Báo cáo khoa học: Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants pdf

Comparative analysis of the site-specific N-glycosylationof human lactoferrin produced in maize and tobacco plants Be´ne´dicte Samyn-Petit1, Jean-Pierre Wajda Dubos2, Fre´de´ric Chirat1,

Comparative analysis of the site-specific N-glycosylation

of human lactoferrin produced in maize and tobacco plants

Be´ne´dicte Samyn-Petit1, Jean-Pierre Wajda Dubos2, Fre´de´ric Chirat1, Bernadette Coddeville1,

Gre´gory Demaizieres2, Sybille Farrer2, Marie-Christine Slomianny1, Manfred Theisen2

and Philippe Delannoy1

1

Unite´ de Glycobiologie Structurale et Fonctionnelle, UMR CNRS 8576, Laboratoire de Chimie Biologique, Universite´ des Sciences

et Technologies de Lille, Villeneuve d’Ascq, France;2Meristem Therapeutics, Clermont-Ferrand, France

We have compared the site-by-site N-glycosylation status of

human lactoferrin (Lf) produced in maize, a

monocotyle-don, and in tobacco, used as a model dicotyledon Maize and

tobacco plants were stably transformed and recombinant Lf

was purified from both seeds and leaves N-glycopeptides

were generated by trypsin digestion of recombinant Lf and

purified by reverse-phase HPLC The N-glycosylation

pat-tern of each site was determined by mass spectrometry

Our results indicated that the N-glycosylation patterns of

recombinant Lf produced in maize and tobacco share

common structural features In particular, both

N-glycosy-lation sites of each recombinant Lf are mainly substituted by typical plant paucimannose-type N-glycans, with b1,2-xy-lose and a1,3-linked fucose at the proximal N-acetylgluco-samine However, tobacco Lf shows a significant amount of processed N-glycans with one or two b1,2GlcNAc linked to the trimannose core, which are weakly expressed in maize Lf Finally, no Lewisaepitope was observed on tobacco Lf Keywords: glycosylation; N-glycopeptides; maize; tobacco; human lactoferrin

Several expression systems including bacteria, yeast, fungi,

insect and mammalian cells, or transgenic animals are used

to produce recombinant human proteins This last decade,

much attention has been paid to the plant expression

systems in order to express mammalian proteins By using

strong promoters, high levels of expression can be achieved

and production costs are relatively low [1] In addition, plant

expression systems are much less likely to harbor human

pathogens than mammalian expression systems This is a

great advantage of the plant system for the production of

therapeutic proteins such as vaccines and antibodies Direct

oral administration of plant material containing

recombin-ant therapeutic molecules has been investigated for delivery

of antigens and antibodies for active or passive

immuniza-tion [2,3] High-level producimmuniza-tion of recombinant human

milk proteins in rice is also investigated as an addition to

infant formula and baby foods [4]

Plant biologists have been able to express recombinant

proteins in various plants including mono- and

dicotyl-edons Moreover, it is possible to direct the expression to

specific parts of the plant, such as fruits, seeds, leaves and

tubers Several examples have shown that plants allow the production of complex human proteins that appear to have biological properties and activities similar to those of the native proteins, such as human collagens [5], human growth hormone [6] and antibodies [7,8]

Most therapeutic proteins are glycoproteins and glyco-sylation is often essential for the stability, the solubility, a proper folding and biological activity In plants, even if the first steps of N-glycosylation that take place in the endoplasmic reticulum are identical to other eukaryotic cells, the Golgi processing of N-glycan chains displays some major differences compared to that of mammalian cells [9,10] High-mannose-type N-glycans of plants are similar to those found in other eukaryotes However, N-glycans found in plants are mostly of the paucimannose-type (Man3GlcNAc2-based structure), even if complex-type N-glycans with a Lewisa terminal sequence (Galb1– 3[Fuca1–4]GlcNAc-R) have been reported [11] First described in sycamore [12,13], the Lewisa epitope is widespread among plants, but several examples have underlined the lack of such complex N-glycans in a number of mono- and dicotyledon species [14,15] These findings indicate that plants do not exhibit the same potential of N-glycosylation, according to the level of expression of several key enzymes involved in the initiation (i.e b1,2-N-acetylglucosaminyltransferases I and II) and the elongation (i.e galactosyltransferases, fucosyltrans-ferases) of antennae of complex N-glycans, but also of the b-hexosaminidase, which governs the paucimannose-type N-glycan pathway [16] Different plant species share similarities in their N-glycosylation, as the absence of N-acetylneuraminic acid residues in the terminal position

Correspondence to P Delannoy, Unite´ de Glycobiologie Structurale

et Fonctionnelle, UMR CNRS no 8576, Laboratoire de Chimie

Biologique, Universite´ des Sciences et Technologies de Lille,

F-59655 Villeneuve d’Ascq, France.

Fax: + 33 320 43 65 55, Tel.: + 33 320 43 69 23,

E-mail: Philippe.Delannoy@univ-lille1.fr

Abbreviations: Lf, lactoferrin; mLf, maize recombinant lactoferrin;

tLf, tobacco recombinant lactoferrin.

(Received 20 March 2003, revised 2 June 2003, accepted 5 June 2003)

of the antennae, and the presence of a bisecting

b1,2-xylose, and of an a1,3-fucose residue instead of

a1,6-fucose, linked to the proximal N-acetylglucosamine

In a previous paper, we described the potential of maize

glycosylation, a monocotyledon expression system, by using

human lactoferrin (Lf) as a model glycoprotein that was

expressed in the endosperm of seeds [17] The molecular

structure of human Lf has been studied in detail This

80 kDa glycoprotein contains three potential

N-glycosyla-tion sites located at Asn138, Asn479 and Asn624,

respect-ively The two first N-glycosylation sites are substituted by

complex-type N-glycans whereas the third one (Asn624) is

mostly unglycosylated [18, 19] Human Lf plays a central

role in numerous biological processes [20] Among them,

the antibacterial and anti-inflammatory activities of human

Lf have led to its large-scale production by recombinant

methods to supplement infant foods In this paper, we

report the site-by-site analysis of the maize recombinant

lactoferrin (mLf) in comparison with the lactoferrin

pro-duced in tobacco (tLf), used as a model for dicotyledons

For that purpose, the recombinant Lf, purified from both

expression systems, was digested by trypsin after reduction

and alkylation Peptides were fractionated by RP-HPLC

and analysed by MALDI-TOF Glycopeptides and the

corresponding peptides, generated from the glycopeptides

by N-glycosidase A treatment, were also analysed by

MALDI-TOF and ES-MS

Materials and methods

Materials

Sequencing-grade modified trypsin was from Promega

(Zu¨rich, Switzerland) HPLC analyses were carried out on

a Spectra Physics apparatus equipped with a

semiprepar-ative Vydac C18ultrasphere (9.4· 250 mm; 5 lm) column

Recombinant peptide-N-glycosidase F (PNGase F) from

Escherichia coliand peptide-N-glycosidase A (PNGase A)

from almonds were purchased from Roche Molecular

Biochemicals (Meylan, France) All other reagents were of

highest quality available

Isolation of hLf cDNA and vector construction

hLf cDNA was according to Salmon et al [21], and

expression vectors containing the lactoferrin sequence, fused

to the sporamin signal peptide from sweet potato for

secretion, were obtained for maize as described in [17] and

for tobacco as described in [21]

Transformation, production and purification

of maize Lf and tobacco Lf

As described previously, three successive generations of

transgenic corn seeds were produced in a greenhouse (T1 to

T3 generations) using self-pollinations and

cross-pollina-tions with an untransformed elite inbred maize variety [17]

To obtain a greater quantity of raw material for extraction

and large scale batch purification of mLf, a field trial was

performed in the south of France throughout summer 1998

on a 0.45 ha plot of land T3 seeds were sown by the end of

May 1998 and T3 transgenic plants were crossed with the

same elite inbred maize variety as mentioned above Mature T4 seeds were then harvested in October 1998, with 32% humidity They were dried at low temperature, cleaned to eliminate the refuse of the ears and bad grains, and stored in big bags Maize Lf was extracted and purified from T4 corn seeds as described previously [17]

For tobacco, plant transformations were carried out according to Salmon et al [21] For extraction and purifi-cation of tLf, fresh tobacco leaves were harvested from the greenhouse and ground in liquid nitrogen The raw material was treated and Lf was purified by the same protocol as for maize, with the following modifications The ratio of biomass to extraction buffer volume was 1/4 and the maceration time was 2 h

Reduction, alkylation and tryptic proteolysis hLf, mLf and tLf (60 nmol of each) were solubilized in 6M guanidinium chloride at a final concentration of 5 mgÆmL)1, reduced and carboxamidomethylated as described previ-ously [22] After extensive dialysis of denaturated proteins against Tris/HCl buffer (100 mM, pH 8.0), sequencing-grade modified trypsin was added to a final enzyme-to-substrate ratio of 1/100 (w/w) and incubated 16 h at 37C Tryptic digestions were stopped by storing the hydrolysates

at)20 C

HPLC analysis of tryptic digests Peptides and glycopeptides generated by tryptic digestions were separated by RP-HPLC with a semipreparative Vydac

C18 ultrasphere (9.4· 250 mm; 5 lm) column and eluted with a linear gradient of 0–80% acetonitrile containing 0.1% (v/v) trifluoroacetic acid for 90 min at a flow rate of

2 mLÆmin)1 Elution was monitored at 214 nm and peaks were collected, lyophilized and stored at)20 C

Peptides and/or glycopeptides (2 nmol) were spotted on a silica gel 60 aluminium sheet (Merck, Germany) and revealed by using 0.2% (w/v) orcinol in a 60% (v/v) sulphuric acid solution

Enzymatic deglycosylation of glycopeptides The N-linked oligosaccharides from hLf glycopeptides (50 pmol) were enzymatically released with 0.25 U PNGase F in ammonium bicarbonate buffer (20 mM,

pH 8.0) whereas those of mLf and tLf were released with 0.0125 mU PNGase A in sodium acetate buffer (100 mM,

pH 5.1) After overnight incubation at 37C, peptides were desalted by C18 phase Sep-Pak cartridges (Waters, MA, USA) and eluted with 80% acetonitrile containing 0.1% trifluoroacetic acid After lyophilization, peptides (10 pmol) were analysed by MALDI-TOF mass spectrometry

Mass spectrometry analyses of peptides and glycopeptides

MALDI-TOF.MALDI-TOF mass spectra were acquired

on a Voyager Elite (DE-STR) linear or reflectron mass spectrometer (Perspective Biosystems, Framingham, MA, USA) equipped with a pulsed nitrogen laser (337 nm) and

a gridless delayed extraction ion source Samples were

analysed in delayed extraction mode using an accelerating

voltage of 20 kV, a pulse delay time of 200 ns and a grid

voltage of 66% Detector bias gating was used to reduce the

ion current below masses of 500 Da

Samples were prepared by mixing directly on the target

1 lL of peptide or glycopeptide solution (10–50 pmol)

with 1 lL of 2,5-dihydroxybenzoic acid matrix solution

(10 mgÆmL)1 in CH3OH/H2O, 70 : 30, v/v) The samples

were allowed to dry for about 5 min at room temperature

Between 150 and 200 scans were averaged for every

spectrum shown

ES-MS and CID-MS-MS Mass spectra were acquired on

Micromass Quattro II triple quadripole mass spectrometer

operating with an API ion source in positive ion electrospray

mode Glycopeptide samples were diluted in CH3CN/H2O

(50/50, v/v), 0.2% (v/v) formic acid, to a final concentration

of about 15 pmolÆlL)1and infused at 8 lLÆmin)1 M ass

spectra were acquired by scanning MS1 with appropriate

mass range, while MS-MS analyses were performed by

transmitting the appropriate precursor ion from MS1 to the

collision cell The collision gas used was argon at a pressure

of 4.9· 10)3mbar with an appropriate collision energy

(25–50 eV) Product ions were scanned with MS2

Peptide sequencing Nano-electrospray mass spectrometric

analyses were performed using a QSTAR Pulsar

quadru-pole time-of-flight (Q-TOF) mass spectrometer (AB/MDS

Sciex, Toronto, Canada) equipped with a nano-electrospray

ion source (Protana, Odense, Denmark) Peptides dissolved

in MeOH/H2O (50/50, v/v), 0.1% (v/v) formic acid at a

concentration of 10 pmolÆlL)1 were sprayed from

gold-coated medium length borosilicate capillaries (Protana,

Odense, Denmark) A potential of ± 800 V was applied to

the capillary tip The declustering potential varied between

± 60 V and ± 110 V and the focusing potential was set at

)100 V

The molecular ions were selected in the quadrupole

analyser and partially fragmented in the hexapole collision

cell, with the pressure of collision gas (N2) 5.3· 10)5Torr

The collision energy was varied between 40 and 110 eV

depending on the sample

QSTAR spectra were acquired by accumulation of 10

MCA scans over the m/z range 700–1000 Da and 900–

2000 Da for MS analyses, and over m/z 150–1000 for

MS-MS analyses Signal detection was performed with a

multichannel plate detector and time to digital conversion

Resolution was measured as the full width at half maximum

and was 7000 in the used mass range This was measured for

both MS and MS-MS modes All signals were

mono-isotopically resolved and TOF calibration was performed

with a solution of 4 pmolÆlL)1 of taurocholic acid in

acetonitrile/H2O (50/50, v/v), 2 mMammonium acetate

Results

Purification of N-glycopeptides from trypsin digestion

of natural and recombinant lactoferrins

The hydrolysates obtained after tryptic digestion of reduced,

alkylated hLf, mLf and tLf were fractionated by RP-HPLC

and eluted with a linear gradient of 0–80% acetonitrile in

0.1% (v/v) trifluoroacetic acid The elution profiles are shown in Fig 1 Even if slight differences can be observed, the three elution profiles were very similar, indicating that the trypsin cleavage sites were identical between natural and both recombinant Lfs The different fractions were collected and analysed by MALDI-TOF The glycopeptide-contain-ing fractions were also confirmed by orcinol stainglycopeptide-contain-ing In each case, we have identified two main glycopeptide fractions, eluted at 47 min for fraction 1 and at 56 min for fraction 2, which correspond to the glycosylation sites Asn479 and Asn138, respectively These fractions were named H1and

H2, M1 and M2, T1 and T2 for hLf, mLf and tLf, respectively, as indicated in Fig 1 In addition, we have also identified a peptide fraction named H3, M3 and T3, which corresponds to the unglycosylated Asn624 site Structural analysis of N-glycopeptides

Glycopeptide-containing fractions were analysed by MALDI-TOF before and after N-glycanase treatments

Fig 1 Fractionation of tryptic digests of natural and recombinant lactoferrins by RP-HPLC Trypsin digests of hLf (A), mLf (B) and tLf (C) were fractionated on a Vidac C 18 ultrasphere column and eluted by

a linear gradient (0–80%) of acetonitrile containing 0.1% (v/v) tri-fluoroacetic acid Peptides were detected at 214 nm The glycopeptide-containing fractions are indicated.

MALDI mass spectra of the glycopeptide fractions reveal

the heterogeneity of these fractions suggesting several

glycoforms and/or peptidic backbone mixtures (Fig 2)

To identify the glycopeptides, PNGase treatment was

carried out (Fig 3) The MALDI mass spectra obtained

after deglycosylation of the fractions H1, M1and T1show

the disappearance of peaks between 3800 and 4600 Da for

hLf, and between 3000 and 3700 Da for the two

recombin-ant lactoferrins In contrast, these spectra reveal the

appearance of peaks exhibiting [M+ H]+ ions at m/z

2049, 2053, 2097 and 2154 The peaks at 2053 and 2097 Da

correspond to the peptide TAGWNIPMGLLFNQTGSCK

(467–485) (Asn479 peptide), which has been identified as the

second N-glycosylation site in hLf, with an oxidized

methionine (expected average mass 2054.37) or the expected

carboxamidomethylated cysteine (expected average mass

2095.42), respectively The ion at m/z 2049 corresponds to

the Asn479 peptide with a carboxamidomethylated cysteine

and an oxidized methionine, which has lost the

methylsul-foxide moiety [23] (expected average mass 2047.37) The

peak at 2154 Da could correspond to this peptide with an

extra carboxamidomethylated amino acid, that sequencing

trials did not allow us to locate either by mass spectrometry

or by Edman degradation

Mass spectra obtained for the N-glycosylation site Asn479 of natural (H1) and recombinant lactoferrins (M1 and T1) are presented in Fig 2 Concerning H1, the mass spectrum displays five main glycopeptides exhibiting [M+ H]+ions at m/z 3921.16, 4066.70, 4212.43, 4358.24 and 4503.76 that are consistent with oligosaccharide struc-tures Hex5(dHex)HexNAc4, NeuAcHex5HexNAc4, Neu-AcHex5(dHex)HexNAc4, NeuAcHex5(dHex2)HexNAc4 and NeuAc2Hex5(dHex)HexNAc4 (Hex, hexose; dHex, deoxyhexose) linked to the Asn479 peptide m/z 2154 (Fig 2A) Three other minor ions at m/z 3863.84, 4008.99 and 4154.98 are also detected, which correspond to the oligosaccharide structures Hex5(dHex)HexNAc4, NeuAc-Hex5HexNAc4, NeuAcHex5(dHex)HexNAc4linked to the Asn479 peptide m/z 2097 As shown in Fig 2B, MALDI mass measurements of M1 indicate one major peak exhibiting [M+ H]+ion at m/z 3220.95 that is consistent with the oligosaccharide structure Hex3(dHex)(Pen)Hex-NAc2 (Pen, pentose) and three minor glycopeptides exhi-biting [M+ H]+ions at m/z 3059.02, 3423.88 and 3626.77 consistent with the structures Hex2(dHex)(Pen)HexNAc2, Hex3(dHex)(Pen)HexNAc3 and Hex3(dHex)(Pen)Hex-NAc4, respectively, all structures being linked to the Asn479 peptide Glycopeptide T MALDI-MS analysis

Fig 2 MALDI-TOF mass spectra of hLf, mLf and tLf Asn479-glycopeptides After HPLC fractionation, glycopeptides (25–50 pmol) were analysed by MALDI-TOF using 2,5-dihydroxybenzoic acid as matrix (A) The hLf spectrum was recorded in positive ion linear mode, while the mLf (B) and tLf (C) spectra were recorded in positive ion reflective mode d, Mannose; j, N–acetylglucosamine;

s, galactose; , a1,3-fucose; , a1,6-fucose;

n, sialic acid; , xylose 5.

(Fig 2C) displays three major glycopeptides at 3222.62,

3425.58 and 3628.60 Da consistent with oligosaccharide

structures Hex3(dHex)(Pen)HexNAc2, Hex3(dHex)(Pen)

HexNAc3 and Hex3(dHex)(Pen)HexNAc4, respectively,

probably linked to the oxidized methionine Asn479 site

The [M+ H]+ions at m/z 3265.04, 3468.08 and 3672.15

correspond to these glycoforms linked to the

carbamido-methylated cysteine Asn479 site

Concerning the glycopeptidic fractions H2, M2 and

T2, we used the same strategy of analysis by MALDI-MS

(data not shown) Spectra obtained after deglycosylation of

H2, M2and T2reveal one major peak corresponding to a

[M+ H]+ ion at m/z 3232.05, 3232.69 and 3232.45,

respectively This peak at 3232 Da corresponds exactly to

the first N-glycosylation site TAGWNVPIGTLRPFL

NWTGPPEPIEAAVAR(123–152) (Asn138) The sequence

of this peptide was also verified by mass spectrometry

MALDI-MS and ES-MS analysis of H2 allowed us to

detect three glycopeptide peaks represented by [M+ H]+

ions at m/z of 5291, 5437 and 5582 consistent with

NeuAc-Hex5(dHex)HexNAc4, NeuAcHex5(dHex2)HexNAc4 and

NeuAcHex(dHex)HexNAc linked to the Asn138 peptide

MALDI mass measurements of M2 indicate two glyco-peptidic [M+ H]+ ions at m/z 4239.38 and 4403.41 matching with Hex2(dHex)(Pen)HexNAc2 -Asn138peptide-and Hex3(dHex)(Pen)HexNAc2-Asn138 peptide- struc-tures Glycopeptide fraction T2MALDI spectrum displays three major ions at 4403.39, 4606.32 and 4809.47 corres-ponding, respectively, with Hex3(dHex)(Pen)HexNAc2 -Asn138 peptide-, Hex3(dHex)(Pen)HexNAc3-Asn138 peptide- and Hex3(dHex)(Pen)HexNAc4-Asn138 peptide-glycopeptidic structures

Analysis of the Asn624 site The MALDI-TOF analysis of the different peptide fractions collected after RP-HPLC fractionation of tryptic hydroly-sates allowed us to detect the potential glycosylation site Asn624 A peptide fraction (H3, M3and T3) eluted at the same elution time (24 min) was shown to correspond to the unglycosylated peptide site NGSDCPDK(624–631) How-ever, we were not able to identify any glycosylated form of this peptide The MALDI spectra obtained for the three lactoferrins were very similar and the spectrum obtained for

Fig 3 MALDI-TOF mass spectra of hLf,

mLf and tLf Asn479-glycopeptides after

PNGase treatment N-glycopeptides from hLf

and recombinant lactoferrins were

deglyco-sylated with PNGase F and PNGase A,

respectively Peptides were then desalted on

C 18 phase Sep-Pak cartridges and analysed by

MALDI mass spectrometry using

2,5-dihy-droxybenzoic acid as matrix (A) The hLf

spectrum was recorded in positive ion linear

mode; mLf (B) and tLf (C) spectra were

recorded in positive ion reflective mode The

masses of Na adducts are indicated in smaller

font.

mLf is shown in Fig 4A Two peaks at m/z of 893.26 and

915.26 were assigned to [M+ H]+and [M+ Na]+ions

of the unglycosylated Asn624 site, respectively Peptide

sequence was analysed by nano-electrospray, by selecting

the dicharged ion at m/z 447.10 that generated by fragmen-tation five [M+ H]+ions at m/z 778.31, 721.21, 634.22, 519.20 and 359.18 (Fig 4B) The mass increments between these peaks, i.e 57, 87, 115 and 160 Da, correspond exactly

to the masses of glycine, serine, aspartic acid and carb-oxamidomethylated cysteine, respectively, amino acid sequence GSDC, that corresponds to the glycosylation site Asn624

Discussion

The present paper reports for the first time the site-by-site N-glycosylation pattern of recombinant human lactoferrin expressed in two different plant expression systems: the endosperm of maize seeds, a monocotyledon expression system allowing full-scale commercial production, and tobacco leaves used as a model of a dicotyledon plant Human Lf is a convenient model to analyse the details of the glycosylation potential of plant expression systems because data are available on the glycosylation of native Lf and of recombinant Lf produced in other systems including mammalian cells [24], lepidopteran cells [25], and transgenic mice [26] N-glycosylation of milk derived human lacto-ferrin has been extensively studied, showing that hLf contains two N-acetyllactosamine-type N-glycans, more

or less fucosylated and sialylated Moreover, a third N-glycosylation site (Asn624) is located in the C-terminal part of the glycoprotein but is mostly unglycosylated [18,19] Human lactoferrin is also an interesting model because it

is a natural defence iron-binding protein that has been found to possess antibacterial, antifungal, antiviral, antineoplastic and anti-inflammatory activity and is considered as a novel therapeutic with broad spectrum potential [27]

The relative proportion of glycans, estimated from the MALDI-TOF spectra of both N-glycopeptides (Asn138 and Asn479) of natural and recombinant Lf, are summar-ized in Table 1 As observed in the natural Lf, Asn138 and Asn479 sites in the recombinant proteins are substituted by

Fig 4 Mass spectrometry analysis of mLf glycosylation site Asn624.

(A) MALDI-TOF mass spectrum of mLf peptide fraction M 3 The

peptide fraction M 3 has been analysed by MALDI-TOF using

2,5-dihydroxybenzoic acid as matrix and spectrum was recorded in

positive ion reflective mode (B) Sequencing by nano-electrospray mass

spectrometry Peptides from fraction M 3 were dissolved in MeOH/

H 2 O and analysed with a QSTAR quadrupole time-of-flight mass

spectrometer The molecular ion at m/z 447.10 was selected and

frag-mented to determine the amino acid sequence of the corresponding

peptide The deduced peptidic sequence is indicated on the spectrum.

Table 1 Relative amounts of N-glycans detected onto hLf, mLf and tLf N-glycosylation sites Asn138 and Asn479.

mature N-glycans Moreover, the third site is

unglycosyl-ated As in human [17], both N-glycosylation sites of tLf

and mLf are N-glycosylated by similar structures

N-gly-cans found on both sites of mLf and tLf are mostly of the

paucimannose-type, substituted by a bisecting b1,2-xylose

and a1,3-fucose residue linked to the proximal GlcNAc

(compound 7) However, the N-glycan structures of tLf

contain a remarkably higher level of terminal GlcNAc than

the corresponding structures isolated from mLf Significant

amounts of compounds 8 (GlcNAc1XylFucMan3

Glc-NAc2) and 9 (GlcNAc2XylFucMan3GlcNAc2) were

iden-tified in the tLf spectrum, whereas these glycans were

virtually absent in the spectrum of mLf (Fig 2 and

Table 1) These results clearly indicated that the first steps

of N-glycosylation are similar in plants and humans and

that the observed differences only arise from the specificity

of the Golgi plant glycosyltransferases and from

post-Golgi degradations of the matured plant N-glycans In

parallel, no complex-type N-glycans with Lewisaterminal

sequence have been found either in mLf or in tLf The lack

of complex type structures with Lewisa determinants has

also been reported for other monocotyledons and

dicotyl-edons endogenous glycoproteins [13] and several studies of

the N-glycosylation of tobacco recombinant glycoproteins

have also shown the absence of such complex-type

N-glycans [28–30]

The transfers of bisecting b1,2-xylose and a1,3-linked

core fucose require the presence of at least one terminal

GlcNAc [31] As the N-glycans identified in both plants

contain both epitopes, the higher proportion of

GlcNAc-containing glycans in tLf mainly reflects differences in

N-acetylglucosaminidase activities that govern the

biosyn-thesis of paucimannose-type glycans, after maturation of

the N-glycans in the Golgi compartment

These changes in glycosylation pattern could be also

related to a difference in glycosylation in seeds and leaves,

a different subcellular localization and/or to a different

developmental stage of the plants Indeed, Elbers et al [28]

have recently shown that the developmental stage of

tobacco leaves influences the N-glycosylation of transgenic

IgG, with a higher proportion of GlcNAc-containing

glycans in older leaves compared to younger ones

The differences in glycosylation patterns of plant and

mammalian cells can represent a limitation for the

produc-tion of some recombinant therapeutic glycoproteins of

mammalian origin in transgenic plants, and efforts are

underway to obtain the in planta conversion of N-glycans

to a human-compatible type Recently, tobacco cells

transformed with human b1,4-galactosyltransferase were

used to evaluate the possibility to galactosylate foreign

glycoproteins such as horseradish peroxidase [32] or mouse

antibody [33] For example, coexpression of human

b1,4-galactosyltransferase and heavy and light chains of mouse

antibody results in the synthesis in tobacco plants, of a

recombinant antibody that exhibits 30% of galactosylated

N-glycans [33]

Even if the terminal GlcNAc content in N-glycans of

maize origin appears to be low and that outcrossing of

transgenic maize could not be excluded, the industrial

advantages of maize seeds as a production system for

recombinant proteins, compared to tobacco leaves, such as

the absence of toxic compounds, the possibility of low cost

storage of biomass and the ease of extracting protein from grains [34] has led us to initiate the engineering of maize N-glycosylation

Acknowledgements

This work was supported in part by the University of Sciences and Technologies of Lille, by a grant (Saut Technologique) by the French Research Ministry and a grant CIFRE of the French ANRT to

B Samyn-Petit We thank our colleagues in Plant Production and the Pilot Unit of Meristem-Therapeutics with help in growing and extracting the lactoferrin plants.

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