| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 276, Issue 33, 30834-30844, August 17, 2001
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
§,§¶,,
**,,,, and§§
From the Bijvoet Center, Department of Bio-organic
Chemistry, Utrecht University, NL-3508 TB Utrecht, The Netherlands, the
Department of Neurobiology, Swiss Federal
Institute of Technology, Höggerberg, CH-8093 Zürich,
Switzerland, and the Department of Chemistry, Carlsberg
Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark
Received for publication, February 2, 2001, and in revised form, June 6, 2001
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
The carbohydrate structures present on the
glycoproteins in the central and peripheral nerve systems are
essential in many cell adhesion processes. The P0 glycoprotein,
expressed by myelinating Schwann cells, plays an important role during
the formation and maintenance of myelin, and it is the most abundant
constituent of myelin. Using monoclonal antibodies, the homophilic
binding of the P0 glycoprotein was shown to be mediated via the
human natural keller cell (HNK)-1 epitope
(3-O-SO3H-GlcUA( The P0 glycoprotein consists of a single immunoglobulin-like
domain in its extracellular part, a transmembranous domain, and a
cytoplasmic tail. It is the most abundant protein constituent of
peripheral myelin. P0 contains a single N-glycosylation site and heterogeneity in its glycosylation pattern that originates from
variable contents of fucose, galactose, and sialic acid residues; sulfate; and the HNK-1 carbohydrate epitope (1-4). P0 appears at the
initial stage of myelination and contributes to the formation and
maintenance of myelin compaction as an adhesion molecule (5). The
essential functional role of P0 in the processes of myelination has
been demonstrated by creating P0 knockout mice, which show severe
hypomyelination and myelin degeneration (5, 6). In humans, several
neurological disorders such as Charcot-Marie-Tooth disease,
Dejerine-Sottas disease, and congenital hypomyelination have been
associated with mutations in the P0 gene (7).
It has been reported that the glycan moiety of P0 plays an important
role in cell-cell adhesion via homophilic binding. This homophilic
binding has been mapped to the SDNGT sequence composing amino acids
91-95, which harbors the single N-glycosylation site on P0
(8). Thus, it was observed that this glycopeptide fragment inhibits
cell adhesion to a greater extent than the corresponding peptide
without glycan (9). Non-glycosylated P0, produced by site-directed
mutagenesis, does not show homophilic adhesion (10). In addition, amino
acids 43-50 and 74-82 may also contribute to the homophilic binding
in a non-carbohydrate-dependent manner (11).
Occurrence of age-dependent alteration in the glycan
moiety of P0 in the peripheral nerve (12) and mammalian spinal cord (5,
6, 13) has been reported, suggesting that the glycan heterogeneity
might be regulated by alterations in physiological conditions, although
details of the structural changes of the carbohydrate chain have not
yet elucidated.
So far, attempts to identify the oligosaccharides on the P0
glycoprotein using mass spectrometry (2, 14) or various chromatographic techniques (3, 15, 16) have not provided conclusive data concerning the
structures of these carbohydrates. Only recently have we described the
detailed structural analysis of a major HNK-1-reactive oligosaccharide
of the bovine peripheral myelin P0 glycoprotein (17).
In this study, a detailed structural analysis of the carbohydrates
contained in the bovine P0 glycoprotein has been performed. The core
structure of the carbohydrates and several epitopes, including HNK-1
(17) and 6-O-sulfosialyl-Lewis X (18), were identified using
highly sensitive techniques for carbohydrate analysis such as high
resolution magic angle spinning
(MAS)1 1H NMR
spectroscopy in a nano probe in combination with both MALDI-TOF and ESI
mass spectrometry.
Isolation of P0 and Release and Isolation of the Carbohydrate
Chains--
The P0 isolation procedure and release of the carbohydrate
chains were described previously by Voshol et al. (17).
After removal of the protein and detergent, fractionation according to
charge was performed on a Mono-Q column (1 ml; Amersham Pharmacia Biotech FPLC system) at a flow rate of 1 ml/min using the following NaCl gradient: 0-4 ml, 0 mM; 4-12 ml, 0-50
mM; and 12-20 ml, 50-500 mM; followed by a
regeneration step at 1 M NaCl. The effluent was monitored
at 214 nm. Further fractionation by high pH anion-exchange chromatography (19) was performed using a CarboPac PA-100 column (Dionex DX-500 system) with a 30-ml gradient from 100 to 600 mM sodium acetate in 0.1 M NaOH. Corresponding
fractions from different runs were pooled, neutralized, and desalted on P2.
NMR Spectroscopy--
Prior to NMR spectroscopic analyses in
2H2O, samples were exchanged twice in 99.9%
2H2O with intermediate lyophilization. For 5-mm
probe and nano probe experiments, samples were finally dissolved in 500 and 40 µl of 99.96% 2H2O (MSD Isotopes),
respectively. One- and two-dimensional 1H NMR measurements
in a 5-mm probe were carried out on a Bruker Daltonik AMX-500 or
AMX-600 spectrometer (Bijvoet Center, Department of NMR Spectroscopy,
Utrecht University). MAS 1H NMR spectra were recorded on a
Varian Unity Inova 500-MHz spectrometer equipped with a 4-mm
observe 1H NMR nano probe (Department of Chemistry,
Carlsberg Laboratory). All spectra were measured at incoming
temperatures of 300 K, which in the nano probe equals 302 K due to the
fast spinning of the sample at ~2 kHz at the magic angle (54.7°).
The one-dimensional spectra were acquired as one-pulse experiments with
pre-saturation of the 1HO2H resonance.
Acquisition data for the one-dimensional spectra were as follows: 2.0-s
acquisition time, 2.0-s pre-saturation delay, and sweep width of
8000 Hz with the number of scans varying between 32 and 1024. One-dimensional experiments in a 5-mm probe were performed at a probe
temperature of 285 K as described by Voshol et al. (17).
1H chemical shifts ( Sialidase Treatment--
Sialidase (Clostridium
perfringens, Oxford Glycosciences) digestion was performed
by incubating lyophilized material in 20 µl of 50 mM
sodium acetate buffer (pH 5.0) with 20 milliunits of
enzyme for 24 h at 37 °C. Subsequently, the reaction mixture was applied to a Millipore MC membrane filter (5000 nmwl
(nominal molecular weight limit), 0.2 cm2) and
centrifuged at 15,000 rpm for 15 min. The effluent was lyophilized and
permethylated as described below.
Permethylation--
After sialidase digestion, the fractions
QN.1 and QN.2 were lyophilized and dried over diphosphorus pentoxide
prior to permethylation. Permethylation was performed essentially as
described by Ciucanu and Kerek (26). After quenching the reaction, the
reaction mixture was washed three times with 700 µl of
CHCl3. The combined CHCl3 fractions were washed
three times with 1 ml of H2O, dried under a stream of
nitrogen, and dissolved in 10 µl of CHCl3. For MALDI-TOF analysis, 0.5 µl was used.
Mass Spectrometry--
Negative-ion mode ESI mass spectrometric
analyses of fractions Q1.5 and Q2.10 were performed on an
Esquire-LCTM quadrupole ion-trap spectrometer (Bruker
Daltonik GmbH, Bremen, Germany). Samples were dissolved in 50 µl
of deionized water/acetonitrile (1:1) and were introduced by infusion
at a flow rate of 1 µl/min. Ions were scanned (scan range of 50-2100
Da) with a scan speed of 13,000 Da/s at unit resolution using resonance
ejection at the hexapole resonance of one-third of the radio frequency
(781,250 Hz). The calibration of the mass spectrometer was performed
using ESI tuning mixture (Hewlett-Packard Co., Palo Alto, CA).
Collision-induced dissociation (CID) tandem mass spectrometric
experiments were performed using the quadrupole ion trap to select the
precursor ion for fragmentation. Helium was used as collision gas, and
the fragmentation energy applied on the end caps varied between 0.5 and
1.8 V. Recorded data were processed using Esquire-NT Version 3.1 software (Bruker Daltonik GmbH). Negative-ion mode mass spectrometric analyses of all other fractions were performed on a Reflex III MALDI-TOF spectrometer (Bruker Daltonik GmbH) equipped with a SCOUT ion
source, a N2 laser (337 nm), and a 2-GHz digitizer. The
spectra were recorded over a mass range of 3000 Da in the reflectron
mode using pulsed ion extraction and an acceleration voltage of 25 kV.
Aliquots (0.5 µl) of fractions QN.1 and QN.2 (dissolved in
CHCl3 after desialylation and permethylation) were deposited on microcrystalline The carbohydrate pool released from P0 was fractionated into three
fractions by anion-exchange chromatography on Mono-Q: QN (15 ± 5% of the total), Q1 (45 ± 5% of the total), and Q2 (40 ± 5% of the total), eluting at starting buffer without NaCl, starting buffer with a gradient of NaCl from 30 to 100 mM, and
starting buffer with a gradient of NaCl from 100 to 400 mM,
respectively. Since the Mono-Q pattern varied slightly from run to run,
due to the remaining SDS in the mixture, we did not attempt to obtain a
better resolution at this stage. Further fractionation by high pH
anion-exchange chromatography (19) resulted in three main fractions for
QN (QN.1-3), four main fractions for Q1 (Q1.4-7), and three main
fractions for Q2 (Q2.8-10) (data not shown). Due to variations in the
pulsed amperometric detection response, no estimation of the
percentages for the individual fractions could be made. These combined
fractions accounted for >90% of all glycans on P0. At this stage of
purification, only three fractions were homogeneous according to
one-dimensional 1H NMR spectroscopy and mass spectrometry;
nevertheless, no further purification was carried out in order to
prevent any loss of material. Instead, the fractions were analyzed by a
combination of mass spectrometry and state-of-the-art NMR spectroscopy.
A description is given below of the characteristic features of each
fraction, and the deduced structures are summarized in Fig.
1.
1-3)Gal(1-4)GlcNAc) present on the N-glycans. We recently described the
structure of the N-glycan carrying the HNK-1 epitope,
present on bovine peripheral myelin P0 (Voshol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and
Schachner, M. (1996) J. Biol. Chem. 271, 22957-22960). In this study, we report on the structural characterization of the detectable glycoforms, present on the single
N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the
hybrid- or biantennary complex-type structures, the variety of
epitopes is remarkable. In addition to the 3-O-sulfate
present on the HNK-1-carrying structures, most of the glycans
contain a 6-O-sulfated N-acetylglucosamine
residue. This indicates the activity of a
6-O-sulfo-GlcNAc-transferase, which has not been described
before in peripheral nervous tissue. The presence of the disialo-,
galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides
evidence for glycosyltransferase activities not detected until now. The
finding of such an epitope diversity triggers questions related to
their function and whether events, previously attributed merely to the
HNK-1 epitope, could be mediated by the structures described here.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) were expressed by reference to
internal acetone ( 2.225) or acetate ( 1.908). Two-dimensional
spectra were recorded in a 5-mm probe with 400-600
t1 experiments, and 96-163 free induction
decays of 2048 data points were collected per t1
experiment. In the nano probe, 468-1024 t1
experiments and 32-128 free induction decays of 2048 complex data
points were collected per t1 experiment.
Two-dimensional TOCSY spectra in the nano probe were acquired with a
pre-saturation delay of 1.05-1.2 5. The mixing time was 80-90 ms
using an adiabatic WURST 8 mixing waveform, with a total length of the
adiabatic pulse of 0.4 ms and a sweep width of 20 kHz used with a
20-step supercycle (20). Two-dimensional TOCSY spectra in a 5-mm probe
were recorded using a "clean" MLEV-17 spin-lock pulse (21-23) of
90-110 ms as described by Voshol et al. (17). A
two-dimensional rotating frame NOESY spectrum in the nano probe was
recorded with a mixing time of 200 ms. The mixing sequence was a
continuous spin-lock pulse of a power level corresponding to a 90°
pulse of 110-µs duration. Two-dimensional rotating frame NOESY
spectra in a 5-mm probe (24) were recorded at spin-lock times of
150-225 ms as described by Voshol et al. (17). A
two-dimensional NOESY spectrum (25) in a nano probe was acquired with a
mixing time of 200 ms and pre-saturation delay of 1.2 s. A
two-dimensional NOE spectrum in
1H2O/2H2O (9:1, v/v)
using a 5-mm probe was obtained at pH 6.0 using phosphate buffer (50 mM Na2HPO4 and 50 mM
NaH2PO4 containing 0.1 mM
NaN3) with offset at 10.01 ppm and a mixing time of 225 ms. Data sets were processed using locally developed software (J. A. van Kuik, Bijvoet Center, Utrecht University) as described by Voshol
et al. (17).
-cyano-4-hydroxycinnamic acid surfaces and allowed to dry at ambient temperature. The
-cyano-4-hydroxycinnamic acid thin-layer preparation was performed
as described by Vorm and Mann (27). The remaining fractions were
dissolved in water, diluted 1:10 with trihydroxyacetophenone (17 g/liter) in 80% acetonitrile and 20% deionized water dosed
with 1 mM diammonium citrate. Aliquots of ~0.5 µl were
spotted on the target and allowed to dry at ambient temperature.
External calibration was performed using a two-peptide mixture, which
was measured using -cyano-4-hydroxycinnamic acid as a matrix. Data
recorded were processed using Bruker Daltonik XMASS/NT Version 5 software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
[in a new window]
Fig. 1.
Structures of the bovine peripheral
glycoprotein P0 glycans. Structures are given with their
corresponding fraction numbers and the numbering of the residues.
Dashed lines indicate that the structures with and without
the substituent were observed in the mass spectra. Percentages were
derived from the high pH anion-exchange chromatography peak areas.
GlcA is GlcUA.
QN.1 and QN.2--
The negative-ion mode MALDI-TOF mass spectra of
QN.1 and QN.2 (data not shown) after sialidase treatment and
permethylation showed two peaks at m/z 1985.0 and 2346.2 corresponding to permethylated Hex4-dHex-HexNAc3 [M 
H]
and Hex5-dHex-HexNAc3 [M H], respectively, and several other pseudo-molecular
ions corresponding to the same structures, but with different degrees
of methylation. The one-dimensional 1H NMR spectra of QN.1
and QN.2 (Fig. 2) revealed the structural reporter group signals (Table I) of a
monosialylated hybrid-type structure as described previously (28). For
QN.1, two methyl doublets were observed at 1.207 and 1.220 (Table
I) belonging to the fucose residue 1,6-linked to the Asn-linked
GlcNAc-1. The core fucosylation resulted in a downfield shift
(~0.043 ppm) of the GlcNAc-2 H-1, which was linked to the GlcNAc-1
-anomer. The two methyl doublets indicated the presence or absence
of a Man-A residue, respectively (29). The observation of two GlcNAc-2 N-acetyl singlets at 2.096 and 2.078 (Fig. 2,
QN.1) corroborated this finding. These structural features were also
observed in most of the other heterogeneous fractions, confining the
structures present in those fractions (Fig. 1) to the hybrid-type
category. In the case of QN.2, the main constituent was a hybrid-type
structure containing N-glycolylneuraminic acid, as evidenced
by the presence of the N-glycolyl singlet at 4.116. In addition, the low intensity signal for N-acetyl at
2.028 (Fig. 2, QN.2) indicated that fraction QN.2 was contaminated
with fraction QN.1. Also in this fraction, part of the structures
carried the Man-A residue.
|
|
QN.3--
The MALDI-TOF mass spectrum of QN.3 (Fig.
3) in the negative-ion mode revealed four
pseudo-molecular ions at m/z 1500.7, 1662.7, 1824.7, and
1986.7 corresponding to
Hex4-dHex-HexNAc3(SO3) [M H] and the Hex5, Hex6, and
Hex7 analogs, respectively. The structures containing
Hex4 and Hex7 were the minor constituents of
this fraction, and the latter structure indicated the existence of a
terminal Hex-Hex sequence. The one-dimensional 1H NMR
spectrum of QN.3 (Fig. 2) showed the reporter group signals of an
asialo hybrid-type structure (30), and the observed chemical shifts are
summarized in Table I. The observation of the downfield shift of the
GlcNAc-5 H-6a and H-6b resonances to 4.405 and 4.348 (Fig. 2, QN.3)
revealed the sulfation of this residue as observed by De Waard et
al. (31). Two additional -anomeric signals were observed with
respect to QN.1 in the NMR spectrum recorded at 300 K (data not shown)
at 4.912 and 5.146. The resonance at 4.912 corresponded to H-1
of Man-B (28), and the signal at 5.146 originated from H-1 of the
Hex(1-)Gal structure (32, 33). In the TOCSY spectrum (Fig.
4, QN.3), the -anomeric spin system at
5.146 displayed a typical set of three cross-peaks of a Gal residue
(Gal-7). Two anomeric resonances were observed for Gal-6 at 4.600 and 4.542. The set of cross-peaks on the anomeric track at 4.600 revealed the substitution at OH-3 of this Gal-6 with Gal-7 (32).
However, the three cross-peaks observed on the anomeric track of Gal-6
at 4.542 clearly indicated that this residue was not substituted
(32). In summary, fraction QN.3 consists of four hybrid-type structures
differing in mass. The Hex4- and
Hex7-containing structures represented a single compound,
whereas the other two could each be present in different glycoforms
(Fig. 1).
|
|
Q1.4--
The MALDI-TOF mass spectrum of Q1.4 (Fig. 3) yielded
mainly one peak in the high mass region at m/z 2099.7 corresponding to the [M H] pseudo-molecular ion
of
Neu5Ac-Hex5-dHex2-HexNAc3(SO3).
In addition, the sodiated ([M + Na 2H],
m/z 2121.7) and potasiated ([M + K 2H], m/z 2137.7) pseudo-molecular ions were
observed, as well as low intensity signals at m/z 2283.8 ([M + Na 2H]) and 1959.7 ([M + Na 2H]), which corresponded to the structures containing
one Hex residue more or less, respectively. The peaks observed below
m/z 1959.7 were identified as metastable ions based on the
characteristic isotope patterns. The NMR data derived from
one-dimensional 1H NMR and two-dimensional TOCSY and
NOESY experiments, as summarized in Table I, showed two major
differences when compared with those of QN.1. In the one-dimensional
spectrum (Fig. 2, Q1.4), the -anomeric proton at 5.112 in
combination with the CH3 doublet at 1.166 evidenced the
presence of 1,3-fucosylation of GlcNAc-5 (34). Moreover, the
observation of the downfield-shifted resonances of the GlcNAc-5 H-6a
and H-6b atoms, as in QN.3, demonstrated the 6-O-sulfation
of this residue (31). The combined data pointed toward the presence of
a 6-O-sulfosialyl-Lewis X structure in this fraction (Fig.
2). The downfield shift of H-3 on the TOCSY track of Gal-6 H-1 (Fig. 4,
Q1.4) indicated that Neu5Ac-7 was 2,3-linked to Gal-6. The
two-dimensional NOESY NMR spectrum (data not shown) recorded using nano
probe MAS NMR spectroscopy, showed NOEs, between Fuc-5' H-1 and Gal-6
H-1 and between Gal-6 H-1 and GlcNAc-5 H-2/3/4, providing evidence for
the existence of this structure.
Q1.5 and Q1.6--
The negative-ion mode ESI mass spectrum of
fraction Q1.5 (Fig. 3) showed one high intensity peak at m/z
976.2 ([M 2H]2) and two low intensity peaks at
m/z 895.2 and 1057.2 ([M 2H]2).
After spectral deconvolution, the pseudo-molecular masses corresponded to Neu5Ac-Hex5-dHex-HexNAc3(SO3)
and the Hex4 and Hex6 analogs, respectively.
CID mass spectrometric analysis (data not shown) of m/z
976.2 yielded several fragments ions. The most important ions could be
assigned to [M dHex-HexNAc] (m/z
801.56) and [M Neu5Ac-Hex2(SO3)-HexNAc-CH6O3]
(m/z 977.12). The negative-ion mode MALDI-TOF spectrum of
fraction Q1.6 (Fig. 3) revealed only one intense peak at m/z
1991.68 corresponding to the [M + Na 2H]
pseudo-molecular ion of
Neu5Gc-Hex5-dHex-HexNAc3(SO3). The
one-dimensional 1H NMR spectrum of fraction Q1.5 (Fig. 2)
was similar to that of QN.1, and the chemical shifts are summarized in
Table I. The main difference was the downfield shift of the GlcNAc-5
H-6a and H-6b resonances as observed for fractions Q1.3 and Q1.4,
indicating 6-O-sulfation at this residue (31). The sulfation
of GlcNAc-5 induced a 0.07-ppm downfield shift of Gal-6 H-1. The
absence of fucosylation at GlcNAc-5, when compared with fraction Q1.4,
resulted in a 0.01-ppm upfield shift of the GlcNAc-5 H-1 resonance and the slightly downfield shift of the GlcNAc-5 N-acetyl signal
of this residue. The one-dimensional 1H NMR spectrum of
fraction Q1.6 (Fig. 2) was similar to that of Q1.5, and the chemical
shifts are summarized in Table I. The peak at 4.113 and the absence
of the N-acetyl resonance at 2.028 in the
one-dimensional NMR spectrum of Q1.6, when compared with that of Q1.5,
demonstrated that in Q1.6, Neu5Gc was present instead of Neu5Ac.
Q1.7--
The negative-ion mode MALDI-TOF mass spectrum of Q1.7
(Fig. 3) showed two pseudo-molecular ions at m/z 2082.81 and
2104.77 ([M H] and [M + Na 2H], respectively) belonging to
Neu5Ac2-Hex4-dHex-HexNAc3(SO3).
The one-dimensional 1H NMR spectrum of this structure (Fig.
2) showed reporter group signals as observed for fraction Q1.5 (Table
I) with the following differences. An additional N-acetyl
signal at 2.064 and additional H-3e ( 2.630) and H-3a ( 1.738) peaks revealed the presence of two Neu5Ac residues, supporting
the mass spectrometric observation and demonstrating the presence of
the Neu5Ac(2-8)Neu5Ac(2-3)Gal structure (35). The presence of
signals at 4.117 and 4.139 stemming from Neu5Ac-7 H-8 and H-9,
respectively, as observed previously for
Neu5Ac(2-8)Neu5Ac(2-)-containing structures (36), confirmed the
aforementioned structure. Since no H-3e signal was observed at 1.666 (37), the presence of a Neu5Ac(2-8)Neu5Ac(2-6)Gal structure could be excluded.
Q2.8--
Fraction Q2.8 was described extensively by Voshol
et al. (17). Interestingly, this fraction
contained only one structure, which carried the HNK-1 epitope at the
Man-4' branch, as evidenced by the NMR data (Table I).
|
Q2.9--
The negative-ion mode MALDI-TOF spectrum of Q2.9 (Fig.
3) revealed the presence of a heterogeneous mixture by the observation of peaks at m/z 1500.00, 1662.84, 1838.73, 2041.80, 2082.78, and 2260.79. The assignments of the peaks to [M H] pseudo-molecular ions was corroborated by the
observation of their [M + Na 2H] analogs. The peak at
m/z 2260.79 corresponded to
Neu5Gc-Neu5Ac-Hex5-dHex- HexNAc3(SO3).
The two peaks at m/z 1500.0 and 1662.84 corresponded to
Hex4-dHex-HexNAc3(SO3) and
Hex5-dHex-HexNAc3(SO3),
respectively, similar to the structures found in fraction QN.3 (Fig.
2), and originated from degradation of
Neu5Gc-Neu5Ac-Hex5-dHex-HexNAc3(SO3) upon storage. The presence of Neu5Ac in Q2.9 was corroborated in the
one-dimensional 1H NMR spectrum (Fig. 2) by the observation
of the low intensity multiplets originating from the H-3a and H-3e
signals. The presence of Neu5Gc was evidenced by the
characteristic N-glycolyl resonance at 4.11. This
signal coincided with H-3 of Gal-6. The anomeric region of the
one-dimensional 1H NMR spectrum (Fig. 2, Q2.9) showed the
presence of the Man-4 and Man-4' signals as observed in fraction Q2.8
(Table I), indicating a chain elongation at the Man-4' branch. In
addition the Man-A and Man-4 signals were observed, revealing a chain
elongation at the Man-4 branch. The presence of a bisecting GlcNAc-9 in
some of the structures was evidenced by the anomeric signal at 4.456 and by the two Man-3 H-1 signals (Table I and Fig. 2, Q2.9) (17). Also, the signals were observed originating from
3-O-SO3H-GlcUA-7 H-1 and H-3 and from
6-O-SO3H-GlcNAc-5 H-6a and H-6b, as in fraction Q2.8 (Table I). The high field region of the one-dimensional 1H NMR spectrum showed at least five N-acetyl
signals (Table I) with different intensities. Furthermore, the methyl
doublet from the core fucose was appreciated. The GlcNAc-2
N-acetyl signals at 2.077 and 2.096 (in a ratio of 5:1)
revealed that ~20% of the structures had the chain elongation at the
Man-4' branch. The peak at m/z at 2028.78 in the mass
spectrum was identified as the structure present in fraction Q2.8 and
possibly its isomer bearing the HNK-1 epitope at the Man-4 branch (Fig.
2). The peak at m/z 2041.78 was then assigned to the
structure containing the bisecting GlcNAc-9 and Man-A, instead of
GlcNAc-5' (Fig. 2), and the peak at m/z 1838.73 was
assigned to the same structure without the bisecting GlcNAc-9 (Fig.
2).
Q2.10--
The negative-ion mode ESI mass spectrum of Q2.10 (Fig.
3) revealed only one peak at m/z 958.6 ([M 2H]2; [M H],
m/z 1918.21). Mass spectra recorded after
collision-induced dissociation (data not shown) of this peak
yielded several fragments suitable for assigning the structure. The ion
at m/z 1662.28 corresponded to [M HexUA(SO3)], and the ion at m/z
1500.23 could be attributed to [M HexUA(SO3)-Hex]. The structure was
identified as
HexUA-Hex5-dHex- HexNAc3(SO3)2. In the one-dimensional 1H NMR spectrum (Fig. 2, Q2.10), the
anomeric region revealed signals, which were also observed for fraction
Q1.6 (Table I) and, in addition, the presence of the signals of GlcUA-7
H-1 and H-3 and the downfield-shifted Gal-6 H-3 signal (Table I). The
downfield-shifted GlcNAc-5 H-6a and H-6b resonances proved the C-6
sulfation of this residue (31). The N-acetyl region showed
three signals, which originated from the GlcNAc residues, since no
Neu5Ac was present. In the two-dimensional TOCSY spectrum (Fig. 4,
Q2.10), the anomeric tracks of GlcNAc-1, -2, and -5 and those of Man-3, -4, and -A were identical to those observed for fraction Q1.6 (data not
shown), allowing the identification of the structure (Fig. 2). The
anomeric tracks of 3-O-SO3H-GlcUA-7 and Gal-6
were identical to those observed for Q2.8, thus proving the position of
the second sulfate group (17). This demonstrated that the HNK-1 epitope
containing a sulfate group on GlcNAc C-6 was situated on Man-4 in
fraction Q2.10.
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this study, we describe the detailed structural characterization of the N-linked glycans present on bovine peripheral myelin P0 using state-of-the-art NMR and mass spectrometry techniques. These glycans occur in addition to the core-fucosylated biantennary structure carrying a bisecting GlcNAc and the HNK-1 epitope on the Man-4' branch that was characterized previously by us (17).
The isolation of myelin P0 proved to be extremely laborious; and therefore, only a limited amount of pure P0 could be obtained. Fractionation of the carbohydrate pool, after being released from the protein backbone, had to be limited as much as possible in order to prevent the loss of precious material. As a result, most of the 10 fractions contained several structurally related glycoforms and had to be analyzed as mixtures. To perform the structural analysis successfully, ultrasensitive analytical methods capable of providing detailed structural information on such complex samples had to be employed. The newly developed nano probe MAS 1H NMR technique allows the structural analysis of nanomolar quantities of material in solution. So far, this technique has scarcely been applied in structural carbohydrate analysis (38-40). The nano tube, which holds a volume of 40 µl, is normally spun at ~2 kHz at the magic angle (54.7°). Spinning at the magic angle removes magnetic susceptibility-induced line broadening and/or contributions from homonuclear dipolar couplings. With the novel probes and the use of gradients, it is possible to apply existing solution state NMR techniques for solving challenging structural problems. In the case of the P0 glycoprotein, the MAS 1H NMR technique was complemented by sensitive mass spectrometric analyses.
The structures identified were for 75 ± 5% of the hybrid-type and for 25 ± 5% of the biantennary complex-type, carrying a distinct variety of structures at the Man-4 branch. All structures were core-fucosylated. Interestingly, Burger et al. (1), who studied P0 from human sciatic nerve by Western blot analysis and affinity chromatography, observed predominantly tri- and tetraantennary oligosaccharides (~80%), in addition to biantennary (10%) and oligomannose and/or hybrid (10%) oligosaccharides. This result may point to species-specific structures. In a study on human material, Field et al. (3) used enzymatic or chemical degradation in conjunction with chromatographic techniques for identification. They described the presence of HNK-1-positive carbohydrates, probably hybrid-type structures, containing either one sulfate residue (80%) or three sulfate residues (20%) in combination with sialylation.
Among the different structures we observed, the HNK-1 epitope
(3-O-SO3H-GlcUA(1-3)Gal(1-4)GlcNAc),
detected in fractions Q2.8-10, has been well documented. This major
carbohydrate epitope is prominently present in the nervous system and
is carried by a variety of cell-surface glycolipids as well as
glycoproteins (41). The antigen has been found to be one of the factors
responsible for the precise cell adhesion and recognition processes
that underlie the interaction of neural cells (42). It has also been
implicated in interactions with laminin and peripheral nerve
regeneration (43, 72-77). This carbohydrate antigen, also found in the
retina and on HNK cells, is the main target of IgM molecules from
patients with peripheral demyelination neuropathies (44, 78).
Especially the sulfate ester within the epitope seems to play an
essential role in the myelin assembly during development (45) and
outgrowth of neurites (46). The sulfation of glucuronic acid, which
occurs at the onset of myelination and regeneration and ceases after development, is mediated by a highly specific HNK-1 sulfotransferase (47, 48). In this context, the presence of the
6-O-sulfo-GlcNAc oligosaccharides (fractions
Q1.4-2.10) and specifically the 6-O-sulfo-HNK-1 epitope,
observed in fraction Q2.10 is interesting since it implies another
sulfotransferase activity. Of the various
6-O-sulfo-GlcNAc-transferases identified thus far (49-52,
79), only one report describes the existence of a nervous
system-involved sulfotransferase (53), which showed a high degree of
homology to a family of 6-O-sulfotransferases, some of which
are expressed also in brain (50, 54, 80, 81).
The observation of the 6-O-sulfosialyl-Lewis X and
non-fucosylated precursors as capping epitopes was surprising. The
terminal Neu5Ac/Gc(2-3)Gal(1-4)[6-O-SO3H]GlcNAc
structure is very uncommon and has been found only in
N-glycans of the glycoprotein family (ZP3) from zona
pellucida (55). The 6-O-sulfosialyl-Lewis X epitope has been
identified as a potent inhibitor of the leukocyte adhesion molecule
L-selectin (18), and its structure, function, and biosynthesis have
been described by Kimura et al. (56). It is intriguing that
we found its presence on the very same molecule that carries the HNK-1
antigen that also binds to L-selectin (57, 58). Previously, it has been
suggested that the sulfoglucuronylparagloboside (containing the HNK-1
epitope) acts as the ligand for L-selectin in inflammatory disorders of
both the central and peripheral nervous systems to regulate the
invasion of activated lymphocytes into the brain (58). It is suggested
that the expression of the 6-O-sulfosialyl-Lewis X structure
serves the same purpose.
The -galactosyl epitope (59) in combination with a
6-O-sulfated GlcNAc
(Gal(1-3)Gal(1-4)6-O-SO3H-GlcNAc), as
observed in fraction QN.3, has not been described before in
glycoproteins. The non-sulfated analog has been found on the
N-linked glycans of mouse oocyte ZP3 (60), porcine
thyroglobulin (31), and IgG1 antibodies produced by murine and
transfectoma cell subclones (61) and has also been observed on
gangliosides (33). The Gal(1-3) linkage to the reducing terminal
GalNAc in O-linked glycans was found to be present in
significant quantities only in brain (62) and nervous tissue
glycoproteins (63). Whether the presence of this -galactosyl
epitope, which has been identified as the major xenoantigen in
pig-to-man transplants, is due only to the availability of the
appropriate enzyme and donor substrate or whether it is functionally
significant remains to be clarified. In fraction Q1.7 a second terminal
structure was observed
(Neu5Ac(2-8)Neu5Ac(2-3)Gal(1-4)6-O-SO3H-GlcNAc), which is completely new in N-linked glycans from
glycoproteins. The disialo terminal epitope has been observed on
glycosphingolipids from bovine brain (35) and frog brain (64) and
in a variety of glycoproteins (65, 82-88), although never in
combination with a sulfated GlcNAc. This structure, when
present in higher polymeric forms as the so-called unusual polysialic
acid associated with the neural cell adhesion molecule (N-CAM), is
known to function in cell adhesion, differentiation, signal
transduction, and surface expression of stage-specific developmental
antigens. It indicates the expression of a sialyltransferase capable of
generating the 2,8 linkage (66). Recently, Sato et al.
(67) demonstrated the presence of this non-sulfated structure on many
mammalian brain glycoproteins, using specific antibodies. It is more
prominent in adult than in embryonic porcine brain tissue, suggesting a developmental regulation.
In summary, the nano probe MAS 1H NMR spectroscopic and
mass spectrometric techniques employed in this study, being at least a
factor of 5 more sensitive than traditional methods, have demonstrated to be very powerful tools for the characterization of the
N-glycans on the P0 glycoprotein from bovine peripheral
myelin. The identification of the epitope library may bring us one step
closer to the understanding of the relationship between the
carbohydrates present and their putative functions in neural tissue
(68-71). The observation of predominantly hybrid- and biantennary
complex-type structures is remarkable. It is, however, very likely that
the already mentioned spatiotemporally regulated and
species-dependent expression causes the variety of epitopes
observed. Although the glycosylation machinery is apparently fully
operational, no higher antennary structures have been observed in
bovine material, which might point toward a very subtle modulation of
the glycan heterogeneity by the down-regulation of specific
glycosyltransferases. The functional significance of the observed
structural diversity poses a new challenging question.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Y. E. M. van der Burgt, A. Schneider, and F. J. Mayer-Posner (Bruker Daltonik GmbH) for allowing the use of the Reflex III and Esquire-LC mass spectrometers and help with the measurements. We acknowledge Dr. B. O. Petersen for help with the interpretation of the nano probe NMR data.
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported by European Commission DG-XII-E Postdoctoral Grant BIO4CT975071 (Biotechnology Program).
** Supported by Danish Technical Council Grant 9900687.
§§ To whom correspondence should be addressed: Bijvoet Center, Dept. of Bio-organic Chemistry, Utrecht University, P. O. Box 80075, NL-3508 TB Utrecht, The Netherlands. Tel.: 31-30-253-2184; Fax: 31-30-254-0980; E-mail: vlieg@pobox.uu.nl.
Published, JBC Papers in Press, June 15, 2001, DOI 10.1074/jbc.M101013200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MAS, magic angle spinning; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; ESI, electrospray ionization; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Burger, D., Simon, M., Perruisseau, G., and Steck, A. J. (1990) J. Neurochem. 54, 1569-1575 |
| 2. | Uyemura, K., and Kitamura, K. (1991) Comp. Biochem. Physiol. C Comp. Pharmacol. 98, 63-72 |
| 3. | Field, M. C., Wing, D. R., Dwek, R. A., Rademacher, T. W., Schmitz, B., Bollensen, E., and Schachner, M. (1992) J. Neurochem. 58, 993-1000 |
| 4. | Badache, A., Burger, D., Villarroya, H., Robert, Y., Kuchler, S., Steck, A. J., and Zanetta, J.-P. (1992) Dev. Neurosci. 14, 342-350 |
| 5. | Giese, K. P., Martini, R., Lemke, G., Soriano, P., and Schachner, M. (1992) Cell 71, 565-576 |
| 6. | Martini, R., Zielasek, J., Toyka, K. V., Giese, K. P., and Schachner, M. (1995) Nat. Genet. 11, 281-286 |
| 7. | Warner, L. E., Hilz, M. J., Appel, S. H., Killian, J. M., Kolodny, E. H., Karpati, G., Carpenter, S., Watters, G. V., Wheeler, C., Witt, D., Bodell, A., Nellis, E., Van Broeckhoven, C., and Lupski, J. R. (1996) Neuron 17, 451-460 |
| 8. | Yoshida, M., and Colman, D. R. (1996) Neuron 16, 1115-1116 |
| 9. | Yazaki, T., Miura, M., Asou, H., Kitamura, K., Toya, S., and Uyemura, K. (1992) FEBS Lett. 307, 361-366 |
| 10. | Filbin, M. T., and Tennekoon, G. I. (1993) Cell Biol. 122, 451-459 |
| 11. | Zhang, K., Merazga, Y., and Filbin, M. T. (1996) J. Neurosci. Res. 45, 525-533 |
| 12. | Brunden, K. R. (1992) J. Neurochem. 58, 1659-1666 |
| 13. | Sato, Y., Kimura, M., Yasuda, C., Nakano, Y., Tomita, M., Kobata, A., and Endo, T. (1999) Glycobiology 9, 655-660 |
| 14. | Jaques, A. J., Opdenakker, G., Rademacher, T. W., Dwek, R. A., and Zamze, S. E. (1996) Biochem. J. 316, 427-437 |
| 15. | Burger, D., Perruisseau, G., Simon, M., and Steck, A. J. (1990) J. Neurochem. 58, 845-853 |
| 16. | Burger, D., Perruisseau, G., Simon, M., and Steck, A. J. (1990) J. Neurochem. 58, 854-861 |
| 17. | Voshol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and Schachner, M. (1996) J. Biol. Chem. 271, 22957-22960 |
| 18. | Galustian, C., Lawson, A. M., Komba, S., Ishida, H., Kiso, M., and Feizi, T. (1997) Biochem. Biophys. Res. Commun. 240, 748-751 |
| 19. | Hardy, M. R., and Townsend, R. R. (1994) Methods Enzymol. 230, 208-225 |
| 20. | Kupce, E., Schmidt, P., Rabce, M., and Wagner, G. (1998) J. Magn. Reson. 135, 361-367 |
| 21. | Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521-528 |
| 22. | Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 65, 355-360 |
| 23. | Griesinger, C., Otting, G., Wüthrich, K., and Ernst, R. R. (1988) J. Am. Chem. Soc. 110, 7870-7872 |
| 24. | Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 63, 207-213 |
| 25. | Jeenerm, J., Meier, B. H., Bachmann, P., and Ernst, R. R. (1979) J. Chem. Phys. 71, 4546-4553 |
| 26. | Ciucanu, I., and Kerek, F. (1984) Carbohydr. Res. 131, 209-217 |
| 27. | Vorm, O., and Mann, M. (1994) J. Am. Soc. Mass. Spectrom. 5, 955-958 |
| 28. | Weisshaar, G., Hiyama, J., and Renwick, A. G. C. (1991) Glycobiolgy 1, 393-404 |
| 29. | Stroop, C. J. M., Weber, W., Nimtz, M., Gutiérrez Gallego, R., Kamerling, J. P., and Vliegenthart, J. F. G. (2000) Arch. Biochem. Biophys. 374, 42-51 |
| 30. | Vliegenthart, J. F. G., Dorland, L., and Van Halbeek, H. (1983) Adv. Carbohydr. Chem. Biochem. 41, 209-374 |
| 31. | De Waard, P., Koorevaar, A., Kamerling, J. P, and Vliegenthart, J. F. G. (1991) J. Biol. Chem. 266, 4237-4243 |
| 32. | Seppo, A., Pentilla, L., Leppanen, A., Maaheimo, H., Niemela, R., Helin, J., Wieruszeski, J.-M., and Rekonen, O. (1994) Glycoconj. J. 11, 217-225 |
| 33. | Dasgupta, S., Hogan, E. L., Glushka, J, and Van Halbeek, H. (1994) Arch. Biochem. Biophys. 310, 373-384 |
| 34. | Michalski, J. C., Wieruszeski, J.-M., Alonso, C., Cache, P., Montreuil, J., and Strecker, G. (1991) Eur. J. Biochem. 201, 439-458 |
| 35. | Dorland, L., Van Halbeek, H., Vliegenthart, J. F. G., Schaurer, R., and Wiegandt, H. (1986) Carbohydr. Res. 151, 233-245 |
| 36. | Michou, F., Brisson, J.-R., and Jennings, H. J. (1987) Biochemistry 26, 8399-8405 |
| 37. | Kitajima, K., Nomoto, H., Inoue, Y., Iwasaki, M., and Inoue, S. (1984) Biochemistry 23, 310-316 |
| 38. | Manzi, A., Salimath, P. V., Spiro, R. C., Keifer, P. A., and Freeze, H. H. (1995) J. Biol. Chem. 270, 9154-9163 |
| 39. | Gilbert, M., Brisson, J.-R., Karwaski, M.-F., Michniewicz, J., Cunningham, A.-M., Wu, Y., Young, N. M., and Wakarchuk, W. W. (2000) J. Biol. Chem. 275, 3896-3906 |
| 40. | Broberg, A., Thomsen, K. K., and Duus, J. Ø. (2000) Carbohydr. Res. 328, 375-382 |
| 41. | Schachner, M., Martini, R., Hall, H., and Orberger, G. (1995) Prog. Brain Res. 105, 183-188 |
| 42. | Schachner, M., and Martini, R. (1995) Trends Neurosci. 18, 183-191 |
| 43. | Hall, H., Lui, L., Schachner, M., and Schimtz, B. (1993) Eur. J. Neurosci. 5, 34-42 |
| 44. | Ilyas, A. A., Chou, D. K., Jungalwala, F. B., Costello, C., and Quarles, R. H. (1990) J. Neurochem. 55, 594-601 |
| 45. | Poduslo, J. F. (1990) J. Biol. Chem. 265, 3719-3725 |
| 46. | Mohan, P. S., Chou, D. K., and Jungalwala, F. B. (1990) J. Neurochem. 54, 2024-2031 |
| 47. | Bakker, H., Friedmann, I., Oka, S., Kawasaki, T., Nifant'ev, N., Schachner, M., and Mantei, N. (1997) J. Biol. Chem. 272, 29942-29946 |
| 48. | Ong, E., Yeh, J.-C., Ding, Y., Hindsgaul, O., and Fukuda, M. (1998) J. Biol. Chem. 273, 5190-5195 |
| 49. | Bistrup, A., Bhakta, S., Lee, J. K., Belov, Y. Y., Gunn, M. D., Zuo, F.-R., Huang, C.-C., Kannagi, R., Rosen, S. D., and Hemmerich, S. (1999) J. Cell Biol. 145, 899-910 |
| 50. | Lee, J. K., Bhakta, S., Rosen, S., and Hemmerich, S. (1999) Biochem. Biophys. Res. Commun. 263, 543-549 |
| 51. | Bowman, K. G., Hemmerich, S., Bhakta, S., Singer, M. S., Bistrup, A., Rosen, S. D., and Bertozzi, C. R. (1998) Chem. Biol. 5, 447-460 |
| 52. | Spiro, R. G., Yasumoto, Y., and Bhoyroo, V. (1996) Biochem. J. 319, 209-216 |
| 53. | Nastuk, M. A., Davis, S., Yancopoulos, G. D., and Fallon, J. R. (1998) J. Neurosci. 18, 7167-7177 |
| 54. | Uchimura, K., Maramatsu, H., Kadomatsu, K., Fan, Q.-W., Kurosawa, N., Mitsuoka, C., Kannagi, R., Habuci, O., and Maramatsu, T. (1998) J. Biol. Chem. 273, 22577-22583 |
| 55. | Noguchi, S. (1992) Eur. J. Biochem. 209, 883-894 |
| 56. | Kimura, N., Mitsuoka, C., Kanamori, A., Hhiraiwa, N., Uchimura, K., Muramatsu, T., Tamatani, T., Kansas, G. S., and Kannagi, R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4530-4535 |
| 57. | Needham, L. K., and Schnaar, R. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1359-1365 |
| 58. | Kanda, T., Yamawaki, M., Ariga, T., and Yu, R. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7897-7901 |
| 59. | Galili, U. (1993) Springer Semin. Immunopathol. 15, 155-171 |
| 60. | Thall, A. P., Maly, P., and Lowe, J. B. (1995) J. Biol. Chem. 270, 21437-21440 |
| 61. | Bergwerff, A. A., Stroop, C. J. M., Murray, B., Holtorf, A. P., Pluschke, G., van Oostrum, J., Kamerling, J. P., and Vliegenthart, J. F. G. (1995) Glycoconj. J. 12, 318-330 |
| 62. | Finne, J. (1975) Biochim. Biophys. Acta 412, 317-325 |
| 63. | Finne, J. (1989) CIBA Found. Symp. 145, 173-183; discussion 183-188 |
| 64. | Bouchon, B., Levery, S. B., Clausen, H., and Hakomori, S.-I. (1992) Glycoconj. J. 9, 27-38 |
| 65. | Finne, J., Krusius, T., and Rauvala, H. (1977) Biochem. Biophys. Commun. 74, 405-410 |
| 66. | Angata, K., Suzuki, M., McAuliffe, J., Ding, Y., Hindsgaul, O., and Fukuda, M. (2000) J. Biol. Chem. 275, 18594-18601 |
| 67. | Sato, C., Fukuoka, H., Ohta, K., Matsuda, T., Koshino, R., Kobayashi, K., Troy, F. A., and Kitajiama, K. (2000) J. Biol. Chem. 275, 15422-15431 |
| 68. | Georgiou, J., and Charlton, M. P. (1999) Glia 27, 101-109 |
| 69. | Griffith, L. S., Schmitz, B., and Schachner, M. (1992) J. Neurosci. Res. 33, 639-648 |
| 70. | Scheinder-Schaulies, J., Von Brunn, A., and Schachner, M. (1990) J. Neurosci. Res. 27, 286-297 |
| 71. | Filbin, M. T., and Tennekoon, G. I. (1991) Neuron 7, 845-855 |
| 72. | Hall, H., Vorherr, T., and Schachner, M. (1995) Glycobiology 5, 435-441 |
| 73. | Hall, H., Deutzmann, R., Timpl, R., Vaughan, L., Schimtz, B., and Schachner, M. (1997) Eur. J. Biochem. 246, 233-242 |
| 74. | Hall, H., Carbonetto, S., and Schachner, M. (1997) J. Neurochem. 68, 544-553 |
| 75. | Martini, R., Bollensen, E., and Schachner, M. (1988) Dev. Biol. 129, 330-338 |
| 76. | Martini, R., Xin, Y., Schmitz, B., and Schachner, M. (1992) Eur. J. Neurosci. 4, 628-639 |
| 77. | Martini, R., Xin, Y., and Schachner, M. (1994) Glia 10, 70-74 |
| 78. | McGarry, R. C., Helfland, S. L., Quarles, R. H., and Roder, J. C. (1983) Nature 306, 376-378 |
| 79. | Degrootte, S., Lo-Guidice, J.-M., Strecker, G., Ducourouble, M.-P., Roussel, P., and Lamblin, G. (1997) J. Biol. Chem. 272, 29493-29501 |
| 80. | Fan, Q.-W., Uchimura, K., Yuzawa, Y., Matsuo, S., Mitsuoka, C., Kannagi, R., Maramatsu, H., Kadomatsu, K., and Maramatsu, T. (1999) Glycobiology 9, 947-955 |
| 81. | Hiraoka, N., Petryniak, B., Nakayama, J., Tsuboi, S., Suzuki, M., Yeh, J.-C., Izawa, D., Tanaka, T., Miyasaka, M., Lowe, J. B., and Fukuda, M. (1999) Immunity 11, 79-89 |
| 82. | Finne, J., Krusius, T., Rauvala, H., and Hemminki, K. (1977) Eur. J. Biochem. 77, 319-323 |
| 83. | Kiang, W. K., Krusius, T., Finne, J., Margolis, R. U., and Margolis, R. K. (1982) J. Biol. Chem. 257, 1651-1659 |
| 84. | Fukuda, M., Dell, A., and Fukuda, M. N. (1984) J. Biol. Chem. 259, 4782-4791 |
| 85. | Fukuda, M., Lauffenburger, M., Sasaki, H., Rogers, M. E., and Dell, A. (1987) J. Biol. Chem. 262, 11952-11957 |
| 86. | Funakoshi, Y., Taguchi, T., Sato, C., Kitajima, K., Inoue, S., Morris, H. R., Dell, A., and Inuoe, Y. (1997) Glycobiology 7, 195-205 |
| 87. | Sato, C., Kitajima, K., Inoue, S., and Inoue, Y. (1998) J. Biol. Chem. 273, 2575-2582 |
| 88. | Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1998) Anal. Biochem. 261, 191-197 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Kizuka, T. Matsui, H. Takematsu, Y. Kozutsumi, T. Kawasaki, and S. Oka Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis J. Biol. Chem., May 12, 2006; 281(19): 13644 - 13651. [Abstract] [Full Text] [PDF] |
, Fuc; 
, GlcNAc; 
, Man; 
, 
,
Neu5Ac; 
, Neu5Gc; 
, GlcUA; S, OSO3; 
,
