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-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

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;
2
Meristem 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 Lewis
a
epitope 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 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

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 (Man
3
GlcNAc
2
-based structure), even if complex-type
N-glycans with a Lewis
a
terminal sequence (Galb1–
3[Fuca1–4]GlcNAc-R) have been reported [11]. First
described in sycamore [12,13], the Lewis
a
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:
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,

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 C
18
ultrasphere (9.4 · 250 mm; 5 lm) column.
Recombinant peptide-N-glycosidase F (PNGase F) from
Escherichia coli and 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 6
M
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 m
M
, 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 37 °C.
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
C
18
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
2mLÆmin
)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 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

1 lL of peptide or glycopeptide solution (10–50 pmol)
with 1 lL of 2,5-dihydroxybenzoic acid matrix solution
(10 mgÆmL
)1
in CH
3
OH/H
2
O, 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 CH
3
CN/H
2
O
(50/50, v/v), 0.2% (v/v) formic acid, to a final concentration
of about 15 pmolÆlL
)1
and infused at 8 lLÆmin
)1
.Mass
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
)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

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/H
2
O (50/50, v/v), 2 m
M
ammonium 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 staining. 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 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

(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.
Ó FEBS 2003 N-glycosylation of maize and tobacco lactoferrin (Eur. J. Biochem. 270) 3237
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 H
1
,M
1
and T
1
show
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 (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

,Neu-
AcHex
5
(dHex)HexNAc
4
,NeuAcHex
5
(dHex
2
)HexNAc
4
and NeuAc
2
Hex
5
(dHex)HexNAc
4
(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)HexNAc
4
,NeuAc-
Hex
5
HexNAc
4
,NeuAcHex
5

(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,

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.
3238 B. Samyn-Petit et al.(Eur. J. Biochem. 270) Ó FEBS 2003
(Fig. 2C) displays three major glycopeptides at 3222.62,
3425.58 and 3628.60 Da consistent with oligosaccharide
structures Hex3(dHex)(Pen)HexNAc
2
, Hex3(dHex)(Pen)
HexNAc
3
and Hex3(dHex)(Pen)HexNAc
4
, 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 H

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

(dHex)HexNAc
4
,NeuAcHex
5
(dHex
2
)HexNAc
4
and
NeuAc
2
Hex
5
(dHex)HexNAc
4
linked to the Asn138 peptide.
MALDI mass measurements of M
2
indicate two glyco-
peptidic [M + H]
+
ions at m/z 4239.38 and 4403.41
matching with Hex2(dHex)(Pen)HexNAc
2
-Asn138peptide-
and Hex3(dHex)(Pen)HexNAc
2
-Asn138 peptide- struc-
tures. Glycopeptide fraction T
2

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

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.
Ó FEBS 2003 N-glycosylation of maize and tobacco lactoferrin (Eur. J. Biochem. 270) 3239
mLfisshowninFig.4A.Twopeaksatm/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.
3240 B. Samyn-Petit et al.(Eur. J. Biochem. 270) Ó FEBS 2003
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 (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

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.
References
1. Doran, P.M. (2000) Foreign protein production in plant tissues
cultures. Curr. Opin. Biotechnol. 11, 199–204.
2. Daniell, H., Streatfield, S.J. & Wycoff, K. (2001) Medical mole-
cular farming: production of antibodies, biopharmaceuticals and
edible vaccines in plants. Trends Plant Sci. 6, 219–226.
3. Tacket, C.O., Mason, H.S., Losonsky, G., Estes, M.K., Levine,
M.M. & Arntzen, C.J. (2000) Human immune responses to a

novel Norwalk virus vaccine delivered in transgenic potatoes.
J. Infect. Dis. 182, 302–305.
4. Lo
¨
nnerdal, B. (2002) Expression of Human Milk Proteins in
Plants. J. Am. Coll. Nutr. 21, 218S–221S.
5. Merle, C., Perret, S., Lacour, T., Jonval, V., Hudaverdian, S.,
Garrone, R., Ruggiero, F. & Theisen, M. (2002) Hydroxylated
human homotrimeric collagen I in Agrobacterium tumefaciens–
mediated transient expression and in transgenic tobacco plants.
FEBS Lett. 515, 114–118.
6. Staub, J.M., Garcia, B., Graves, J., Hajdukiewicz, P.T., Hunter,
P., Nehra, N., Paradkar, V., Schlittler, M., Carroll, J.A., Spatola,
L., Ward, D.Ye, G. & Russell, D.A. (2000) High-yield production
of a human therapeutic protein in tobacco chloroplasts. Nat.
Biotechnol. 18, 333–338.
7. Peeters, K. De Wilde, C. De Jaeger, G. Angenon, G. & Depicker,
A. (2001) Production of antibodies and antibody fragments in
plants. Vaccine 19, 2756–2761.
8. Sto
¨
ger, E. Vaquero, C. Torres, E. Sack, M. Nicholson, L.
Drossard, J. Williams, S. Keen, Perrin, D. & Christou, Y. (2000)
Cereal crops as viable production and storage systems for
pharmaceutical scFv antibodies. Plant Mol. Biol. 42, 583–590.
9. Lerouge, P., Cabanes-Macheteau, M., Rayon, C., Fitchette-Laine
´
,
A.C., Gomord, V. & Faye, L. (1998) N-glycoprotein biosynthesis
in plants: recent developments and future trends. Plant. Mol. Biol.

38, 31–48.
10. Jenkis, N., Parekh, R.B. & James, D.C. (1996) Getting the gly-
cosylation right. implications for the biotechnology industry. Nat.
Biotechnol. 14, 975–981.
11. Staudacher, E., Altmann, F., Wilson, I.B. & Marz, L. (1999)
Fucose in N-glycans: from plant to man. Biochim. Biophys. Acta
1473, 216–236.
12. Fitchette-Laine, A.C., Gomord, V., Cabanes-Macheteau, M.,
Michalski, J.C., Saint Macary, M., Foucher, B., Cavelier, B.,
Hawes, C., Lerouge, P. & Faye, L. (1997) N-glycans harboring the
Lewis a epitope are expressed at the surface of plant cells. Plant J.
12, 1411–1417.
13. Wilson, I.B., Zeleny, R., Kolarich, D., Staudacher, E., Stroop,
C.J., Kamerling, J.P. & Altmann, F. (2001) Analysis of Asn-linked
glycans from vegeTable foodstuffs. widespread occurrence of
Lewis a, core a1,3-linked fucose and xylose substitutions. Glyco-
biology 11, 261–274.
14. Yang, B.Y., Gray, J.S.S. & Montgomery, R. (1996) The glycans of
horseradish peroxydase. Carbohydr. Res. 287, 203–212.
Ó FEBS 2003 N-glycosylation of maize and tobacco lactoferrin (Eur. J. Biochem. 270) 3241
15. Makino, Y., Shimazaki, A., Omichi, K., Odani, S. & Hase, S.
(2000) Processing pathway deduced from the structures of
N-glycans in Carica papaya. J. Biochem. 127, 1121–1126.
16. Altmann, F., Schwihla, H., Staudacher, E., Glo
¨
ss, J. & Ma
¨
rz, L.
(1995) Insect cells contain an unusual, membrane-bound
b-N-acetylglucosaminidase probably involved in the processing of

protein N-glycans. J. Biol. Chem. 270, 17344–17349.
17. Samyn-Petit, B., Gruber, V., Flahaut, C., Wajda-Dubos, J.P.,
Farrer, S., Pons, A., Desmaizieres, G., Slomianny, M.C., Theisen,
M. & Delannoy, P. (2001) N-glycosylation potential of maize: the
humanlactoferrinusedasamodel.Glycoconjugate J. 18, 519–527.
18. Spik, G., Strecker, G., Fournet, B., Bouquelet, S. & Montreuil, J.
(1982) Primary structure of the glycans from human lacto-
transferrin. Eur. J. Biochem. 121, 413–419.
19. Van Berkel, P.H.C., Van Veen, H.A., Geerts, M.E.J., De Boer,
H.A. & Nuijens, J.H. (1996) Heterogeneity in utilization of
N-glycosylation sites Asn
624
and Asn
138
in human lactoferrin: a
study with glycosylation-site mutants. Biochem. J. 319, 117–122.
20. Brock, J.H. (2002) The physiology of lactoferrin. Biochem. Cell.
Biol. 80, 1–6.
21. Salmon, V., Legrand, D., Slomianny, M C., El Yazidi, I., Spik,
G., Gruber, V., Bournat, P., Olagnier, B., Mison, D., Theisen, M.
&Me
´
rot, B. (1998) Production of human lactoferrin in transgenic
tobacco plants. Protein Express. Purif. 13, 127–135.
22. Flahaut, C., Capon, C., Balduyck, M., Ricart, G., Sautiere, P. &
Mizon, J. (1998) Glycosylation pattern of human inter-a-inhibitor
heavy chains. Biochem. J. 333, 749–756.
23. Qin, J., Fenyo, D., Zhao, Y., Hall, W.W., Chao, D.M., Wilson,
C.J., Young, R.A. & Chait, B.T. (1997) A strategy for rapid, high-
confidence protein identification. Anal. Chem. 69, 3995–4001.

24. Legrand, D., Salmon, V., Coddeville, B., Benaissa, M., Plancke,
Y. & Spik, G. (1995) Structural determination of two N-linked
glycans isolated from recombinant human lactoferrin expressed in
BHK cells. FEBS Lett. 365, 57–60.
25. Salmon, V., Legrand, D., Georges, B., Slomianny, M C.,
Coddeville, B. & Spik, G. (1997) Characterization of human
lactoferrin produced in the baculovirus expression system. Protein
Expr. Purif. 9, 203–210.
26. Nuijens, J.H., van Berkel, P.H., Geerts, M.E., Hartevelt, P.P., de
Boer, H.A., van Veen, H.A. & Pieper, F.R. (1997) Characteriza-
tion of recombinant human lactoferrin secreted in milk of trans-
genic mice. J. Biol. Chem. 272, 8802–8807.
27. Weinberg, E.D. (2001) Human lactoferrin: a novel therapeutic
with broad spectrum potential. J. Pharm. Pharmacol. 53, 1303–
1310.
28. Elbers, I.J., Stoopen, G.M., Bakker, H., Stevens, L.H., Bardor,
M., Molthoff, J.W., Jordi, W.J., Bosch, D. & Lommen, A. (2001)
Influence of growth conditions and developmental stage on
N-glycan heterogeneity of transgenic immunoglobulin G and
endogenous proteins in tobacco leaves. Plant Physiol. 126, 1314–
1322.
29. Cabanes-Macheteau, M., Fitchette-Laine, A.C., Loutelier-Bour-
his,C.,Lange,C.,Vine,N.D.,Ma,J.K.,Lerouge,P.&Faye,L.
(1999) N-Glycosylation of a mouse IgG expressed in transgenic
tobacco plants. Glycobiology 9, 365–372.
30. Dirnberger, D., Steinkellner, H., Abdennebi, L., Remy, J.J. & van
de Wiel, D. (2001) Secretion of biologically active glycoforms of
bovine follicle stimulating hormone in plants. Eur. J. Biochem.
268, 4570–4579.
31. Strasser, R., Mucha, J., Mach, L., Altmann, F., Wilson, I.B.,

Glossl, J. & Steinkellner, H. (2000) Molecular cloning and func-
tional expression of b1,2-xylosyltransferase cDNA from Arabid-
opsis thaliana. FEBS Lett. 472, 105–108.
32. Fujiyama, K., Palacpac, N.Q., Sakai, H., Kimura, Y., Shinmyo,
A., Yoshida, T. & Seki, T. (2001) In vivo conversion of a glycan to
human compatible type by transformed tobacco cells. Biochem.
Biophys. Res. Commun. 289, 553–557.
33. Bakker, H., Bardor, M., Molthoff, J.W., Gomord, V., Elbers, I.,
Stevens,L.H.,Jordi,W.,Lommen,A.,Faye,L.,Lerouge,P.&
Bosch, D. (2001) Galactose-extended glycans of antibodies
produced by transgenic plants. Proc. Natl Acad. Sci. USA 98,
2899–2904.
34. Mison, D. & Curling, J. (2000) The industrial production cost of
recombinant therapeutic proteins in transgenic corn. Biopharma-
cology 13, 48–54.
3242 B. Samyn-Petit et al.(Eur. J. Biochem. 270) Ó FEBS 2003