Comparative analysis of the site-specific N-glycosylation
of human lactoferrin produced in maize and tobacco plants
Be
´
ne
´
dicte Samyn-Petit
1
, Jean-Pierre Wajda Dubos
2
, Fre
´
de
´
ric Chirat
1
, Bernadette Coddeville
1
,
Gre
´
gory Demaizieres
2
, Sybille Farrer
2
, Marie-Christine Slomianny
1
, Manfred Theisen
2
and Philippe Delannoy
1

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 production 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

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:
Abbreviations: Lf, lactoferrin; mLf, maize recombinant lactoferrin;
tLf, tobacco recombinant lactoferrin.
(Received 20 March 2003, revised 2 June 2003, accepted 5 June 2003)
Eur. J. Biochem. 270, 3235–3242 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03706.x
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,

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

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 m
M
,
pH 8.0) whereas those of mLf and tLf were released with
0.0125 mU PNGase A in sodium acetate buffer (100 m
M
,
pH 5.1). After overnight incubation at 37 °C, peptides were
desalted by C
18
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
3236 B. Samyn-Petit et al.(Eur. J. Biochem. 270) Ó FEBS 2003
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

of 4.9 · 10
)3
mbar 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/H
2
O (50/50, v/v), 0.1% (v/v) formic acid at a
concentration of 10 pmolÆlL
)1
were sprayed from gold-
coated Ômedium lengthÕ 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 (N
2
)5.3· 10
)5
Torr.
The collision energy was varied between 40 and 110 eV
depending on the sample.
QSTAR spectra were acquired by accumulation of 10

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 H
1
and
H
2
,M
1
and M
2
,T
1
and T
2
for hLf, mLf and tLf,
respectively, as indicated in Fig. 1. In addition, we have
also identified a peptide fraction named H
3
,M
3
and T
3
,
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

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 (H
1
) and recombinant lactoferrins (M
1
and T
1
) are presented in Fig. 2. Concerning H
1
,themass
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)HexNAc
4
,NeuAcHex
5
HexNAc
4

(dHex)HexNAc
4
linked to the
Asn479 peptide m/z 2097. As shown in Fig. 2B, MALDI
mass measurements of M
1
indicate one major peak
exhibiting [M + H]
+
ion at m/z 3220.95 that is consistent
with the oligosaccharide structure Hex3(dHex)(Pen)Hex-
NAc
2
(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)HexNAc
2
,
Hex3(dHex)(Pen)HexNAc
3
and Hex3(dHex)(Pen)Hex-
NAc
4
, respectively, all structures being linked to the
Asn479 peptide. Glycopeptide T
1
MALDI-MS analysis
Fig. 2. MALDI-TOF mass spectra of hLf,

2
,M
2
and
T
2
, we used the same strategy of analysis by MALDI-MS
(data not shown). Spectra obtained after deglycosylation of
H
2
,M
2
and T
2
reveal 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 H
2
allowed us to
detect three glycopeptide peaks represented by [M + H]
+
ions at m/z of 5291, 5437 and 5582 consistent with NeuAc-
Hex
5

MALDI spectrum displays
three major ions at 4403.39, 4606.32 and 4809.47 corres-
ponding, respectively, with Hex3(dHex)(Pen)HexNAc
2
-
Asn138 peptide-
,
Hex3(dHex)(Pen)HexNAc
3
-Asn138
peptide- and Hex3(dHex)(Pen)HexNAc
4
-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 (H
3
,M
3
and T
3
)elutedatthe
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

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

(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 (GlcNAc
1
XylFucMan
3
Glc-
NAc
2
)and9(GlcNAc
2
XylFucMan
3
GlcNAc
2
)wereiden-
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 Lewis
a
terminal
sequence have been found either in mLf or in tLf. The lack
of complex type structures with Lewis
a

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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