role in the prolongation of
half-life (Perlman et al., 2003). Additional sialic acid residues increase in size and charge, rendering the glycoprotein metabolically more
stable (Kanwar, 1984; Wide, 1986).
genicity may raise concerns regarding the systemic
applications of plant-derived therapeutics in humans. The
plant-specific N-glycans, α(1,3)-fucose and β(1,2)-xylose
residues linked to the core Man3GlcNAc2 on glycoproteins
in plants, differ from their natural counterparts (Fig. 1).
These glycans induce the generation of IgE and IgG
antibodies specific for α(1,3)-fucose and β(1,2)-xylose
glycoepitopes (Gomord et al., 2005; van Ree et al., 2000).
However, no glycan-specific antibody was generated in
BALB/c mice when exposed to a plant-derived antibody
harboring the α(1,3)-fucose and β(1, 2)-xylose (Chargelegue
et al., 2000), whereas rats, rabbits, and goats generated
anti-glycan antibodies (Bardor et al., 2003; Faye et al., 1993;
Kuroska et al., 1991). Indeed, immunoreactivity specific for
these glycan epitopes appears to occur in a species-dependent manner (Gomord et al., 2005). The potential
allergenic or immunogenic responses against plant glycan
epitopes are considered to be a weak point of the plant
production system, although humans are constantly exposed
to plant glycoproteins in the diet. Therefore, in order to
receive potential benefits from a plant biopharmaceutical
production system, it may be necessary to determine whether
humans evidence immunogenicity of glycan epitopes prior to
the administration of plant-derived biopharmaceuticals.
Furthermore, the initial removal of such plant specific
glycoepitopes resulting in humanized N-glycans with nonimmungenicity should clearly be considered. The majority of
therapeutic proteins are glycoproteins which harbor a variety
of glycan structures, which influence their folding, stability,
and functional activities. It has been previously shown that
recombinant monoclonal antibodies with oligosaccharides
harboring a bisecting N-acetylglucosamine evidence
efficacy superior to that of antibody-dependent cell cyto-
toxicity (ADCC) (Umana et al., 1999). In addition, the
removal of fucose from the primary N-acetylglucosamine
residue on antibody induced a 40- to 50-fold increase in the
efficacy of FcγRIII-mediated ADCC (Shields et al., 2002).
The improvement in ADCC was correlated with the
increased affinity of the IgG-Fc fragment to the FcγRIIIa
receptor (Okazaki et al., 2004). The alteration of glycan
structures on therapeutic monoclonal antibodies generated in
plant systems can potentially alter their activity. Therefore,
when any therapeutic glycoprotein is expressed in a plant,
altered glycosylation should be characterized, even the
favorable modifications of the intended glycoform, which
are crucial to the increase in their efficacy on biological
characteristics. For instance, the antibodies can be tuned up
to improve the efficacy of antibody-based immunotherapy
via the modification of glycosylation (Fig. 3).
Plant glycoengineering to modify N-glycosylation It is
currently possible to tailor the plant glycosylation machinery toward the ideal design of glycosylation patterns on
glycoproteins (Gomord et al., 2005). The glycosylation
processes are not identical in plants and animals, and consequently their glycosylated proteins differ in terms of
structure, biological activity, and immunogenicity (Bakker
et al., 2001b). The plant-specific oligosaccharide structures,
namely α(1,2)-xylose and β(1,3)-fucose residues linked to
the core Man3GlcNAc2 attached to glycoproteins generated in transgenic plants differ from their natural counterparts. Despite the economically efficient generation of biologically active recombinant therapeutic glycoproteins using plant systems, their potential immunogenicity and allergenicity may have implications with regard to their use as
plant-specific glycoepitopes for systemic applications of
Kisung Ko et al.
plant-derived therapeutics in humans. Recent approaches to
eliminate the plant specific glyco-eptiopes are to knock out
the expression of the α(1,3)-fucosyltransferase and β(1,2)xylosyltransferase with knockout plant lines or antisense
technology (Koprivova et al., 2004; Schahs et al., 2007).
However, so far the approaches were able to only decrease
the enzyme activities in N-glycan biosynthesis in plants
(Wenderoth and von Schaewen, 2000).
The simplest method to avoid such plant glycoepitopeinduced immunogenicity is aglycosylation via the removal of
the glycosylation sites of the gene sequences of glycoproteins (Fig. 4). Aglycosylated human IgG has no effect on the
direct interaction between the Fc receptors and Fc of human
IgG, which is crucial to the activity of biological effector
molecules. However, aglycosylation results in subtle conformational changes, which can exert a profound effect on
local sites (Walker et al., 1989). In human IgG1, the monoclonal antibody with lack of glycosylation lost its ADCC
activity, but not a significant level of CDC activity and serum half-life and bio-distribution compared to the native
mAb in mice (Dorai et al., 1991). Thus, this limiting approach may prove useful to certain glycoproteins only, as it
requires antigen binding activity of the variable region of
Fab portions and also requires CDC-driven activity (Ko and
Koprowski, 2005). As plants are currently being recognized
as an important system for the production of recombinant
therapeutic glycoproteins, it is crucial to optimize ideal glycoforms on the proteins for therapeutic purposes. Indeed,
recent studies presented the first detailed analysis of the glycosylation of a functional mammalian glycoprotein expressed in transgenic plants (Ko et al., 2003; 2005; Tekoah et
al., 2004). The structures of the N-linked glycans attached to
the heavy chain of the monoclonal antibodies generated in
transgenic tobacco plants were identified and compared to
those detected in the parental monoclonal antibodies (Bardor
et al., 2006). The strategy employed to attenuate the risk of
immunogenicity against plant-specific glycan residues involved the localization of the glycoproteins in the plant endoplasmic reticulum (ER), yielding high-mannose type Nglycans lacking α(1,2)-xylose and β(1,3)-fucose residues
(Fig. 4). The fusion of the ER retrieval motif, KDEL (LysAsp-Glu-Leu), to proteins allows for their efficient retention in the ER (Schouten et al., 1996). In fact, the signal
peptides and the KDEL sequences were antibody targeted
and retained in ER, thereby yielding oligomannose (Man5-9
GlcNAc2) type N-glycans in plants, coupled with an improvement in the level of protein accumulation (Conrad
and Fielder, 1998; Ko et al., 2003; Sharp and Doran, 2001;
Wright et al., 2001) (Fig. 4). Monoclonal antibodies fused
to the KDEL sequences at the heavy chains expressed in
plants principally harbored the high- mannose type glycans
(Ko et al., 2003). However, 90% of monoclonal antibodies
harbored high-mannose type glycans, whereas 10% of the
monoclonal antibodies were shown to exhibit complex Nglycans, thereby indicating that the KDEL-fused mono-
499
clonal antibody was secreted partially (Ko et al., 2003;
Petruccelli et al., 2006). By way of contrast, the fusion of
KDEL to both heavy and light chains exclusively harbored
high-mannose type N-glycans (Bardor et al., 2006; Sriraman
et al., 2004). Antibodies with high-mannose type N-glycans
evidenced shorter half-lives as compared to mammalian
antibodies with complex N-glycans (Ko et al., 2003). The
shorter half-life of the antibody provides a certain advantage for antibody-vaccine rabies prophylaxis, where the
longevity of antibody can present interference between
passive and active immunity (Ko et al., 2003). Its rapid in
vivo clearance may reduce T-cell activation, in which the
IgG-Fc mediated effector function is crucial for anti-tumor
activity (Gomord et al., 2005), resulting in negative immunotherapeutic effects. One approach to the avoidance of
such clearance involves the addition of the terminal sialic
acid residues to plant N-glycan via the expression of a
mammalian sialyltransferase in plants (Wee et al., 1998), as
sialylated glycoproteins can be avoided from hepatic
asialoglycoprotein receptor-dependent clearing processes
(Kelm and Schauer, 1997) (Fig. 4). The sialylation of endogenous oligosaccharides cannot occur in plants, because
the precursor, CMP-N-acetylneuraminic acid (CMPNeu5Ac), does not exist in the Golgi complexes of plants
(Séveno et al., 2004). Thus, the plant-made glycoproteins
are synthesized with oligosaccharides which do not harbor
terminal sialic acid residues (Séveno et al., 2004). They detected no sialic acids in plants, and proposed that even if they
exist, such sialic acids are not associated with N-glycans
(Zeleny et al., 2006a). However, Shah et al. (2003) verified
the presence of sialylated glycosylation in A. thaliana
suspension-cultured cells. It remains unclear as to whether
sialylation machinery exists in plants. Nevertheless, several
research trials are currently underway to synthesize Neu5Ac
in plants. One of these trials involves the expression of
Neu5Ac lyase, which combines D-ManNAc and pyruvate,
thereby yielding Neu5Ac, and the other approach involves
the expression of Neu5Ac synthase, which condenses DManNAc to phopshoenol pyruvate (PEP), thus generating
Neu5Ac in plants. This suggests that Neu5Ac can be synthesized in vitro by both plant-expressed enzymes (Paccalet
et al., 2007). However, what still requires consideration is
how the endogenous D-GlcNac or UDP-GlcNAc are
converted into D-ManNAc as a substrate for the synthesis
of Neu5Ac (Paccalet et al., 2007). Thus far, CMP-Neu5Ac
synthase (Misaki et al., 2006), sialic acid transporter
(Misaki et al., 2006), and sialyltransferase (Wee et al.,
1998) have been expressed as components of the biosynthetic pathway of sialylated glycans in plants. These previous studies show that the establishment of the sialylation
machinery in plants is very possible via the expression of
multiple genes for the human glycosylation machinery.
Indeed, it appears that sialylation on galactose is not always
essential, as the Fc portion of monoclonal antibody with
sialaylation on galactose can abrogate the interaction with
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Plant Glycosylation
Fig. 4. Glyco-engineering strategies for the modulation of N-glycosylation reducing the immunogenicity and humanizing complex Nglycans in plants. Aglycosylation via the elimination of the N-glycosylation site of the amino acid sequences of antibody and high mannose-type glycosylation via the fusion of KDEL and the ER retention motif to antibody can prevent immunogenic plant specific complex
N-glycans (Ko et al., 2003). Knock-out strategies with α(1,3)FucT and β(1,2)XylT can remove plant specific glycan residues,
β(1,2)xlyose and α(1,3)fucose avoiding immunogenicity/allergenicity (Gomord et al., 2005; Koprivova et al., 2004). Knock-in strategies
using human specific glycosyltransferases have emerged for the humanization of N-glycosylation in plants (Bakker et al., 2001a). Mammalian Golgi-specific α(2,6)sialyltransferase has been utilized successfully for the targeting of its expression in the plant Golgi apparatus
(Wee et al., 1998). However, engineering the addition of sialic acid on plant N-glycans is challenging, as many sialic acid precursors and
non-existing glycosyltransferases should be expressed for the establishment of sialylation machinery in plants (Zeleny et al., 2006a;
2006b). Abbreviations: β(1,2)XyT, β(1,2)xylosyltransferase α(1,3)FucT, α(1,3)fucosyltransferase; β(1,3)GalT, (1,3)galactosyltransferase;
α(1,4)FucT, α(1,4)fucosyltransferase; β(1,4)GalT, β(1,4)galactosyltransferase.
the Fc receptor (Kaneko et al., 2003). This attenuated interaction between them results in decreased levels of ADCC,
which is indispensable to monoclonal antibody-derived
anti-tumor activity (Koprowski and Croce, 1980). In addition, monoclonal antibody lacking galactose augments the
interaction between the Fc and the Fc receptors, thus resulting in the killing of cells (Li et al., 2006). Furthermore,
monoclonal antibody without fucosylation did not evidence
abrogated anti-tumor activity (Ko et al., 2005) and actually
enhanced ADCC by a factor of approximately 50 to 100 as
compared to its parental mAb (Niwa et al., 2004;
Shinkawa et al., 2003). Thus, although the regulatory con-
siderations regarding non-humanized glycosylation on
monoclonal antibody remain, the humanization of glycosylation is not always a promising approach to monoclonal
antibody. When glycosylation on mAb is designed, the types
of therapeutic effects observed in conjunction with a given
mAb should be considered.
Conclusion
The advantages of plant systems in the production of therapeutic proteins have been well established, and already
Kisung Ko et al.
offer an opportunity to meet the enormously increasing
demand for therapeutic proteins. In particular, the time
scale for the production of transgenic plants is quite similar
to that of other production systems, including mammalian
systems, but plants have an advantage due to the flexibility
of their production scales. Many of the leading pharmaceutical companies have begun mAb production research, and
it is speculated that the mAb market should reach US$34
billion by the end of the decade. Thus, transgenic plants are
generally considered to represent a promising alternative
production system for particular mAb. Glycosylation engineering in plants is expected to become a subject of increasing interest. It is essential to investigate plant physiological alteration by humanization of glycosylation processing in plant cells. Removal of plant specific fucosyltransferase and xylosyltransferase genes with addition of
human specific glycosyltransferase genes might affect plant
cell wall formation and cause aberrant defense responses
against phytopathogens. Cell walls have an architectural
role in plant cell division and growth, and xylose and
fucose are components of the plant cell wall polysaccharides, which are important factors in host-pathogen
interactions. Thus, further consideration on any prospecting
plant physiology concerns from plant glyco-engineering
that we have not yet revealed is worth to acquire its full
appreciation.
Acknowledgments We are grateful to Dr. Hilary Koprowski and
Dr. Maxim Golovkin for discussions and comments. This study
was supported by a grant from the BioGreen 21 R&D Project of
Rural Development Administration, Korea (2007041034026).
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