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Plant Physiol. (1999) 119: 725-734
Characterization of N-Glycans from
Arabidopsis.
Application to a Fucose-Deficient Mutant1
Catherine Rayon,
Marion Cabanes-Macheteau,
Corinne Loutelier-Bourhis,
Isabelle Salliot-Maire,
Jérome Lemoine,
Wolf-Dieter Reiter,
Patrice Lerouge, and
Loïc Faye*
Laboratoire des Transports Intracellulaires, Centre National de la
Recherche Scientifique (CNRS)-ESA 6037 (C.R., M.C.-M., P.L., L.F.),
Spectroscopie de Masse Bioorganique (C.L.-B.), and Laboratoire de
Résonance Magnétique Nucléaire, CNRS-ESA 6014 (I.S.-M.), IFRMP 23, Université de Rouen, 76821 Mont Saint
Aignan, France; IFRMP 23, Université de Rouen, 76821 Mont Saint
Aignan, FranceLaboratoire de Chimie Biologique, CNRS-Unite, Mixte de
Recherche 111, Université de Lille, 59655 Villeneuve d'Ascq,
France (J.L.); and Department of Molecular and Cell Biology,
University of Connecticut, Storrs, Connecticut 06269 (W.-D.R.)
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ABSTRACT |
The structures of glycans
N-linked to Arabidopsis proteins have been fully
identified. From immuno- and affinodetections on blots,
chromatography, nuclear magnetic resonance, and glycosidase sequencing data, we show that Arabidopsis proteins are
N-glycosylated by high-mannose-type
N-glycans from Man5GlcNAc2 to
Man9GlcNAc2, and by xylose- and fucose
(Fuc)-containing oligosaccharides. However, complex biantenary
structures containing the terminal Lewis a epitope recently reported in
the literature (A.-C. Fitchette-Lainé, V. Gomord, M. Cabanes,
J.-C. Michalski, M. Saint Macary, B. Foucher, B. Cavalier, C. Hawes, P. Lerouge, and L. Faye [1997] Plant J 12: 1411-1417) were not
detected. A similar study was done on the Arabidopsis
mur1 mutant, which is affected in the biosynthesis of
L-Fuc. In this mutant, one-third of the Fuc residues of the xyloglucan has been reported to be replaced by L-galactose
(Gal) (E. Zablackis, W.S. York, M. Pauly, S. Hantus, W.D. Reiter,
C.C.S. Chapple, P. Albersheim, and A. Darvill [1996] Science 272:
1808-1810). N-linked glycans from the mutant were
identified and their structures were compared with those isolated from
the wild-type plants. In about 95% of all N-linked
glycans from the mur1 plant, L-Fuc residues were absent and were not replaced by another monosaccharide. However, in the remaining 5%, L-Fuc was found to be replaced by a
hexose residue. From nuclear magnetic resonance and mass spectrometry data of the mur1 N-glycans, and by analogy with data
reported on mur1 xyloglucan, this subpopulation of
N-linked glycans was proposed to be
L-Gal-containing N-glycans resulting from
the replacement of L-Fuc by L-Gal.
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INTRODUCTION |
Mutants have been widely used for biochemical studies of
N-glycosylation in yeast and mammals. Although a rapidly
increasing number of mutants are available in several plant species and
particularly in Arabidopsis, only one of them presents a clearly
identified mutation that affects the biosynthesis of
N-linked glycans. This Arabidopsis cgl mutant
lacks the N-acetyl glucosaminyltransferase I and is unable
to synthesize complex-type N-glycans (von Schaewen et al.,
1993 ). Another Arabidopsis mutant, mur1, does not
synthesize L-Fuc in the leaves (Reiter et al.,
1993). This mutant was found to be affected in the gene encoding for a
GDP-D-Man-4,6-dehydratase, a key enzyme in the
biosynthesis of L-Fuc (Bonin et al., 1997). The
structure of cell wall polysaccharides from plants carrying the
mur1 mutation has been investigated recently (Zablackis et al., 1996) and compared with cell wall polysaccharides from wild-type plants (Zablackis et al., 1995). This study has shown that in the
xyloglucan of this Fuc-deficient mutant, L-Fuc is
replaced by L-Gal, a monosaccharide structurally
similar to L-Fuc, without affecting the
biological activity of Fuc-containing oligosaccharides derived from
these polymers.
L-Fuc is a monosaccharide that is found not only in
primary cell wall polysaccharides but also in the plant
N-linked oligosaccharides. Consequently, the study of
N-glycosylation in the mur1 mutant could provide
information on the biosynthesis and roles of glycans N-linked to proteins when L-Fuc is not
available in Arabidopsis. In plants the
N-glycosylation of proteins starts by the transfer in the ER
of the oligosaccharide precursor
Glc3Man9GlcNAc2,
which is subsequently modified during the transport of the glycoprotein by glycosidases and glycosyltransferases located in the ER, the Golgi
apparatus, and the vacuole. These enzymes convert the precursor to
high-mannose-type N-glycans ranging from
Man9GlcNAc2 to
Man5GlcNAc2 and eventually
to complex-type N-glycans having an  (1,3)-Fuc and/or a
 (1,2)-Xyl residue (for review, see Lerouge et al., 1998). Recently,
some complex N-linked glycans have been described as having
two antennae containing a Lewis a epitope that is usually found on
cell-surface mammalian glycoconjugates (Fitchette-Lainé et al., 1997; Melo et al., 1997). These structures result
from the elongation of the intermediate complex-type
N-glycan by action of
(1,4)-fucosyltransferase and (1,3)-galactosyltransferase. Complex-type N-glycans have been described as
being N-linked to extracellular glycoproteins. In
contrast, vacuolar glycoproteins are N-glycosylated by
modified oligosaccharides having only an (1,3)-Fuc and/or a
(1,2)-Xyl residue linked to the core
Man3GlcNAc2 and are
named paucimannosidic-type N-glycans (Lerouge et al., 1998).
These oligosaccharides arise from the elimination from complex-type
N-glycans of terminal residues in the vacuole (Vitale and
Chrispeels, 1984).
We report in this paper the first complete structural analysis, to our
knowledge, of N-linked glycans isolated from leaves of
Arabidopsis. The strategy developed in the first part of our study was
applied to the N-glycosylation analysis of proteins isolated
from the mur1 mutant to analyze the effects of the absence of L-Fuc on the structure and the biosynthesis of
N-linked oligosaccharides. We show that in wild-type
Arabidopsis, the proteins are N-glycosylated by
high-mannose-type N-glycans and by Xyl- and Fuc-containing N-glycans previously described in other plants. Complex
biantennary structures containing terminal Lewis a epitopes were not
detected. Furthermore, as found previously in the cell wall xyloglucan
(Zablackis et al., 1996), the analytical data suggest a partial but
less prominent replacement of L-Fuc by
L-Gal in the core of the N-glycans in
the mur1 mutant.
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MATERIALS AND METHODS |
Enzymes, Chemicals, and Plant Material
PNGase A from almond was purchased from Boehringer-Mannheim.
Pepsin and -N-acetylglucosaminidase from Jack bean were
from Sigma. BioGel P4 and AG50W-X2 were from Bio-Rad.
C18 Bond Elut cartridges were from Varian
(Sugarland, TX). 2-aminopyridine (99% purity) was from Aldrich.
Wild-type Arabidopsis and mutant plants were grown under long-day
conditions in the greenhouse, and leaf material was harvested from
plants in early bolting stages.
SDS-PAGE and Western-Blot Analyses
Proteins isolated from leaves (as described in the next paragraph)
were resolved by SDS-PAGE in a homogeneous 15% polyacrylamide gel
under reducing conditions according to the method of Laemmli (1970),
and electrophoretically transferred to nitrocellulose membrane.
Affinodetection using the concanavalin A/peroxidase method (Faye and
Chrispeels, 1985) and immunodetection with antibodies directed against
Fuc- or Xyl-containing plant N-glycans were carried out,
respectively, as described in Faye et al. (1993).
Preparation of N-Linked Glycans from Protein Extracts
Isolated from Wild-Type and mur1 Plants
Protein extracts from Arabidopsis were obtained by homogenizing
100 g of freeze-dried leaf material in 2 L of 50 mM
Hepes (pH 7.5) and 2 mM sodium bisulfite containing 0.1%
SDS. Insoluble material was eliminated by centrifugation at
4400g for 15 min at 4°C. A second protein extraction was
performed using the pellet. The supernatants were pooled and filtered
through Miracloth (Calbiochem). Proteins were precipitated at 0°C
overnight with 12.5% TCA (final concentration), and the pellet was
then washed three times with 90% (v/v) acetone. The proteins were
digested with pepsin in 30 mL of 0.01 N HCl, pH
2.2, at 37°C for 48 h. Pepsin (20 mg) was dissolved in 1 mL of
buffer and one-half of the enzyme solution was added at 0 and 24 h, respectively. The pepsin digest was neutralized with NaOH, heated at
100°C for 5 min, and lyophilized. The sample was desalted on a BioGel
P4 column (70 × 2.6 cm) equilibrated with 0.1 N acetic acid. A portion of each fraction was
assayed for monosaccharide composition. Glycopeptide fractions were
pooled and then deglycosylated with 1 milliunit of PNGase A in a 100 mM sodium acetate buffer (pH 5.0) overnight at
37°C (Tomiya et al., 1987). N-glycans were purified by
successive elutions through a AG50W-X2 column (5 × 1 cm) and a
C18 Bond Elut cartridge. Complex-type N-glycans were separated from high-mannose-type
oligosaccharides by affinity chromatography on a concanavalin
A-Sepharose 4B column as described in Montreuil et al. (1986).
Fluorescent Derivatization of N-Glycans with
2-Aminopyridine
Freeze-dried N-linked oligosaccharides were coupled to
2-aminopyridine as described previously by Hase et al. (1984). PA
oligosaccharides were separated from the excess of reagent by gel
permeation on a BioGel P4 column (40 × 1.6 cm) equilibrated in 50 mM ammonium acetate buffer, pH 6.0.
HPAEC Analysis
Chromatography of N-linked glycans was achieved by
HPAEC-PAD. Analytical HPAEC-PAD was performed on a DX500 system
equipped with a GP 40 gradient pump, an ED 40 detector, and a CarboPac PA1 column (250 × 4.6 mm, Dionex, Sunnyvale, CA). Analysis
of the monosaccharide composition was carried out by hydrolysis of the
glycan fraction with 2 N trifluoroacetic acid at
100°C for 2 h, followed by analysis of the resulting
monosaccharides using 16 mM NaOH at 1 mL
min 1 as described in Hardy (1989). Elution of
the high-mannose-type N-glycans was carried out using a
linear gradient from 0 to 100 mM NaOAc in 100 mM NaOH at 1 mL min1 over
30 min.
HPAEC of PA oligosaccharides was achieved by dissolving the sample in
0.1 N NaOH and elution on a CarboPac PA1 column using a
gradient from 0 to 100 mM NaOAc in 100 mM NaOH
at 1 mL min1 over 60 min. PA oligosaccharides
were detected using a FL 2000 system (Spectra-Physics, San Jose,
CA) fluorescence detector with excitation and emission
wavelengths at 320 and 400 nm, respectively. PA oligosaccharides were
collected and desalted by gel filtration on a Sephadex G-10 column
(30 × 1 cm) equilibrated and eluted in 50 mM sodium
acetate, pH 6.0.
Reverse-Phase HPLC
A liquid chromatograph equipped with a SFM-25 fluorescence
spectrophotometer (Kontron, Milan, Italy) was used. PA
oligosaccharides were separated on a Spherisorb ODS2
C18 column (250 × 4.6 mm, Prolabo, Creteil,
France). Elution of PA oligosaccharides was performed at a flow
rate of 1 mL min1 at 55°C using solvent A (10 mM sodium phosphate buffer, pH 3.8), and solvent B (10 mM sodium phosphate buffer, pH 3.8, 0.5% 1-butanol) (Tomiya et al., 1988). After injection of the sample, the ratio of
solvent B to A increased with a linear gradient from 20:80 to 50:50
over 60 min. PA oligosaccharides were detected by fluorescence using
excitation and emission wavelengths of 320 and 400 nm, respectively.
-N-Acetylglucosaminidase Digestion of PA
Oligosaccharides
PA oligosaccharides were isolated by HPAEC and then digested with
1 unit of -N-acetylglucosaminidase in a 50 mM sodium acetate buffer, pH 5.0, at 25°C. The
digest was analyzed by HPAEC with fluorescence detection after 1 and
18 h of incubation time.
NMR
1H-NMR was performed at 600 MHz on a
spectrometer (model DMX600, Bruker, Billerica, MA). The spectrum was
recorded at 297 K in 2H oxide
(2H2O 99.97). Chemical
shifts were expressed in parts per million downfield from
internal tetrain etnyl silare and with an 2H
water presaturation sequence.
Electrospray MS
The electrospray ionization mass experiments were carried out on a
triple-quadrupole mass spectrometer (Quattro II, Micromass, Manchester,
UK). Samples were dissolved in water:methanol:acetic acid
(49:49:2) at a concentration of approximatively 50 pmol
µL1 and the solution was infused into the
electrospray ion source by a syringe pump (Harvard Apparatus, South
Natick, MA). Optimization of the parameters was performed both
in the MS and MS-MS mode using (Glc)n-PA. Typically, best conditions
were defined for a cone voltage of about 60 V, a collision energy range
between 30 and 40 eV, and an Ar gas pressure at 4.0 mTorr. Spectra were
recorded at a scan speed of 10 s and were smoothed 1 time.
| |
RESULTS |
Western-Blot Analysis of Protein Extracts from Wild-Type and
mur1 Arabidopsis Plants
A first analysis of the N-linked glycans associated to
proteins isolated from leaves of the wild-type and the mur1
mutant of Arabidopsis was obtained by affino- and immunodetections on blots using specific probes. We used antibodies of well-established specificities for the different oligosaccharide epitopes that are found
in plant N-glycans, i.e. the (1,2)-Xyl residue and the
(1, 3)-Fuc residue linked to the chitobiose unit (Faye et al.,
1993). Glycoproteins from wild-type plants were detected on western
blots by affinodetection using concanavalin A (Faye and Chrispeels,
1985) (Fig. 1B, lane 1), as well as by
immunoblotting with antibodies directed against the (1,2)-Xyl
residue (Fig. 1C, lane 1) or directed against the -1,3 Fuc residue
(Fig. 1D, lane 1) linked to the core
Man3GlcNAc2 (Faye et al.,
1993). Similar results were obtained on the protein extract from
mur1 plants, except that no protein was detected by the
antibodies specific for the (1,3)-Fuc residue linked to the proximal
glucosamine unit (Fig. 1D, lane 2). Some differences in the detection
level of some glycoproteins (see the glycoprotein band at 35 kD) were observed, but the identity of these glycoproteins was not investigated further. Protein extracts from both the wild-type and mur1
plants were also analyzed by immunoblotting with antibodies directed against Lewis a epitopes (data not shown). In contrast to results obtained in other plants, only very weak signals were detected in both
extracts, confirming that such epitopes are almost completely absent in
Arabidopsis leaves, as previously reported in Fitchette-Lainé et
al. (1997). As a consequence, these results indicate that Arabidopsis leaf proteins are N-glycosylated by both high-mannose and
paucimannosidic or complex-type N-glycans of limited size
(no Lewis a-containing N-glycans). Similar results were
obtained with the mur1 mutant, except for the absence of
detection of the (1,3)-Fuc epitope as expected from the previous
results on this mutant (Reiter et al., 1993).
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| Figure 1.
SDS-PAGE and western-blot analysis of protein
extracts from leaves of wild-type Arabidopsis (lanes 1) or
mur1 mutant (lanes 2). A, Silver staining; B,
affinodetection with concanavalin A; C, immunodetection with antibodies
specific for (1,2)-Xyl-containing plant N-glycans; D,
immunodetection with antibodies specific for (1,3)-Fuc-containing
plant N-glycans.
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Structural Identification of N-glycans Synthesized by
Arabidopsis Wild-type and mur1 Plants
The complete structural analysis of N-linked glycans
associated with proteins from leaves of wild-type Arabidopsis and
mur1 plants was performed to investigate whether the partial
replacement of L-Fuc by
L-Gal observed in the cell wall xyloglucan
(Zablackis et al., 1996) also occurs in the glycoproteins of the
mur1 mutant. Since the N-glycosylation of
proteins in the wild-type Arabidopsis has not yet, to our knowledge,
been investigated in detail, N-linked glycans from both the
wild-type and the Fuc-deficient mutant were analyzed. After TCA
precipitation proteins from both wild-type and mur1 leaves
were digested with pepsin. The resulting glycopeptides were isolated by
size-exclusion chromatography on a BioGel P4 column and the fraction
corresponding to glycopeptides from the wild-type and the
mur1 plants were further treated with PNGase A. PNGase A
from sweet almond is able to release both high-mannose-type and
modified plant N-glycans presenting or not presenting an
(1,3)-Fuc residue linked to the proximal glucosamine unit (Taga et
al., 1984). The reducing N-linked oligosaccharides released
from the wild-type Arabidopsis and mur1 leaves were
fractionated by affinity chromatography on a concanavalin
A-Sepharose 4B column to separate paucimannosidic and complex-type
N-glycans (concanavalin A fraction)
from high-mannose-type N-glycans (concanavalin
A+ fraction). The HPAEC-PAD profile of the
concanavalin A+ fraction isolated from the
wild-type plants (Fig. 2) shows a mixture
of high-mannose-type N-glycans, which were identified as
structures from Man5GlcNAc2
to Man9GlcNAc2 (Man-5 to
Man-9, see Fig. 3) by comparison of their
retention times in HPAE-PAD chromatography with standard
oligosaccharides from porcin thyroglobulin as previously described
(Rayon et al., 1996). The high-mannose-type N-glycans
isolated from the mur1 plants were found to be identical and
in the same ratio as those of the wild-type plants (data not shown).
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| Figure 2.
HPAEC-PAD profile of high-mannose-type
N-glycans Man5GlcNAc2 to
Man9GlcNAc2 (Man-5 to Man-9) isolated from
wild-type Arabidopsis leaves. Corresponding structures are represented
in Figure 3.
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| Figure 3.
Abbreviations and structures of high-mannose-type
N-glycans Man5GlcNAc2 to
Man9GlcNAc2 (Man-5 to Man-9) isolated from
wild-type Arabidopsis leaves.
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The N-glycans contained in the concanavalin
A fraction from both the wild-type and the
mur1 plants were analyzed by HPAEC-PAD. The profiles were
composed of a complex mixture of unresolved peaks. As a consequence,
these oligosaccharides were reductively aminated with 2-aminopyridine,
and the resulting PA oligosaccharides were then analyzed with HPAEC
using a fluorescence detector. Under these conditions all labeled
glycans from both preparations were found to be well resolved (Fig.
4). The elution profile of the PA
oligosaccharides obtained from wild-type plants showed the presence of
six major compounds (Fig. 4A). The structures of these oligosaccharides
were identified by comparison with standard glycans and by
glycosidase sequencing. Compounds a through h were collected separately. Glycans a and e were identified as the PA derivatives of
Man3XylFucGlcNAc2
(M3XFGN2, Fig.
5) and
Man3XylGlcNAc2
(M3XGN2, Fig. 5)
oligosaccharides, respectively, by comparison of the retention time
with reference standards. The structures of the other
oligosaccharides were further determined after digestion with
-N-acetylglucosaminidase, an enzyme that specifically
releases terminal -GlcNAc residues. The material collected
in peak d was found to be progressively converted to compounds b+c and
then to compound a during -N-acetylglucosaminidase treatment (Fig. 6).
-N-acetylglucosaminidase action on the material in peak
b+c released a single -GlcNAc residue, leading to compound a. As a
consequence, compounds b+c and d were identified as Xyl and
Fuc-containing oligosaccharides bearing one or two terminal glucosamine
residues, respectively. Peaks f+g and h were analyzed in the same
manner and were found to correspond to Xyl-containing glycans bearing
one and two terminal glucosamine residues, respectively. Peaks b+c and
f+g were subjected to a second chromatography analysis using a
reverse-phase C18 column. In this case, both
fractions were shown to be split further into two components (Fig. 4A,
including boxed insets). PA derivatives of plant complex-type
N-glycans bearing a single terminal GlcNAc residue have been
described to be efficiently separated on a reverse-phase column. The
isomers having the glucosamine unit associated to the mannose arm
(1,3)-linked to the -mannose residue
(GN3-M3XFGN2 and
GN3-M3XGN2) are eluted sooner in these conditions than N-linked glycans having a
terminal GlcNAc residue on the (1,6)-antennae (Takahashi et al.,
1986; Hayashi et al., 1990). In conclusion, paucimannosidic and
complex-type oligosaccharides N-linked to glycoproteins from
Arabidopsis can be classified into two groups: Xyl- and
Fuc-containing oligosaccharides having zero, one, or two terminal
glucosamine residues
(M3XFGN2 to
GN2M3XFGN2,
Fig. 5) and analogous nonfucosylated structures (M3XGN2 to
GN2M3XGN2,
Fig. 5).
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| Figure 4.
HPAEC profiles of the PA derivatives of
paucimannosidic and complex-type N-glycans.
N-glycans isolated from wild-type Arabidopsis (A) and
mur1 mutant (B) leaves. The chromatograms presented in
the boxed insets correspond to the C18 reverse-phase
profiles of peaks b+c and f+g purified by HPAEC. Abbreviations
refer to N-linked glycans represented in Figure 5.
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| Figure 5.
Abbreviations and structures of paucimannosidic
and complex-type N-glycans isolated from Arabidopsis.
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| Figure 6.
HPAEC profiles of the -N-acetylglucosaminidase
digest of compound d after different incubation times. Peak a, PA
derivative of M3XFGN2; peak b+c, PA derivatives of
GN6-M3XFGN2 and
GN3-M3XFGN2 (see Fig. 5).
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Paucimannosidic and complex-type N-glycans isolated from the
mur1 plants were submitted to the same analysis. The
nonfucosylated PA structures e to h (Fig. 4B), identical to those of
the minor group found in the wild-type extract, were detected and
identified using the same approach as
M3XGN2 to
GN2M3XGN2
oligosaccharides represented in Figure 5. To investigate the presence
in the pool of mur1 N-glycans of minor oligosaccharides
co-migrating with the xylosylated oligosaccharides and containing an
additional hexose residue, the PA derivatives of complex and
paucimannosidic-type N-glycans were then analyzed by
electrospray MS (Fig. 7A). Three major
(M+H)+ ions at m/z = 1121, 1324, and 1527 were assigned to the major xylosylated PA oligosaccharides
M3XGN2 to
GN2M3XGN2
identified by chromatography. However, a second family of ions was also
detected at m/z = 1283, 1486, and 1689, having an
additional 162 mass units. These ions were not detected in the wild
type (data not shown) and were estimated to represent about 5% to 10%
of the total oligosaccharides (Fig. 7A). These ions were supposed to
correspond to (M+H)+ ions of the PA
L-Gal-containing N-glycans
M3XGalGN2 to
GN2M3XGalGN2 represented in Figure 8. To demonstrate
that these ions arise from the replacement of
L-Fuc by L-Gal, the most
abundant (M+H)+ at m/z = 1283 was
submitted to a CID-MS-MS analysis (Fig. 7B). This MS analysis allows
the observation of fragment ions specifically arising from the cleavage
of glycosidic linkages of the selected (M+H)+ ion
of PA oligosaccharides. As a consequence, some information on the
oligosaccharide sequence can be deduced from the CID-MS-MS spectrum (Gu
et al., 1994). In the CID-MS-MS of the ion at m/z = 1283, the successive loss of three mannose residues and one Xyl residue
from the nonreducing end is observed. Furthermore, the detection of the
fragment ion at m/z = 462 is strong evidence that the
additional hexose unit is linked to the proximal GlcNAc residue (Fig.
7B). This fragmentation pattern is consistent with data reported in the
literature on fragmentation of PA N-glycans either having or
not having an additional residue linked to the proximal glucosamine
residue (Gu et al., 1994). The mixture of labeled glycans was then
analyzed by 1H-NMR. In addition to the H-1 signal
of the -Xyl at 4.43 ppm, the NMR spectrum shows, in the 4.5 to 5.2 ppm region, four signals that were assigned to the H-1 of
mannose residues of the different N-glycans (Fig.
9). In addition to these signals, a minor
doublet at 5.422 ppm (J = 3.5 Hz) was assigned to the H-1
of the anomer of the additional hexose residue detected by MS. The
chemical shift of this signal is in agreement with NMR data on terminal -Galp residues (Zablackis et al., 1996). The amount of
L-Gal containing N-glycans was
estimated to represent about 5% by integration of the H-1 signal
of L-Gal to H-1 signal of Man-4 (Fig. 9, signals a and b).
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| Figure 7.
MS analysis of the mur1 N-glycans.
A, Electrospray mass spectrum of the PA derivatives of the
paucimannosidic and complex-type N-glycans isolated from
the mur1 mutant.  , (M+H)+ ions of the PA
derivatives of M3XGN2,
GNM3XGN2, and
GN2M3XGN2 (see Fig. 5).  ,
(M+H)+ ions of the PA derivatives of the
L-Gal-containing oligosaccharides
M3XGalGN2, GNM3XGalGN2,
and GN2M3XGalGN2 (see Fig. 8). B,
CID-MS-MS of the (M+H)+ ion of the PA derivative of
M3XGalGN2 (m/z = 1283).
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| Figure 8.
Abbreviations and structures of
L-Gal-containing pauci-mannosidic and complex-type
N-glycans isolated from the mur1
mutant.
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| Figure 9.
1H-NMR spectrum of the mixture of PA
derivatives of the paucimanosidic and complex-type
N-glycans isolated from the mur1 mutant
leaves. Numbers and letters refer to the corresponding protons (for
example, 14 = signal of H-1 proton of residue 4). A, H1
signal of the nonsubstituted Man 4 of M3XGN2
and GN6-M3XGN2. b, H1 signal of substituted Man
4 of GN3-M3XGN2 and
GN2M3XGN2.
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Taken together, these results indicate that the second family of
N-linked glycans detected in the mur1 mutant
contains an -Gal linked to the proximal GlcNAc residue. The presence
of Gal in the mur1 N-glycan preparation was confirmed by
sugar composition analysis. However, due to the low amount of material,
the absolute configuration of the Gal and its location on the GlcNAc
were not determined. However, since L-Fuc was
found to be replaced by L-Gal in the
mur1 cell wall xyloglucan, the MS and NMR data strongly suggest that the same process occurs in the N-glycans of
this mutant. As a consequence, the partial replacement of
L-Fuc by L-Gal leads to the
formation of plant N-glycans with a novel core structure
represented in Figure 8. Thus, we conclude that the mur1
mutation results in the accumulation of major nonfucosylated structures (95%) and minor
L-Gal-containing-oligosaccharides (5%) without
affecting, in a detectable manner, the processing of
N-linked glycans.
| |
DISCUSSION |
The structures of N-linked glycans associated to
proteins isolated from Arabidopsis have been elucidated by affino- and
immunodetection on blots with specific probes, as well as by structural
identification of reducing oligosaccharides released from glycoproteins
by treatment with a specific peptide N-glycosidase.
High-mannose-type N-glycans from Man-5 to Man-9 have been
identified. Furthermore, paucimannosidic and complex-type
N-glycans having an (1,3)-Fuc, a (1,2)-Xyl residues
have been fully identified. However, the total
N-glycosylation in Arabidopsis presents a lower diversity
than observed in other plants (Takahashi et al., 1986;
Fitchette-Lainé et al., 1997). No complex-type
N-glycans containing terminal Lewis a epitopes have been
detected, indicating that these glycans either are not synthesized in
Arabidopsis plants, or they are present only in very low amounts in
this plant or only in some tissues.
N-linked glycans associated to proteins from the Fuc-deficient
mur1 mutant have been isolated and identified using the same approach as was developed for the wild-type plants. This study has
shown that mostly nonfucosylated oligosaccharides are
N-linked to proteins of the mur1 mutant. No
modification in the processing of the N-glycans was observed
in the mutant. Major nonfucosylated oligosaccharide structures have
been identified, bearing zero, one, or two terminal glucosamine
residues in a ratio similar to that found in wild-type plants. This
result confirms that the addition of the (1,3)-Fuc residue is a late
event in the processing of plant N-linked glycans. As a
consequence, the absence of L-Fuc transfer does
not disturb the biosynthesis of these oligosaccharides. In addition to
the nonfucosylated oligosaccharides, a second family of
oligosaccharides was detected by MS in the total protein extract from
the mur1 plants. From analytical data these
oligosaccharides, representing about 5% of the N-glycans,
were found to have an additional hexose residue (-galactose from NMR
data) linked to the proximal glucosamine residue. Since the chitobiose
unit of N-glycans in plants and other organisms has been
reported so far as only substituted by Fuc residues, such data strongly
suggest that the minor population of N-glycans, identified
by MS, result from the mur1 mutation. By analogy with
previously reported data on the mur1 xyloglucan (Zablackis
et al., 1996), we propose that the minor 5% subpopulation of
N-glycans found in this mutant are new plant
N-glycans having (1,3)-L-Gal linked to the
proximal glucosamine and resulting from the replacement of
L-Fuc by L-Gal.
The replacement of one-third of the terminal L-Fuc residues
in the xyloglucan by L-Gal, a monosaccharide
structurally related to L-Fuc, indicates that
the (1,2)-fucosyltransferase involved in the xyloglucan biosynthesis
is able to transfer L-Gal from GDP-L-Gal to the
xyloglycan backbone in the absence of GDP-L-Fuc. This
phenomenon also seems to occur with the (1,3)-fucosyltransferase involved in N-linked glycan biosynthesis, which is localized
in the same compartment as the xyloglucan (1,2)-fucosyltransferase, i.e. the trans cisternae of the Golgi apparatus (Driouich et
al., 1993; Fitchette-Lainé et al., 1994). However, the difference in level of L-Gal incorporation suggests that the
two fucosyltransferases show different efficiencies in the replacement
of L-Fuc by L-Gal.
| |
FOOTNOTES |
1
This work was supported in France by CNRS (no.
ESA 6037), the University of Rouen, and the M.E.N.E.S.R. (Actions
Concertées Coordonnées des Sciences du Vivant ACCSV 14, réseau G-Trec), and by grants from the Région
Haute-Normandie. This work was supported in the United States by the
Department of Energy Biosciences Program (grant no. DE-FG02-95ER20203).
C.R. and M.C.-M. are recipients of Region Haute-Normandie and Biopole
fellowships, respectively.
*
Corresponding author; e-mail lfaye{at}crihan.fr; fax
33-2-35-14-67-87.
Received July 16, 1998;
accepted October 26, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CID-MS-MS, collision-induced dissociation MS.
HPAEC, high pH anion-exchange chromatography.
HPAEC-PAD, HPAEC coupled
to pulsed amperometric detection.
PA, pyridylamino derivative.
PNGase
A, peptide N-glycosidase A.
| |
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Top
Abstract
Introduction