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: qiqingsheng@sdu.edu.cnandpwang@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.

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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.