Modification of plant N-glycans processing: The future of producing therapeutic protein by...
Transcript of Modification of plant N-glycans processing: The future of producing therapeutic protein by...
Modi¢cation of Plant N-glycansProcessing:The Future of Producing
Therapeutic Protein byTransgenic Plants
Min Chen, Xianwei Liu, Zhankun Wang, Jing Song, Qingsheng Qi,Peng George Wang
The State Key Laboratory of Microbial Technology, School of Life Science, Shandong University,
Jinan, Shandong 250100, P.R. China
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/med.20022
!
Abstract: Transgenic plants are regarded as one of the most promising systems for the production
of human therapeutic proteins. The number of therapeutic proteins successfully produced in plants
is steadily arising. However, the glycoproteins normally produced from plants are not the same as
native therapeutic proteins produced from mammals or humans. In addition to in vitro enzymatic
modeling glycoproteins, there are two gene manipulation strategies to humanize plant N-glycans
connected to the glycoproteins. One is retaining the recombinant glycoproteins in endoplasmic
reticulum (ER), the site where few specific modifications of N-glycans occurs. The other is
inhibiting the plant endogenous Golgi glycosyltransferase and/or adding new glycosyltransferase
from mammalians. In this review, the biosynthesis of N-glycans in plants, the modification of the
plant N-glycans processing will be discussed. � 2004Wiley Periodicals, Inc. Med Res Rev, 25, No. 3, 343–
360, 2005
Key words: therapeutic protein; transgenic plants; glycosylation
1 . I N T R O D U C T I O N
The number of protein-based therapeutics entering the preclinical and clinical evaluation has shown
robust growth and been expected to increase in the years to come. Transgenic plants are regarded as
one of the most promising systems for the production of human therapeutic proteins. In the past
decades, more than 100 recombinant proteins have been produced in different species. At the same
time, transgenic plant derived proteins have already entered into the market.1–3
Correspondence to: Qingsheng Qi and Peng GeorgeWang,The State Key Laboratory of Microbial Technology, School of Life
Science,ShandongUniversity, Jinan,Shandong 250100,P.R.China.E-mail: [email protected]@sdu.edu.cn
Medicinal Research Reviews, Vol. 25, No. 3, 343^360, 2005
� 2004 Wiley Periodicals, Inc.
Most therapeutic proteins are glycoproteins,N-glycosylation of the proteins is often essential for
their stability, folding, and biological activity. Transgenic plants have the inherent capacity to
produce glycoproteins similar to those ofmammalian cells.4 However,N-glycoproteins derived from
plants are different from those of mammalians. In this review, the modification of transgenic plant
glycoproteins towards the humanized proteins will be discussed.
A. Benefits of Transgenic Plants and Commercial Application
Compared to other expression systems, transgenic plants have many advantages in producing
therapeutic proteins4,5 (see Table I).
The most important feature of transgenic plants is that they can produce recombinant proteins
with very low investment and operating costs and have obvious economic advantages than the
traditional fermentation methods. For transgenic plants, the scales of production only depend on
usingmore or less land as required, while fermentation systems and transgenic animals are limited in
this respect.
Plants also havemany advantages in downstream processing. Several methods have been used to
reduce the cost of the therapeutic proteins produced by plant. One way is directing the protein
synthesis to seed endosperm,6 from where the recombinant proteins can be easily extracted. For
example, avidin extracted from transgenic maize seed endosperm was estimated to be 10-fold
Table I. Features of Different Expression Systems for Recombinant Protein
Production
TP, totalprotein;TSP, total soluteprotein.
344 * CHEN ET AL.
cheaper than that purified from eggs, and is now marketed by Sigma-Aldrich.1 It is estimated that
recombinant proteins can be produced in plants at 2–10% of the cost of microbial fermentation
systems and at 0.1% of the cost of mammalian cell cultures.7,8 Another way is depositing
recombinant proteins in specific organs of the plant.2–4 In cereals, proteins in seeds can remain stable
at room temperature for months or years without loss of biological activity. Meanwhile, the
purification requirement can be eliminated when the plant tissue containing the recombinant protein
is used as food.9,10 As a recent example, one single-chain Fv fragment (scFv) antibody in pea seeds
was reported by using the seed specific USP promoter11 and the high stability of a recombinant scFv
antibody was also confirmed in tobacco seeds over a period of 1.5 years.12
In addition, plants are considered to bemore secure than bothmicrobes and animals because they
usually do not contain human or animal pathogens, oncogenic DNA sequence, or endotoxins.
In brief, the advantages of using transgenic plants to produce therapeutic proteins are: (1)
significantly lower production costs than other expression systems; (2) plants do not contain any
known human pathogens that could contaminate the final product; (3) higher plants can synthesize
proteins with correct folding, glycosylation, and activity; and (4) plant cells can direct proteins to
environments that reduce degradation and, therefore, increase stability.13
The first functional antibodies produced in transgenic plants were reported in 1989.14 Since then,
many therapeutically valuable proteins, such as food allergens15 and biopharmaceutical proteins,
have been expressed in transgenic plants.7,16–19 Proteins currently being produced in plants can be
categorized into four broad areas: (1) vaccines, (2) monoclonal antibodies (MAbs), (3) biopharma-
ceutical proteins, and (4) industrial proteins (e.g., enzymes) (Table II).
The first reported clinical trial of plant-produced antibody was carried out by Planet
Biotechnology, Inc. (Mountain View, CA). The drug CaroRxTM is a recombinant antibody against
Streptococcus mutans, a causal agent of human tooth decay.7 At present, there are about 100 small
companies producing therapeutic proteins by transgenic plants. Each company focuses on a
few products.8 Several proteins have been put into the market now, such as avidin, b-glucuronidase,and hirudin (see Table III). Besides the proteins described above, there are at least nine products
thought to be close to reach commercial market in the next 5 years, such as aprotinin, lipase,
lysozyme, and so on.15
B. Disadvantage and The Resolvent
Although there aremany successful examples, several limitations restrict the commercial application
of recombinant proteins produced by plants.
First, the expression levels of recombinant proteins are often very low. For example, the amount
of human protein C produced in plant is lower than 0.01% of total soluble protein41 while that of
Guy’s 13 (secretory IgA) is only 0.003% of fresh weight leaves.33,34
This problem can be settled by selection of stronger promoters, better plant host, or other
transformation systems. For example, a peroxidase gene promoter isolated from sweet potato
(Ipomoea batatas) was used to drive the gusA reporter gene in transgenic tobacco. This promoter
produced 30 times more b-glucuronidase (GUS) activity than the cauliflower mosaic virus (CaMV)
35S promoter did.46,47 An alternative to nuclear gene transfer is the transformation of organelles,48
such as the chloroplast transformation methodology. Expression of recombinant proteins in the
chloroplast genome has some advantages compared with nuclear gene transfer (e.g., high levels of
expression and containment). Amultimeric vaccine gene49 and a rAb50 have been recently expressed
in chloroplasts. Specific targeting signals are also used to increase the recombinant protein
accumulation. For example, although the ability of some scFv antibodies to accumulate in the cytosol
seems to be dependent on their intrinsic properties, most scFv antibodies accumulate to higher levels
when expression is targeted to the apoplast or the endoplasmic reticulum (ER), rather than to the
cytosol.51,52
MODIFICATION OF PLANT N-GLYCANS * 345
Table II. Proteins Produced in Transgenic Plants
FW, freshweight;TSP, totalsoluteprotein.
346 * CHEN ET AL.
Second, there are some difficulties in down-stream processing, which will lead to inconsistent
product quality. In earlier time, oleosin fusion system was used. The fusion protein can be recovered
from oil bodies using a simple extraction procedure and the recombinant protein separated from its
fusion partner by endoprotease digestion. Now, affinity tags are used to facilitate the recovery of
proteins as long as the tag can be removed after purification to restore the native structure of the
protein. Recently the expression of His-tagged GUS-fusion proteins in tobacco chloroplasts, the
extraction of His-tagged proteins by foam fractionation, and the release of recombinant proteins
using a modified intein expression system have been reported.47,53–55
In 2003, a recombinant murine monoclonal antibody specific for the hepatitis B surface antigen,
expressed in transformed transgenic tobacco and protein A streamline chromatography was
successfully used in the purification process yielding a recovery of about 60% and a plantibody SDS–
PAGE purity of over 90%. This work verified the potentiality of plants to replace animals or
bioreactors for large-scale production of monoclonal antibody.56
Third, there are non-authentic glycan structures presented on recombinant human proteins.57
Most therapeutic proteins are glycoproteins, which include O-glycoprotein and N-glycoprotein. N-
Table III. Products on the Market
MODIFICATION OF PLANT N-GLYCANS * 347
glycosylation of the protein is often essential for reasons including stability, solubility, folding, and
biological activity. Glycosylation of the protein in plants and mammalian systems differ in fine
details. These differences can induce undesirable immune response inmammals or reduce activity of
recombinant proteins. Recently, it is found that plants are able to introduce N-glycans on complex
recombinant mammalian proteins in a sufficient way for production of biologically activemolecules.
C. N-glycosylation in Plants
Amajor limitation shared with other heterologous expression systems like bacteria, yeast, and insect
cells is their different glycosylation profiles compared with that of mammals. In bacteria expression
systems, recombinant proteins are not glycosylated. In yeast cells, high-mannose structures are added
to proteins. Insect cells put on shorter mannose structure than yeast. In contrast to bacteria and yeasts,
plants are able to produce proteins with complex N-linked glycan having a core substituted by two
N-acetylglucosamine (GlcNAc) residues as observed in mammals.4 However, the N-glycan in plant
lacks galactose and terminal sialic acids, and with plant specific a (1,3)-fucose and b (1,6)-xylose
residues (Fig. 1).
N-glycans in plants are usually classified in three groups (Fig. 2). One is high-mannose-type
N-glycan (Man5–9GlcNAc2), the other is complex-type N-glycan, and the last one is paucimanno-
sidic-typeN-glycans having only one a (1,3)-fucose or one b (1,2)-xylose residues linked to the core
Man2-3GlcNAc2 without GlcNAc residues or larger antennae b (1,2)-linked to the a (1,6)- or the a(1,3)-mannose constitutive of the core. Paucimannosidic-type N-glycans are found similar with
N-linked glycans in insect.58,59
Complex-glycans are further divided into two sub-groups. One is GlcNAc1–2Man3XylFuc0–1GlcNAc2, the other is Man3XylFucGlcNAc2 with antennae constituted of Galb1-3 (Fuca1–4)GlcNAc sequences,which are namedLewisa. These structures, known asLewisa (Lea) antigen,which
are usually found on cell surface glycoconjugates in mammals and involved in cell recognition and
cell-to-cell communication processes, had been characterized60,61 (Fig. 2). The presence of Lewisa is
still ambiguous in Cruciferae family, because the Lea motif was characterized in papaya fruit62,63 but
not detected in Arabidopsis plant64 and cultured BY2 tobacco cells.65 b (1,2)-Xyl and a (1,3)-Fuc
epitopes are known to be highly immunogenic and might play a role in allergenicity.66 So the
modification of glycosylation activities in transgenic plants is important for medical and bio-
technological purposes.
D. Biosynthesis of N-linked Glycans in Plants
In order to modify the glycoproteins, it is necessary to know the biosynthesis of N-linked glycans in
transgenic plant.
As in other eukaryotes, the N-glycosylation of plant proteins begins in endoplasmic reticulum
(ER) with the transfer of the oligosaccharide precursor Glc3Man9GlcNAc2 from a dolichol lipid
carrier to specific Asn residues on the nascent polypeptide chain.58 The precursor then will be
subsequently modified by glycosidase and glycosyltransferase located in ER and Golgi apparatus
(Fig. 3).
The process in endoplasmic reticulum (ER) is conserved amongst almost all species and
restricted to oligomannose (Man5–9GlcNAc2) type N-glycans. First the terminal glucose units in the
oligosacchride precursor Glc3Man9GlcNAc2 are eliminated by terminal glucosidase I and II in
ER.67,68 Then the mannose residues are removed. ER-mannosidase, which specifically removes a
single mannose residue to yield Man8GlcNAc2 in mammals, has not been detected in plants so far.
AlthoughNavazzio et al. suggested that a specificmannosidase could be involved in the processing of
plant N-linked glycan within ER.69
Further modification of glycans in Golgi is highly diverse between plant and animal. In plant,
the first modification of N-glycan is that one to four a-(1,2)-mannose residues are removed by
348 * CHEN ET AL.
a-mannosidase I (a-Man I) and thus produces Man5GlcNAc2.70,71 After that the first N-
acetylglucosamine residue is added to the a (1,3) mannose branch of Man5GlcNAc2 by N-
acetylglucosamingltransferase I (GNT I) and then produces GlcNAcMan5GlcNAc2.72,73 After
two additional mannose units are removed from GlcNAcMan5GlcNAc2 by the a-mannosidase II
Figure 2. Structure of plantN-glycans. A:High-mannose-typeN-glycans; (B,C) complexN-glycans; and (D,E) paucimanosidic-
typeN-glycans. [Color figure canbe viewed inthe online issue, which is availableat www.interscience.wiley.com.]
Figure 1. Glycosylation in different expressingsystems. [Color figure canbe viewed in the online issue, which is available at www.interscience.wiley.com.]
MODIFICATION OF PLANT N-GLYCANS * 349
(a-Man II),74 another outer N-acetylglucosamine residue will then be transferred by N-
acetylglucosaminyltransferase II (GNT II) to a (1,6)-mannose branch.73,75 In this stage, a (1,3)-
fucose and b (1,2)-xylosemay are added toMan3GlcNAc2. But the sequences of these two events are
not completely understood. Royon et al. demonstrated that these two events were independent,
because they mostly occurred in the medial and the trans Golgi cisternae, respectively.
Complex-typeN-glycans can be furthermodified byb (1,3)-galactosyltransferase [b (1,3)-GalT]and a (1,4)-fucosyltransferase [a (1,4)-FucT] on terminal N-acetylglucosamine residues producing
mono- and bi-antennary plant complex N-glycans. This kind of complex-type N-glycans is often
found as extracellular glycoproteins. They may be partially degraded by exoglycosidases in the
extracellular compartment.73
After being modified in ER and Golgi apparatus, the complex-type N-glycans can be further
modified either during its transportation to, or in the compartment of, its final location.
Post-Golgi maturation of intermediate complex-type N-glycans occurs mostly in the vacuole
and rapidly leads to the formation of paucimannosidic oligosaccharides.59 In vacuolar, the
terminal glucosamine residues attached to complex-glycans, especially complex-type with Lea will
be removed by N-acetylglucoseaminidase.76 The complex glycans may also be degraded by
exoglycosidase.
E. Modification of plant N-glycans
As we discussed above, general eukaryotic protein synthesis pathway seems to be very well
conserved between plants and animals, so plants can fold and assemble full-sized antibodies,77,78
secretory IgAs,34,79 and so on. However, post-translational modifications are not identical in plants
and animals. There are also some structural differences, the most apparent obstacle is the presence of
the plant-specific residuesa (1,3)-fucose andb (1,2)-xylose.80 The presence of plant specific residues
Figure 3. ThebiosynthesisprocessingofplantN-glycans in ERandGolgi. [Color figure canbe viewed intheonline issue, which isavailableat www.interscience.wiley.com.]
350 * CHEN ET AL.
a (1,3)-fucose and b (1,2)-xylose glyco-epitopes on N-linked glycans to therapeutic glycoproteins
has raised the questions of their immugenicity in human therapy.
For example, Muriel Bardor81 extracted N-glycans in pea, rice, and maize and reinvestigated
their immunogenicity in rodents. They demonstrated that about 50% of nonallergic blood donors
contain in their sera Abs specific for core xylose, whereas 25% have Abs against core (1,3)-fucose.
The presence of such Abs might be some limitations to the use of plant-derived biopharmaceutical
glycoproteins. However, studies of the immune response of mice to a systemically administered
recombinant IgG (Guy’s 13) isolated from plants showed that, although there were some differences
in the glycan groups present on the recombinant antibody, neither the antibody nor the glycans were
immunogenic in mice.82 In this case, differences in protein glycosylation in plants and mammals did
not provoke an immune response.83
Although there are several successful examples in produce glycoprotein by transgenic plant,
such as mAbGuy’s 13 in tobacco78 and phytase in alfalfa,84 people tried to findways to humanize the
glycoprotein in plant.
In addition to the in vitro enzymatic remodeling of glycoproteins being carried out by companies
such as Neose, several strategies have been developed to humanize the glycan patterns generated by
transgenic plants.
The first strategy is retaining the recombinant glycoprotein in endoplasmic reticulum (ER) so
that it can avoid further modification of the glycoproteins in the Golgi apparatus, with specific plant
oligosaccharides. The second is modifying the enzymatic machinery of the Golgi apparatus by
knocking out enzymes or the genes that relate b (1,2)-xylosylation and a (1,3)-fucosylation and/or byadding new glycosyltransferase to modify the processing of N-glycans.
F. Retaining the Recombinant Glycoprotein in Endoplasmic Reticulum (ER)
Plants and mammalians have the same N-glycosylation site: specific asparagines residues of the
nascent polypeptide (Asp-x-Ser/Thr), x can be any residue except Pro and Asn. High-mannose type
N-glycans in plants is identical to those found in mammalian cells, but their complex N-linked
glycans differ substantially. The processing of high-mannose-type to complex-type N-glycans is not
required for transport and secretion of extracellular glycoproteins in plants.85,86 The first report of
targeting a cytoplasmic protein to ER of plant applied a signal peptide of patatin to b-glucuronidase.Denecke et al. 87 also found that non-secretory enzymes phosphinothricin acetyl transferase,
neomycin phosphotransferase II, and b-glucuronidase were secreted when targeted to the lumen of
the ER by signal peptide-mediated translocation.
From then on, K/HDEL and other signal peptides are also found to lead the protein located in ER.
KDEL (Lys-Asp-Glu-Leu) sequence is now often used to produce oligomannose (Man5–9GlcNAc2)
type N-glycans. In 2001, A Fab fragment expressed in Arabidopsis with an N-terminal leader peptide
and aC-terminal KDEL sequencewas found to retain in ER, whereas the same Fab fragment devoid of
any C-terminal sequence was efficiently secreted in leaves and roots.88 In 2003, Ko et al. used KDEL
sequence to link C-terminus of the heavy chain of mAb (monoclonal antibodies). They expected that
glycans attached to proteins containing aC-terminal KDEL sequencewould be restrictedmainly to the
oligomannose type.89 About 90% of the total glycan pool was found to be oligomannose-type
oligosaccharides in transgenic plants, no Fuc or Gal residues derived glycan structures were found.
ER retention of proteins in transgenic plants usually can improve the production level.90,91 In
some cases,7,92,93 the highest expression levels were achieved when protein was targeted to ER.
Antibodies expression level can be increased if the protein is retained to ER lumen using an H/KDEL
C-terminal tetrapeptide tag.8,90 As with vaccines, retention of rAbs in ER can increase expression
level from 0.35% to 2%TSP, and some even higher.7,94–96 For example, different signals were added
to HbsAg (hepatitis B surface antigen) in transgenic potatoes and the expression levels can be
improved by adding ER signals.25
MODIFICATION OF PLANT N-GLYCANS * 351
However, there are also some limitations in this ER signal additionmethod. Issartel et al. used the
sequenceKDEL to retain the recombinant dog’s gastric lipase (rGL) in the ER of plant. They found at
least three individual bands by SDS analysis around 49 kDa while the molecular weight of native
protein is 50 kDa, which indicated that heterogeneous forms of rGL existed after different
glycosylation.97
Results obtained with a hybrid immunoglobulin (IgA/G) suggested that the sorting of complex
molecules within the secretory pathway was not only dependent on particular signal sequences, but
also on the protein itself.98 Tissue-specific differences in protein deposition have been observed with
scFv containing an ER retention signal. This scFvwas detected in ER-derived protein bodies and also
in protein storage vacuoles of transgenic rice endosperm cells.99 Results from Ramirez N100 also
drew a similar conclusion.
G. Interrupting ��� (1,2)-linked Xylose and ��� (1,3)-linked Fucose
PlantN-glycanswith b (1,2)-xylose anda (1,3)-fucose are regarded as themajor class of the so-called
‘‘carbohydrate cross-reactive determinants’’ reactivewith IgE antibodies in the sera of many allergic
patients.101–105 The presence of a (1,2)-linked xylose and b (1,3)-linked fucose in therapeutic
glycoproteins are often thought to produce immunogenicity in human therapy.
Mutant and gene knocking out technologies are often used to avoid the special modification
processing ofN-glycans in plant. Arabidopsis cglmutant lacks theN-acetyl glucosaminyltransferase
I and is unable to synthesize complex-typeN-glycans.106 Another Arabidopsismutant,mur1, did not
synthesize L-Fuc in the leaves because of the disruption of the gene encoding for a GDP-b-Man-4,6-
dehydratase, a key enzyme in the biosynthesis of L-Fuc. In allN-linked glycans from thisArabidopsis
mutant, L-Fuc residueswere absent. The absence of L-Fuc did not disturb the biosynthesis ofN-linked
oligosaccharides.107 The result indicated that knocking out the genes related to the b (1,2)-
xylosylation and a (1,3)-fucosylation to produce more mammalian-like glycoproteins is possible. In
2003, a (1,3)-fucosyltransferase and b (1,2)-xylosyltransferase genes were knocked out in
Physcomitrella patens and prevented the production of plant-specific glyco-epitopes without
affecting the secretion of the protein.108 These results paved the way to use these strategies in higher
plants. In 2004, Strasser et al. knocked out b (1,2)-xylosyltransferase (XylT) and two a (1,3)-
fucosyltransferase (FucTA and FucTB) in Arabidopsis.105 Knockout plants were generated with
complete deficiency of XylT and FucT. These plants can produce N-glycans with two b-N-acetyglucosamine residues but lacking b (1,2)-linked xylose and a (1,3)-linked fucose (Table IV).
Table IV. Majority Glycans of Knockout and Mutant Plant
352 * CHEN ET AL.
Besides knocking out XylT and FucT, b (1,2)-xylosidase can also be used to eliminate b (1,2)-
xylose in plant. b (1,2)-xylosidase can releases xylose residues b (1,2)-linked to the beta-mannose of
anN-glycan core, if the 3-position of this mannose is not occupied.109 Potatoes are a cheap and easily
available source for the preparation of this degradative enzyme and should be an important tool for the
analysis of N-glycans and in the modification of N-glycans.
Addition of human glycosyltransferases is also necessary for the production of humanized
proteins. b (1,4)-galactosyltransferases is competed for the same acceptor substrate as XylT and
FucT. The overexpression of b (1,4)-galactosyltransferases is used to eliminated a (1,2)-xylose and b(1,3)-fucose,4,5 but the complete elimination of a (1,2)-xylose and b (1,3)-fucose has not been
achieved in this case.
All known Golgi glycosyltransferases are N-in/C-out (type II) memberane proteins with a short
amino-terminal cytoplasmic(C) tail, a hydrophobic transmembrane (T) domain, and a luminal stem
(S) region.110–113 Dirnberger et al. found that the Golgi localization of Arabidopsis thaliana b (1,2)-
xylosyltransferase in plant cells is dependent on its cytoplasmic and transmembrane sequences.114
This means that disruption of this domain will hinder the addition of xylose in Golgi.
H. Adding Penultimate Galactose
The terminal b (1,4)-galactose residues are thought to contribute to the immunological functions and
the correct folding of antibodies.115 Thus, galactose may be critical for pharmacokinetic activity of
certain therapeutic antibodies.
To determine whether mammalian glycosyltransferase could extend and modify the N-linked
glycoprotein processing pathway in plants, human b (1,4)-galactosyltransferase (GalT) gene was
transferred intoNicotiana tabacumL.cv. Bright Yellow (BY2) cells lackingGalT gene.5 It was found
that this human enzyme extended some complex-type sugar chains from nongalactose- into
galactose-containing types. The analysis of the oligosaccharide structures indicated that the galacto-
sylated N-glycans account for 47.3% of the total sugar chains.
Horseradish peroxidase isozyme C (HRP) was used to evaluate the capacity of tobacco cells
transformedwithGT6 gene tomodify and galactosylate a foreign glycoprotein. Cells transformedwith
the HRP gene are designated as BY2-HRP and GT6-HRP, for wild type BY2 and GT6 transformed
cells, respectively. HRP with galactosylated N-glycans are only presented in GT6-HRP cells.116
After that, Bakker et al. expressed a plantibody with 30% galactosylated N-glycans, which was
approximately as abundant as that produced by hybridoma cells, by crossing a tobacco plant
expressing human b (1,4)-galatosyltransferasewith a plant expressing the heavy and light chains of amouse antibody. Misaki R. found that plant cultured cells expressing human beta1,4-galactosyl-
transferase can secrete glycoproteins with galactose-extended N-linked glycans. However, the
distribution of proposed N-glycan structures of GT6-secreted glycoproteins is different from that
found in intracellular glycoproteins. Sialic acid was transferred to sugar chains of extracellular
glycoproteins from the GT6 spent medium.117
Thefinal productGalGlcNAcMan5GlcNAc2produced inplants differs fromGalGlcNAc2Man3Glc-
NAc2, which was found in mammals.4 The expression of mammalian beta1,4-galactosyltransferase in
plants, which would complete and/or competewith the endogenousmachinery forN-glycansmaturation
in the plant Golgi apparatus is an useful strategy to humanize plant N-glycans. The efficiency of
heterologous glycosyltransferases could be increased by improved control of their targeting in Golgi
subcompartments. The efficient targeted expression of heterologous glycosyltransferases using the fusion
of a sequence responsible for the targeting of A. thaliana b (1,2)-xylosyltransferase in the Golgi to the
catalytic domain of the human b (1,4)-galactosyltransferase has been patented.118
I. Adding Terminal Sialic Acid
Glycoproteins in plants lack the terminal sialic acid, which is an important component in human
glycoproteins.
MODIFICATION OF PLANT N-GLYCANS * 353
Production of sialylate therapeutic proteins in plants will be more difficult because plants lack
most of themachinery required to synthesize Neu5Ac and to transfer it from cytidinemonophosphate
(CMP)-Neu5Ac to b (1,4)-Gal-terminatedN-glycans.117 Few reports describe efforts to add terminal
sialic acid to glycoproteins produced by transgenic plants.
In 1998, Edmund et al. transferred a a (2,6)-sialyltransferase gene from rat into A. thaliana and
targeted the enzyme correctly to plant Golgi apparatus. A significant change in glycosylation activity
was obtained by expressing the mammalian glycosyltransferase in Golgi, but to obtain sialylation in
plants, the enzymes of CMP-sialic acid synthesis and the Golgi sugar nucleotide transporter need to
be coexpressed.119
2 . P R O S P E C T I V E
The number of protein-based therapeutics entering preclinical and clinical evaluation has shown
robust growth and is expected to increase in the years to come. Fueled by advances in proteomics and
genomics as well as the ability to engineer and humanize monoclonal antibodies, there are about 500
protein-based therapeutic candidates currently in clinical trials. The majority of the therapeutic
proteins require additional posttranslational modifications to obtain full biological function.
However, this posttranslational modification differs between plant and mammals. To overcome this
drawback, metabolic engineering of the plant N-glycan biosynthesis pathway is necessary. Except
knocking out the genes encoding enzymes responsible for the specific addition of plant sugars, many
human specific glycosyltransferases need to be supplied. Successful production of the complex
human glycoproteins in yeast provided an useful information on how to engineer the plant metabolic
pathways towards the formation of the humanized glycoproteins.
On the other hand, most of the therapeutic glycoproteins produced in alternative systems have
been produced inexpensively in plants. This would reduce the cost of treatment and increase the
number of patients with access to suchmedicines. The absolute yield of a given recombinant protein,
in particular plant species and plant cell compartment, is unpredictable. Some recombinant proteins
already reached very high expression levels, for example, apoplast targeted recombinant phytase
accumulated to almost 14% total soluble protein in tobacco leaves. However, the expression levels of
most of the recombinant proteins were often very low. Thus, in the future, for the production of
humanized glycoprotein from plant, three key problems need to be solved: metabolic engineering
of N-glycan biosynthesis pathway towards the formation of humanized protein; improvement of the
production yield in the plant; and establishment of the extraction and purificationmethod from plant.
R E F E R E N C E S
1. Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M, Flynn P, Register J, Marshall L,Bond D, Kulisek E. Commercial production of Avidin from transgenic maize: Characterization oftransformant, production, processing, extraction, and purification. Mol Breed 1997;3:291–306.
2. Witcher DR, Hood E, Peterson D, Bailey M, Marshall L, Bond D, Kusnadi A, Evangelista R, Nikolov Z,Wooge C, Mehigh R, Kappel B, Ritland D, Register J, Howard J. Commercial production of b-glucuronidase (GUS): A model system for the production of proteins in plants. Mol Breed 1998;4:301–312.
3. Giddings G, Allison G, Brooks D, Carter A. Transgenic plants as factories for biopharmaceuticals. NatBiotechnol 2000;18:1151–1155.
4. BakkerH,BardorM,Moltho JW,GomordV,Elbers I, StevensLH, JordiW,LommenA, FayeL, Lerouge P,Bosch D. Galactose-extended glycans of antibodies produced by transgenic plants. Proc Natl Acad SciUSA 2001;98:2899–2904.
5. PalacpacNQ,Yoshida S, Sakai H, KimuraY, FujiyamaK,Yoshida T, Seki T. Stable expressing of human b1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc Natl Acad Sci1999;96:4692–4697.
354 * CHEN ET AL.
6. WrightKE, Prior F, SardanaR,Altosaar I, DudaniAK,Ganz PR, Tackaberry ES. Sorting of glycoproteinBfrom human cytomegalovirus to protein storage vesicles in seeds of transgenic tobacco. Transgenic Res2001;10:177–181.
7. Giddings G. Transgenic plants as protein factories. Curr Opin Biotechnol 2001;12:450–454.8. Twyma RE, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: Host systems and
expression technology. Trends Biotechnol 2003;21:570–578.9. Larrick JW, Thomas DW. Producing proteins in transgenic plants and animals. Curr Opin Biotechnol
2001;12:411–418.10. Chadd HE, Chamow SM. Therapeutic antibody expression technology. Curr Opin Biotechnol 2001;
12:188–194.11. Saalbach I, Giersberg M, Conrad U. High-level expression of a single-chain Fv fragment (scFv) antibody
in transgenic pea seeds. Plant Physiol 2001;158:529–533.12. RamirezN,Oramas P,AyalaM,RodriguezM, PerezM,Gavilondo J. Expression and long-term stability of
a recombinant single-chain Fv antibody fragment in transgenic Nicotiana tabacum seeds. Biotechnol Lett2001;23:47–49.
13. Horn ME, Woodard SL, Howard JA. Plant molecular farming: Systems and products. Plant Cell Reports2004; online: 28 February.
14. Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants. Nature 1989;342:76–78.15. Obermeyer G, Gehwolf R, SebestaW, Hamilton N, Gadermaier G, Ferreira F, Commandeur U, Fischer R,
Bentrup FW. Over-expression and production of plant allergens bymolecular farming strategies. Methods2004;32:235–240.
16. Salmon V, Legrand D, Slomianny MC, Yazidi IE, Spik G, Gruber V, Bournat P, Olagnier B, Mison D,Theisen M, Me’rot B. Production of human lactoferrin in transgenic tobacco plants. Protein Expr Purif1998;13:127–135.
17. Spik1 G, Theisen M. Characterization of the post-translational biochemical processing of humanlactoferrin expressed in transgenic tobacco. Bundesgesundheitsbl Gesundheitsforsch Gesundheitsschutz2000;43:104–109.
18. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: Production of antibodies,biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 2001;6:219–226.
19. Fischer R, Emans N. Molecular farming of pharmaceutical proteins. Transgenic Res 2000;9:279–299.20. GaoY,MaY,LiM,ChengT, Li SW,Zhang J,XiaNS.Oral immunization of animalswith transgenic cherry
tomatillo expressing HbsAg. World J Gastroenterol 2003;9:996–1002.21. Streatfield SJ, Lane JR, Brooks CA, Barker DK, PoageML,Mayor JM, Lamphear BJ, Drees CF, Jilka JM,
Hood E, Howard JA. Corn as a production system for human and animal vaccines. Vaccine 2003;21:812–815.
22. Silva JV, Garcia AB, Flores VMQ, Macedo ZS, Medina AE. Phytosecretion of enteropathogenicEscherichia coli pilin subunit A in transgenic tobacco and its suitability for early life vaccinology. Vaccine2002;20:2091–2101.
23. Jani D, Singh Meena L, Mohammad Rizwan-ul-Haq Q, Singh Y, Sharma AK, Tyagi AK. Expression ofcholera toxin B subunit in transgenic tomato plants. Transgenic Res 2002;11:447–454.
24. Daniell H, Lee SB, Panchal T, Wiebe PO. Expression of the native cholera toxin B subunit gene andassembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 2001;311:1001–1009.
25. Richter LJ, Thanavala Y, Arntzen CJ, Mason HS. Production of hepatitis B surface antigen in transgenicplants for oral immunization. Nat Biotechnol 2000;18:1167–1171.
26. Kapusta J, Modelska A, Figlerowicz M, Pniewske T, Letellier M, Lisowa O, Yusibov V, Koprowske H,PlucienniczakA, Legocki AB.A plant-derived edible vaccine against hepatitis B virus. FASEB J 1999;13:1796–1799.
27. Yu J, Langridge W. Expression of rotavirus capsid protein VP6 in transgenic potato and its oralimmunogenicity in mice. Transgenic Res 2003;12:163–169.
28. Matsumura T, Itchoda N, Tsunemitsu H. Production of immunogenic VP6 protein of bovine group Arotavirus in transgenic potato plants. Arch Virol 2002;147:1263–1270.
29. Bouquin T, Thomsen M, Nielsen LK, Green TH, Mundy J, Hanefeld Dziegiel M. Human anti-rhesus DIgG1 antibody produced in transgenic plants. Transgenic Res 2002;11:115–122.
30. Staub J, Garcia B, Graves J, Hajdukiewicz P, Russell D, Hunter P, Nehra N, Paradkar V, SchlittlerM. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 2000;42:583–590.
31. Khoudi H, Laberge S, Ferullo JM, Bazin R, Darveau A, Castonguay Y, Allard G, Lemieux R, Vezina LP.Production of a diagnostic monoclonal antibody in perennial alfalfa plants. Biotechnol Bioeng 1999;64:135–143.
MODIFICATION OF PLANT N-GLYCANS * 355
32. Zeitlin L, Olmsted SS, Moench TR, CoMS,Martinell BJ, Paradkar VM, Russell DR, Queen C, Cone RA,WhaleyKJ. A humanizedmonoclonal antibody produced in transgenic plants for immunoprotection of thevagina against genital herpes. Nat Biotechnol 1998;16:1361–1364.
33. Ma JK, Hiatt A, HeinM, Vine ND,Wang F, Stabila P, van Dolleweerd C, Mostov K, Lehner T. Generationand assembly of secretory antibodies in plants. Science 1995;268:716–719.
34. Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L. Characterization of a recombinant plantmonoclonal secretory antibody and preventive immunotherapy in humans. Nat Med 1998;4:601–606.
35. Masarik M, Kizek R, Kramer KJ, Billova S, Brazdova M, Vacek J, Bailey M, Jelen F, Howard JA.Application of avidin-biotin technology and adsorptive transfer stripping square-wave voltammetry fordetection of DNA hybridization and avidin in transgenic avidin maize. Anal Chem 2003;75:2663–2669.
36. Nandi S, Suzuki YA, Huang J, Yalda D, Pham P, Wu L, Bartley G, Huang N, Lonnerdal B. Expression ofhuman lactoferrin in transgenic ricegrains for the application in infant formula. PlantSci 2002;163:713–722.
37. Leite A, Kemper EL, da Silva MJ, Luchessi AD, Siloto RMP, Bonaccorsi ED, El-Dorry HF, Arruda P.Expression of correctly processed human growth hormone in seeds of transgenic tobacco plants. MolBreed 2000;6:47–53.
38. Zhong GY, Peterson D, Delaney D, BaileyM,Witcher D, Register J. Commercial production of Aprotininin transgenic maize seeds. Mol Breeding 1999;5:345–356.
39. Lee JS, Choi SJ, Kang HS, OhWG, Cho KH, Kwon TH, Kim DH, Jang YS, YangMS. Establishment of atransgenic tobacco cell suspension culture system for producing murine granulocyte—Macrophagecolony stimulating factor. Mol Cell 1997;7:783–787.
40. Cramer CL, Weissenborn DL, Oishi KK, Grabau EA, Bennett S, Ponce E, Grabowski GA, Radin DN.Bioproduction of human enzymes in transgenic tobacco. Ann NYAcad Sci 1996;792:62–71.
41. Parmenter DL, Boothe JG, Van Rooijen GJ, Teung EC, Moloney MM. Production of biologically activehirudin in plant seeds using oleosin partitioning. Plant Mol Biol 1995;29:1167–1180.
42. KumagaiMH, Turpen TH,Weinzett N, Della CG, TurpenAM,Donson J, HilfME,GranthamGL,DawsonWO,ChowTP.Rapid, high level expression of biologically active alpha-trichosanthin in transfected plantsby an RNAviral vector. Proc Natl Acad Sci USA 1993;90:427–430.
43. Sijmons PC,Dekker BM, Schrammeijir B, Verwoerd TC, Elzen PJM,HoekemaA. Production of correctlyprocessed human serum albumin in transgenic plants. Bio Technol 1990;8:217–221.
44. Bailey MR, Woodard SL, Callaway E, Beifuss K, Magallanes-Lundback M, Lane JR, Horn ME,Mallubhotla H, Delaney DD, Ward M, Van Gastel F, Howard JA, Hood EE. Improved recovery of activerecombinant laccase from maize seed. Appl Microbiol Biotechnol 2004;63:390–397.
45. Hood EE, BaileyMR, Beifuss K,Magallanes-LundbackM, HornME, Callaway E, Drees C, Delaney DE,CloughR,Howard JA.Criteria for high-level expression of a fungal laccase gene in transgenicmaize. PlantBiotechnol J 2003;1:129–140.
46. Kim KY, Kwon SY, Lee HS, Hur Y, Bang JW, Kwak SS. A novel oxidative stress-inducible peroxidasepromoter from sweet potato: Molecular cloning and characterization in transgenic tobacco plants andcultured cells. Plant Mol Biol 2003;51:831–838.
47. Fischer R, Stoger E, Schillberg S,Christou P, TwymanRM.Plant-based production of biopharmaceuticals.Curr Opin Plant Biol 2004;7:152–158.
48. Stoger E, Sack M, Fischer R, Christou P. Plantibodies: Applications, advantages and bottlenecks. CurrOpin Biotechnol 2002;13:161–166.
49. Daniell H, Lee SB, Panchal T, Wiebe PO. Expression of the native cholera toxin B subunit gene andassembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 2001;311:1001–1009.
50. Daniell H, Wycoff K. Production of antibodies in transgenic plastids. Patent Application NumberWO 01/64929.
51. De JG, Fiers E, Eeckhout D, Depicker A. Analysis of the interaction between single-chain variablefragments and their antigen in a reducing intracellular environment using the twohybrid system. FEBSLett2000;467:316–320.
52. WornA, PluckthunA. Stability engineering of antibody single-chain � Fv fragments. JMolBiol 2001;305:989–1010.
53. Leelavathi S, Reddy VS. Chloroplast expression of His-tagged GUS fusions: A general strategy tooverproduce and purify foreign proteins using transplastomic plants as bioreactors. Mol Breed 2003;11:49–58.
54. Crofcheck C, Loiselle M, Weekly J, Maiti I, Pattanaik S, Bummer PM, Jayt M. Histidine-tagged proteinrecovery from tobacco extract by foam fractionation. Biotechnol Prog 2003;19:680–682.
55. Morassutti C, De Amicis F, Skerlavaj B, Zanetti M, Marchetti S. Production of a recombinantantimicrobial peptide in transgenic plants using a modified VMA intein expressionsystem. FEBS Lett2002;519:141–146.
356 * CHEN ET AL.
56. Valdes R, Gomez L, Padilla S, Brito J, Reyes B, AAlvarez T, Mendoza O, Herrera O, Ferro W, Pujol M,Leal V, LinaresM, Hevia Y, Garcıa C, Mila L, Garcıa O, Sanchez R, Acosta A, Geada D, Paez R, Vega JL,Borroto C. Large-scale purification of an antibody directed against hepatitis B surface antigen fromtransgenic tobacco plants. Biochem Biophys Res Commun 2003;308:94–100.
57. Stein KE, Webber KO. The regulation of biologic products derived from bioengineered plants. Curr OpinBiotechnol 2001;12:308–311.
58. Rayon C, Lerouge P, Faye1 L. The protein N-glycosylation in plants. J Exp Bot 1998;326:1463–1472.59. Altmann F. More than silk and honey—Or, can insect cells serve in the production of therapeutic
glycoproteins? Glycoconj J 1997;14:643–646.60. Fitchette-Laine’ AC, Gomord V, Cabanes M, Michalski JC, Saint-Macary M, Foucher B, Cavelier B,
Hawes C, Lerouge P, Faye L. N-glycans harboring the lewisa epitope are expressed at the surface of plantcells. Plant J 1997;12:1411–1417.
61. Melo NS, Nimtz M, Conradt HS, Feveiro PS, Costa J. Identification of the human Lewis a carbohydratemotif in a secretory peroxidase from a plant cell suspension culture Vaccinium myrtillus L. FEBS Lett1997;415:186–191.
62. Fitchette AC, Cabanes-Mancheteau M, Marvin L, Martin B, Satiat-Jeunemaitre B, Gomord V, Crooks K,Lerouge P, Faye L, Hawes C. Biosythesis and immunolacalization of Lewisa-containing N-glycans in theplant cell. Plant Physiol 1999;121:333–343.
63. Wilson IBH, ZelenyR,KolarichD, Staudacher E, StroopCJM,Kamerling JP,Altmann F.Analysis ofAsn-linked glycans from vegetable foodstuffs: Widespread occurrence of Lewis a, core a (1, 3)-fucose andxylose substitutions. Glycobiology 2001;12:276–284.
64. Joly C, Leonard R, Maftah A, Riou KC. 4-Fucosyltransferase is regulated during flower development:Increases in activity are targeted to pollen maturation and pollen tube elongation. J Exp Bot 2002;53:1429–1436.
65. Palacpac NQ, Kimura Y, Fujiyama K, Yoshida T, Seki T. Structures of N-linked oligosaccharides ofglycoproteins from tobacco BY2 suspension cultured cells. Biosci Biotechnol Biochem 1999;63(1):35–39.
66. Bardor M, Faveeuw C, Fitchette AC, Gilbert D, Galas L, Trottein F, Faye L, Lerouge P. Immunoreactivityin mammals of two typical plant glyco-epitopes,core a(1,3))-fucose and core xylose. Glycobiology2003;13:427–434.
67. Szumilo T,Kaushal GP, ElbeinAD. Purification and properties of glucosidase I frommung bean seedlings.Arch Biochem Biophys 1986;247:261–271.
68. Kaushal GP, Pastuszak I, Hatanaka KI, Elbein AD. Purification to homogeneity and properties ofglucosidase II from mung bean seedlings and suspension-cultured soybean cells. J Biol Chem 1990;265:16271–16279.
69. Navazzio L, Baldan B, Mariani P, Gerwig GJ, Vliegenthart JFG. Primary structure of the N-linkedcarbohydrate chains of calreticulin from spinach leaves. Glycoconj J 1996;13:977–983.
70. Szumilo T, Kaushal GP, Hori H, Elbein AD. Purification and properties of a glycoprotein processing a-mannosidase from mung bean seedling. Plant Physiol 1986;81:383–389.
71. SturmA, JohnsonKD, Szumilo T, Elbein AD, ChrispeelsMJ. Subcellular localization of glycosidases andglycosyl-transferases involved in the processing of N-linked oligosaccharides. Plant Physiol 1987;85:741–745.
72. Johnson KD, Chrispeels MJ. Substrate specificities of N- acetylglucosaminyl-, fucosyl-, andxylosyltransferases that modify glycoproteins in the Golgi appparatus of bean. Plant Physiol 1987;84:1301–1308.
73. Tezuka K, Hayashi M, Ishihara H, Akazawa T, Takahashi N. Studies on synthetic of xylose-containing N-linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells(Acer pseudoplatanus L). Eur J Biochem 1992;203:401–413.
74. Kaushal GP, Szumilo T, Pastuszak I, Elbein AD. Purification to homogeneity and properties ofmannosidase II from mung bean seedlings. Biochemistry 1990;29:2168–2176.
75. Staudacher E, Dalik T, Wawra P, Altmann F, Marz L. Functional purification and characterization of aGDPfucose:b-N-acetylglucosamine (Fuc to Asn linked GlcNAc) a-1,3-fucosyltransferase from mungbeans. Glycoconjugate 1995;12:780–786.
76. Vitale A, Chrispeels MJ. Transient N-acetylglucosamine in the biosynthesis of phytohaemagglutinin:Attachment in the Golgi apparatus and removal in protein bodies. J Cell Biol 1984;99:133–140.
77. Vaquero C, Sack M, Chandler J, Drossard J, Schuster F, Monecke M, Schillberg S, Fischer R. Transientexpression of a tumor-specific single chain fragment and a chimeric antibody in tobacco leaves. Proc NatlAcad Sci USA 1999;96:11128–11133.
78. Ma J, Lehner T, Stabila P, Fux CI, Hiatt A. Assembly of monoclonal antibodies with IgG1 and IgA heavychain domains in transgenic tobacco plants. Eur J Immunol 1994;24:131–138.
MODIFICATION OF PLANT N-GLYCANS * 357
79. Larrick JW, Yu L, Naftzger C, Jaiswal S, Wycoff K. Production of secretory IgA antibodies in plants.Biomol Eng 2001;18:87–94.
80. Cabanes-Macheteau M, Fitchette-Laine AC, Loutelier-Bourhis C, Lange C, Vine ND, Ma JK, Lerouge L,Faye L. N-glycosylation of a mouse IgG expressed in transgenic tobacco plants. Glycobiology 1999;9:365–372.
81. Bardor M, Faveeuw C, Fitchette AC, Gilbert D, Galas L, Trottein F, Faye L, Lerouge P. Immunoreactivityinmammals of two typical plant glyco-epitopes, core (1,3)-fucose and core xylose. Glycobiology 2003;13:427–434.
82. Chargelegue D, Vine N, Dolleweerd CV, Drake PM, Ma J. A murine monoclonal antibody producedin transgenic plants with plant-specific glycans is not immunogenic in mice. Transgen Res 2000;9:187–194.
83. Schillberga S, Fischera RB, Emansb N. Molecular farming of recombinant antibodies in plants CMLS.Cell Mol Life Sci 2003;60:433–445.
84. Ullah A HJ, Sethumadhavan K, Mullaney EJ, Ziegelhoffer T, Phillips SA. Cloned and expressed fungalphyA gene in alfalfa produces a stable phytase. Biochem Biophys Res Commun 2002;290:1343–1348.
85. Driouich A, Gonnet P, Makkie M, Laine AC, Faye L. The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins. Planta 1989;180:96–104.
86. Lerouge P, Fitchette-LaineAC,ChekkafiA,AvidgorV, Faye L.N-linked oligosaccharide processing is notnecessary for glycoprotein secretion in plants. Plant J 1996;10:101–107.
87. Denecke J, Botterman J, Deblaere R. Protein secretion in plant cells can occur via a default pathway. PlantCell 1990;2:51–59.
88. Peeters K, DeWilde C, Depicker A. Highly efficient targeting and accumulation of a Fab fragment withinthe secretory pathway and apoplast of Arabidopsis thaliana. Eur J Biochem 2001;268:4251–4260.
89. Ko K, Tekoah Y, Rudd PM, Harvey DJ, Dwek RA, Spitsin S, Hanlon CA, Rupprecht C, Dietzschold B,Golovkin M, Koprowski H. Function and glycosylation of plant-derived antiviral monoclonal antibody.Proc Natl Acad Sci USA 2003;100:8013–8018.
90. ConradU, FiedlerU. Compartment-specific accumulation of recombinant immunoglobulins in plant cells:An essential tool for antibody production and immunomodulation of physiological functions and pathogenactivity. Plant Mol Biol 1998;38:101–109.
91. Sharp JM, Doran PM. Characterization of monoclonal antibody fragments produced by plant cells.Biotechnol Bioeng 2001;73:338–346.
92. Perrin Y, Vaquero C, Gerrard I, SackM, Drossard J, Stoger E, Christou P, Fischer R. Transgenic pea seedsas bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosisand therapy. Mol Breed 2000;6:345–352.
93. Stoger E, Vaquero C, Torres E, SackM,Nicholson L, Drossard J,Williams S, KeenD, Perrin Y, Christou P,Fischer R. Cereal crops as viable production and storage systems for pharmaceutical scFvantibodies. PlantMol Biol 2000;42:583–590.
94. FischerR,HoffmannK,SchillbergS,EmansN.Antibodyproductionbymolecular farming in plants. JBiolRegul Homeost Agents 2000;14:83–92.
95. WrightKE, Prior F, SardanaR,Altosaar I, DudaniAK,Ganz PR, Tackaberry ES. Sorting of glycoprotein Bfrom human cytomegalovirus to protein storage vesicles in seeds of transgenic tobacco. Transgenic Res2001;10:177–181.
96. Perrin Y, Vaquero C, Gerrard I, SackM, Drossard J, Stoger E, Christou P, Fischer R. Transgenic pea seedsas bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosisand therapy. Mol Breed 2000;6:345–352.
97. Issartel NM, Bouchon B, Farrer S, Laparra PBH, Madelmontb JC, Theisen MA. transient tobaccoexpression system coupled to MALDI-TOF-MS allows validation of the impact of di.erential targeting onstructure and activity of a recombinant therapeutic glycoprotein produced in plants. FEBS Lett 2003;552:170–176
98. Frigerio L, Vine ND, Pedrazzini E, HeinMB,Wang F,Ma JK, Vitale A. Assembly, secretion, and vacuolardelivery of a hybrid immunoglobulin in plants. Plant Physiol 2000;123:1483–1494.
99. Torres E, Gonzalez-Melendi P, Stoger E, Shaw P, Twyman RM, Nicholson L, Vaquero C, Fischer R,Christou P, Perrin Y. Native and artificial reticuloplasmins co-accumulate in distinct domains of theendoplasmic reticulum and in post-endoplasmic reticulum compartments. Plant Physiol 2001;127:1212–1223.
100. Ramirez N, Rodriguez M, Ayala M, Cremata J, Perez M, Martinez A, Linares M, Hevia Y, Paez R, ValdesR, Gavilondo JV, Selman-Housein G. Expression and characterization of an anti-(hepatitis B surfaceantigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in theimmunopurification of its target antigen. Biotechnol Appl Biochem 2003;38:223–230.
358 * CHEN ET AL.
101. BencUrova M, Hemmer W, Focke TM, Wilson IBH, Altmann F. Specificity of IgG and IgE antibodiesagainst plant and insect glycoprotein glycans determined with artificial glycoforms of human transferring.Glycobiology 2004;581–590.
102. Aalberse RC, Koshte V, Clemens JG. Immunoglobulin E antibodies that crossreact with vegetable foods,pollen, and Hymenoptera venom. J Allergy Clin Immunol 1981;68:356–364.
103. Ree RV, Macheteau MC, Akkerdaas J, Milazzo JP, Bourhis CL, Rayon C, Villalba M, Koppelman S,Aalberse R, Rodriguez R, Faye L, Lerouge P. Beta(1,2)-xylose and alpha(1,3)-fucose residues have astrong contribution in IgE binding to plant glycoallergens. J Biol Chem 2000;275:11451–11458.
104. Foetisch K, Westphal S, Lauer I, Retzek M, Altmann F, Kolarich D, Scheurer S, Vieths S. Biologicalactivity of IgE specific for cross-reactive carbohydrate determinants. J Allergy Clin Immunol 2003;111:889–896.
105. Strasser R, Altmann F, Mach L, Glossl J, Steinkellner H. Generation of Arabidopsis thaliana plants withcomplex N-glycans lacking a1,2-linked xylose and core b1,3-linked fucose. FEBS Lett 2004;561:132–136.
106. von Schaewen A, Sturm A, O’Neill J, Chrispeels MJ. Isolation of a mutant Arabidopsis plant that lacksN-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans.Plant Physiol 1993;102:1109–1118.
107. Rayon C, Macheteau MC, Bourhis CL, Maire IS, Lemoine J, Reiter WD, Lerouge P, Faye L.Characterization ofN-Glycans fromArabidopsis. Application to a Fucose-DeficientMutant. Plant Physiol1999;119:725–733.
108. Koprinovova A, Lienhart O, Decker EL, Stemmer C,Wagner S, Gorr G.Modifying glycosylation patternsin moss. Conference on Plant-Made Pharmaceuticals: 2003 March 16–19; Quebec City, California.CPMP, 2003.
109. Peyer C, Bonay P, Staudacher E. Purification and characterization of a beta-xylosidase from potatoes(Solanum tuberosum). Biochim Biophys Acta 2004;1672:27–35.
110. Munro S. Sequencewithin and adjacent to the transmemberane segment of a-2,6-sialyltransferase specifyGolgi retention. EMBO 1991;10:3577–3588.
111. Nilsson T, Lucocq JM, Mackay D, Warren G. The membrane spanning domain of b-1, 4-galacto-syltransferase specifies trans Golgi localization. EMBO 1991;10:3567–3575.
112. Tang BL, Wong SH, Low SH, Hong W. The transmemberane domain of N-glucosaminyltransferase Icontains a Golgi retention signal. J Bio Chem 1992;267:10122–10126.
113. Wong SH, Low SH, Hong W. The 17-residue transmemberane domain of b-galactoside a2, 6-sialyl-transferase in sufficient for Golgi retention. J Cell Biol 1992;117:245–258.
114. Dirnberger D, Bencur P, Mach L, Steinkellner H. The Goligi locaton of Arabidopsis thaliana b1,2-xylosyltransferase in plant cells is dependent on its cytoplasmic and transmembrane sequences. Plant MolBiol 2002;50:273–281.
115. Wright A, Morrison SL. Effect of C2-associated carbohydrate structure on Ig effector function: Studieswith chimeric mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary cells.J Immunol 1998;160:3393–3402.
116. Kazuhito F,NirianneQ. P,Hiromi S,YoshinobuK,Atsuhiko S, ToshiomiY, Tatsuji S. Invivo conversion ofa glycan to human compatible type by transformed tobacco cells. Biochem Biophys Res Commun 2001;289:553–557.
117. Misaki R, Kimura Y, Palacpac NQ, Yoshida S, Fujiyama K, Seki T. Plant cultured cells expressing humanbeta1,4-galactosyltransferase secrete glycoproteins with galactose-extended N-linked glycans. Glyco-biology 2003;13:199–205.
118. Gomord V, Faye L. Posttranslational modification of therapeutic proteins in plants. Curr Opin Plant Biol2004;7:171–181.
119. WeeEG, SherrierDJ, PrimeTA,Dupree P. Targeting of active sialyltransferase to the plant golgi apparatus.Plant Cell 1998;10:1759–1768.
Min Chen received her Ph.D. degree in College of Biological Sciences, China Agricultural
University. She is now a researcher in the State Key Laboratory for Microbial Technology (SKLMT)
at Shandong University. Her major research interests include N-glycosylation in recombinant
glycoproteins produced in transgenic plants.
MODIFICATION OF PLANT N-GLYCANS * 359
Qingsheng Qi received his Ph.D. degree in Microbiology from the University of Muenster in
Germany. He then worked as a postdoctoral fellow at Technical University of Chemnitz, Germany.
He is now a professor in Shandong University in China. His major research interests include the
cloning and expression of glycosyltransferases and the biosynthesis of glycoproteins invarious hosts.
Peng George Wang obtained a B.S. degree in 1984 in Chemistry from Nankai University,
China. In 1985 he came to the USA for his graduate education and obtained a Ph.D. degree at the
University of California, Berkeley in 1990. Then he did postdoctoral research at the University of
California, Berkeley and the Scripps Research Institute. In 1994 he started his independent research
career at University of Miami as Assistant Professor. He moved to Wayne State University in 1997
and there he became full Professor in 2001. Currently Dr. Wang is a Professor in the Departments of
Biochemistry andChemistry atOhio StateUniversity.Dr.Wang’smain research focus is in the area of
glycoscience with emphasis on microbial glycobiology and glycochemistry, glycomics, glyco-
immunology, carbohydrate chemistry, and glycopharmaceutical science.
360 * CHEN ET AL.