From the
Studies from this laboratory showed that 85-90% of the
[6-
The mixture of labeled and unlabeled material was dialyzed
extensively against water using 3500 M
Electrospray mass
spectrometric analysis of remaining anionic sample IB in the negative
ion mode (Fig. 2 D) showed the presence of some remaining
major product ( m/z 1011). In addition, other pseudomolecular
ions seen are consistent with the following structures:
HexNAc
The connectivities of the anomeric protons in the
two-dimensional TOCSY NMR spectrum of the same sample are shown in
Fig. 4
. It should be pointed out that the signal at approximately
4.4-4.5 ppm observed in the one-dimensional experiment is not
found here. Since the spinning rate for this particular experiment was
2240 Hz, this corresponds to a spinning side band located at 2240 Hz
(about 4.4 ppm) from the origin (4,4-dimethyl-4-silapentane-1-sulfonate
signal). In the two-dimensional TOCSY experiment, since the spinning
rate is different, this is producing the t
As indicated by the one-dimensional
1) The sample still contains
N-acetylgalactosamine, as indicated by the presence of a
N-acetyl methyl signal in the one-dimensional spectrum (not
shown), together with a clear TOCSY pattern (not shown), which chemical
shifts are listed in I. Furthermore, a second
2) Only one set of xylose signals was found with a
3) The
region of the TOCSY spectrum corresponding to the
This paper extends our previous work showing that human
melanoma and Chinese hamster ovary cells incubated with MU- or
p-nitrophenol-
Since sulfate and phosphate esters have been
identified in the core regions of some chondroitin sulfate chains
(24, 25, 26, 27) , either of these
groups might account for the negative charge in the before mentioned
molecules. However, we found no evidence for sulfate by metabolic
labeling, or for acid-labile phosphodiesters or E. coli alkaline phosphatase-sensitive phosphomonoesters. The other
candidate was an acidic sugar residue, the most likely choice being
GlcA. However, the resistance of the
[
This result was unexpected
since mature chondroitin/dermatan sulfate chains contain
Our discovery of an
Although terminal
The
Electrospray mass spectrometric analysis also
showed evidence for at least five distinct types of structures made on
Xyl
Several
conclusions can be drawn from the electrospray analysis. The molecule
HexNAc
The broad array of minor xyloside products identified
by NMR and mass spectrometric analyses raises questions about their
significance and relationship to the known route of GAG core
biosynthesis. Xylose residues are primarily, but not exclusively, found
in the GAG core region of mammalian cells. For instance, bovine
coagulation factor IX contains
Xyl
Is the presence of the
Chemical shifts are given in
ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonate
We thank Deepak Sampath for preliminary experiments,
Delia Matriano for technical assistance, and Drs. Herman Van Halbeek
and Claudio Schteingart for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Xylosides compete with endogenous proteoglycan core
proteins and act as alternate acceptors for synthesizing protein-free
glycosaminoglycan chains. Their assembly on these alternate acceptors
utilizes the same glycosyltransferases that make the protein-bound
chains. Most studies using alternate acceptors focus on the production
of sulfated glycosaminoglycan chains that are thought to be the major
products. However, we previously showed that labeling melanoma cells
with [6-
H]galactose in the presence of
4-methylumbelliferyl (MU) or p-nitrophenyl (pNP)
-xylosides led to the synthesis of mostly di- to tetrasaccharide
products including incomplete core structures. We have solved the
structure of one of the previously unidentified products as,
GalNAc
(1, 4) GlcA
(1, 3) Gal
(1, 3) Gal
(1, 4) Xyl
MU,
based on compositional analysis by high performance liquid
chromatography, fast atom bombardment, electrospray mass spectrometry,
and one-dimensional and two-dimensional
H NMR spectroscopy.
The novel aspect of this molecule is the presence of a terminal
-GalNAc residue at a position that is normally occupied by
-GalNAc in chondroitin/dermatan sulfate or by
-GlcNAc in
heparin or heparan sulfate chains. An
-GalNAc residue at this
critical location may prevent further chain extension or influence the
type of chain subsequently added to the common tetrasaccharide core.
-Xylosides serve as primers for glycosaminoglycan
(GAG)
(
)
chain synthesis in animal cells
(1, 2, 3, 4, 5, 6) .
These acceptors diffuse into cells and into their Golgi apparatus where
they compete with endogenous core proteins and make protein-free GAG
chains. Previous studies of these acceptors typically focused on the
physiological effects of disrupting normal proteoglycan synthesis and
on the analysis of the large, highly sulfated GAG chains
(1, 2, 4, 5, 6, 7) .
Surprisingly, however, several recent studies showed that the major
products were not large GAG chains, but rather a series of relatively
small, non-sulfated molecules
(8, 9, 10) . Some
of these were the expected intermediates in the synthesis of the core
linkage region of GAG chains,
GlcA
(1, 3) Gal
(1, 3) Gal
(1, 4) Xyl
-O-3Ser/Thr.
Other structures were novel and appeared unrelated to the known route
of GAG chain synthesis
(8, 9, 10) .
Significantly, synthesis of some of these smaller structures also
caused selective inhibition of glycolipid synthesis
(9) .
H]Gal-labeled products made on
4-methylumbelliferyl-
-xyloside (Xyl
MU) by human melanoma
cells consisted of Gal
(1, 4) Xyl
MU,
Gal
(1, 3) Gal
(1, 4) Xyl
MU,
GlcA
(1, 3) Gal
(1, 3) Gal
(1, 4) Xyl
MU,
and primarily an unexpected novel compound,
Sia
(2, 3) Gal
(1, 4) Xyl
MU
(9) . About 10-15% of the anionic material remained
uncharacterized in this study because it was resistant to a variety of
enzymatic digestions used to successfully analyze the others. Here we
report the purification and structural analysis of one of the
components of this material. The results explain its resistance to
enzymatic digestions and demonstrate the existence of a previously
unidentified core-related structure. In addition, we present evidence
that Xyl
MU also primes the synthesis of molecules that lack the
typical tandem galactosyl residues found in the core oligosaccharide
region of GAG chains.
Materials
Human melanoma cells UACC-903 were
obtained from Dr. J. M. Trent, University of Michigan, Ann Arbor, MI.
Arthrobacter ureafaciens sialidase was obtained from Oxford
Glycosystems, and bovine -glucuronidase was the generous gift of
Dr. Phil Stahl, Washington University, St. Louis, MO. C-18 (SPICE)
cartridges (400 mg and 2 g) were purchased from Analtech Inc.
[6-
H]Galactose (15 Ci/mmol) was obtained from
American Radiolabeled Chemicals; and QAE-Sephadex, Xyl
MU, and
routine chemicals were obtained from Sigma or Fisher.
Isolation of Anionic Material
Six T450 culture
flasks each containing 1.8
10
melanoma cells
(UACC-903) at 80% confluency were incubated for 5 days at 37 °C in
7% CO
atmosphere. Each flask contained 200 ml of serum-free
Dulbecco's modified Eagle's medium supplemented with 1
m
M Xyl
MU. The cells were removed by centrifugation and
the medium was lyophilized and then reconstituted in 40 ml of water. A
mixture of 54,000 cpm of [
H]Gal-labeled anionic
MU-
-xylosides previously purified from another batch of these
cells was added to the reconstituted medium to monitor purification.
The radiolabeled material had been purified by C-18 chromatography and
ion-exchange chromatography on QAE-Sephadex as described below and in
previous work
(9) . The material contained a single negative
charge as determined by ion-exchange HPLC on a Varian AX5 column.
cut-off
dialysis tubing. The material retained within the tubing was
chromatographed on a 5-ml column of QAE-Sephadex and the anionic
molecules were eluted with 100 m
M NaCl. The eluate was
desalted on a 2-g bed C-18 cartridge (Alltech) by washing with water
and the MU-
-xyloside-based products were eluted with 40% MeOH. The
sample was concentrated by evaporation to 0.5 ml and digested with 40
milliunits of A. ureafaciens sialidase and 50 milliunits of
bovine testicular
-glucuronidase together in 0.1
M sodium
acetate, pH 6.0, with 4 m
M CaCl
at 37 °C for
16 h. It was again chromatographed on QAE-Sephadex to remove the
neutralized material and the remaining anionic material was eluted with
100 m
M NaCl. This was desalted on a C-18 cartridge, eluted
with 40% MeOH, dried, reconstituted with 40% MeOH, and stored at 4
°C.
Digestion of Anionic Material with
About 15
µg of the anionic material described above was digested with a
combination of 0.3 units of -N-Acetylgalactosaminidase and
-Glucuronidase
-glucuronidase and 25 milliunits of
- N-acetylgalactosaminidase in 100 m
M sodium
citrate-phosphate buffer, pH 4.0 (Supplied by Oxford Glycosystems), in
a volume of 50 µl for 20 h at 37 °C. Following digestion the
neutral and anionic xylosides were separated on QAE-Sephadex as
described above, desalted on a C-18 cartridge, and dried for analysis.
Compositional Analysis
Compositional analyses were
carried out at the UCSD Glycobiology Core. The sample was hydrolyzed in
2
M trifluoroacetic acid, 100 °C, for 4 h and the
hydrolysate was analyzed by HPAEC-PAD on a Dionex DX500 (Dionex Corp.)
on a CarboPac PA-1 column. All monosaccharide components and the MU
residue were eluted using a linear gradient of sodium acetate from 0 to
100 m
M between 5 and 25 min with constant, 16 m
M
sodium hydroxide concentration throughout the run. The elution position
and detector response were determined with authentic standards. Initial
qualitative analysis of the hydrolysate was done on silica gel plates
developed in EtAC:Pyr:AcOH:HO, 5:5:1:3, solvent system.
Sugars were detected either by silver nitrate spray or by ninhydrin
spray for amino sugars.
The
samples analyzed were: I, H NMR Spectroscopy
25 µg of the undigested anionic
xyloside; IA,
15 µg of the material neutralized by the
combined
- N-acetylgalactosaminidase and
-glucuronidase digestions; IB,
10 µg of the material
which remained anionic after the combined enzyme digestion. Each sample
was repeatedly exchanged in D
O (99.96%, Aldrich Chemical
Co.) with intermediate lyophilization, and finally dissolved in 40
µl of 99.996% D
O. Spectra were recorded on a Varian
Unity Plus 500 MHz spectrometer (Varian Applications Laboratories, Palo
Alto, CA) using a Nano-NMR probe. The Nano-NMR probe spins samples
rapidly (1-2 kHz) at the magic angle to remove the
magnetic-susceptibility contributions to the
H NMR line
widths
(11) . This unique technology produces high-resolution
spectra in sample volumes <40 µl, thus lowering solvent
contaminants and background. The probe temperature was 25 or 30 °C
(as indicated in the figure legends) and the spin rate was typically
about 1850 Hz. Proton one-dimensional NMR spectra were obtained in
about 1000 scans using presaturation of the HOD peak. TOCSY
two-dimensional spectra
(12) were obtained using a 10-kHz
MLEV17 spin lock of either 50- or 100-ms duration, 1.6 s (sample I), or
1.0 s (samples IA, and IB) of presaturation, 88 (sample I) or 48
(sample IA and IB) scans per t
data point, and 300
complex data points in t
; total measuring time was
30 h for sample I, and 13 h for samples IA and IB. Double
quantum-filtered COSY (DQFCOSY) spectra
(13) were obtained
using either 300 (samples I and IA) or 350 (sample IB) complex
t
data points having 48 scans each; measurement
times were 18 and 21 h, respectively. ROESY two-dimensional data
(14) for sample I were obtained using a 2-KHz pulsed spinlock of
200 ms duration, using 192 scans per 300 complex t
data points (60 h experiment). All two-dimensional spectra were
obtained in a phase-sensitive mode using hypercomplex sampling in
t
, and included presaturation of the HOD
resonance. Line broadening (0.2 Hz) was applied to one-dimensional
spectra, while two-dimensional spectra were weighted with gaussian
functions. Chemical shifts are given relative to
4,4-dimethyl-4-silapentane-1-sulfonate added as internal standard to
xyloside I. In the case of xylosides, IA and IB were actually measured
relative to the residual HOD peak at 4.80 ppm. When analyzing
Me
SO- d
solutions chemical shifts were
referenced to the Me
SO multiplet at 2.49 ppm.
LSI-MS and Electrospray MS
LSIMS analysis was
performed with a VG 70-SE (VG Instruments, United Kingdom) magnetic
sector mass spectrometer, in both positive and negative ion modes. The
instrument was equipped with a cesium ion gun operated at 23 kV and
with an emission current of 2-3 µA, in the positive mode and
18 kV, with an emission current of 4-5 µA in the negative
mode. Spectra were recorded in the mass range 200-2000 at constant
magnetic field. The xyloside (10 µg) dissolved in 5 µl of
40% methanol was applied to the stainless steel target holding 2 µl
of glycerol/thioglycerol matrix. The scans were repeated after addition
of sodium chloride to confirm the identity of the molecular ion.
Electrospray experiments were performed at the Scripps Research
Institute Mass Spectrometry Facility using an API III Perkin Elmer
Sciex triple-quadrupole mass spectrometer with an upper mass range of
m/z 2400. The ion spray interface was used for sample
introduction with a potential of the interface sprayer at 3.4 kV. All
solvents and reagents were of 99.9% purity and were obtained from
Aldrich or Curtin Matheson. Water was purified on a Nanopure filtration
system. The sample was dissolved in a methanol/water solvent at
20-50 µ
M and injected into the spectrometer at 4.0
µl/min.
Preliminary Analysis of the
In a previous study we labeled and purified a group of
[-Xyloside-based
Products
H]Gal-labeled
-xyloside-based products that
were secreted into the medium by human melanoma cells
(9) . We
determined the structures of
90% of these products by sequential
enzymatic digestions and HPLC analysis, as reported. Applying the same
methods to analyze the size and charge of the remaining 10-15% of
anionic species showed that this labeled material contains 3-5
monosaccharides and a single negative charge by anion-exchange HPLC
(data not shown). In the previous study, one of the keys to solving the
structures of the other components was sequential enzymatic or chemical
degradations of the anionic molecules. However, the negative charge on
the uncharacterized material was resistant to all treatments and
digestions which included: mild acid hydrolysis that cleaves sialic
acids, Newcastle Disease Virus sialidase, A. ureafaciens sialidase, bovine
-glucuronidase, Escherichia coli alkaline phosphatase, sequential mild acid hydrolysis and alkaline
phosphatase digestion, jack bean
-galactosidase digestion, or a
sequential digestion with human placental
-hexosaminidase A
followed by
-glucuronidase, and treatment with mercuric acetate
(or chloride). The last procedure cleaves the glycosidic linkage of
terminal uronic acids that contain a 4,5-unsaturated bond
(15, 16) . In addition, this material cannot be
metabolically labeled when cells are incubated for 12 h with up to 1
mCi/ml
SO
under conditions that heavily label
chondroitin sulfate chains at the same time (data not shown). Thus, the
molecule(s) does not appear to contain sulfate esters. Since these
treatments failed to provide any structural information, we turned to
the purification of larger amounts of nonradiolabeled material for
direct chemical analysis.
Purification of Unknown Xyloside-based
Product(s)
Human melanoma cells were grown in the presence of 1
m
M XylMU for 5 days as described under
``Experimental Procedures.'' To monitor purification and the
completeness of sialidase and
-glucuronidase digestions of the
nonradiolabeled material, 54,000 cpm of
[
H]Gal-labeled
-xylosides with one negative
charge were added to the culture medium at this point. This labeled
tracer was purified from a separate batch of cells by C-18 and
ion-exchange chromatography on QAE-Sephadex and a small portion was
characterized by prior
-glucuronidase and sialidase digestions.
This provided an estimate of the amount of the tracer material that
should be resistant to the digestion in the mixture and, therefore, a
measure of the expected yield. The flow chart in Fig. 1shows the
purification strategy based on the methods used to obtain the
radiolabeled material. A combination of dialysis, C-18, and
QAE-Sephadex anion-exchange chromatographies coupled with
-glucuronidase and sialidase digestions gave nearly the expected
yields based on our previous results
(9) and prior analysis of
the radiolabeled tracer itself (see ). At this point, we
could not determine the purity of the nonlabeled material in the
preparation because its isolation was solely based on the use of the
radioactive tracer.
Figure 1:
Purification of anionic xyloside
products. Flow chart of the purification of the XylMU products by
C-18 and QAE-Sephadex chromatographies combined with exoglycosidase
digestions. The final product was desalted on a C-18 cartridge and used
for the analyses described here. Details are given under
``Experimental Procedures'' and in the
text.
Compositional Analysis
The monosaccharide
composition of the preparation was determined as described under
``Experimental Procedures.'' MU, Xyl, Gal, GlcA, and
GalNHwere detected in molar ratios of 1.0:1.1:2.1:0.6:1.6.
Stronger hydrolysis conditions (6
N trifluoroacetic acid, 18
h, 100 °C) did not show the presence of any other monosaccharide.
FAB-MS and Electrospray MS Analysis of the Major
Component
To further characterize the molecule, a portion of the
sample was subjected to FAB-MS analysis in both positive ion and
negative ion modes. These analyses indicated that the major component
has a native molecular mass of 1011 (data not shown). The sample was
then analyzed by electrospray mass spectrometry in both negative and
positive ion modes. The results in the negative ion mode
(Fig. 2 A) showed a major pseudomolecular ion at m/z 1010 and minor peaks at m/z 1032 ([M -
2H+ Na
]
)
and 1054 ([M -
H
+
2Na
]
) resulting from the
substitution with Na
ions (22 µm each). Detailed
analysis of the structure of the minor components (peaks between 1068
and 1196 atomic mass units) is in progress. Fig. 2 B,
shows the results in positive ion mode with the expected [M +
H
]
( m/z 1012), [M
+ Na
]
( m/z 1034),
and [M + 2Na
-
H
]
( m/z 1056). Thus,
the native molecular mass for the major component by electrospray mass
spectrometry is 1011 and agrees with that obtained by FAB-MS. This mass
is consistent with the following structure,
HexNAc
HexA
Hex
Pent
MU
.
Figure 2:
Electrospray mass spectrometry. The
purified xylosides I, IA, and IB were subjected to electrospray mass
spectrometry as described under ``Experimental Procedures.''
A, negative ion mode spectrum of xyloside I; B,
positive ion mode spectrum of xyloside I; C, positive ion mode
spectrum of xyloside IA; and D, negative ion mode spectrum of
xyloside IB. The molecular mass of the major compound in sample I is
calculated to be 1011 atomic mass units, which is consistent with a
molecule composed of HexNAc-HexA-Hex-Hex-Xyl-MU.
Minor Components of Xyloside I Detected by Electrospray
MS Analysis
Several other minor components were detected (less
than 10% each): m/z 994, corresponding to
HexNAcPent
MU
; m/z 818,
corresponding to
HexNAc
HexA
Pent
MU
; and
m/z 747 and 769, corresponding to
HexA
Pent
MU
pseudomolecular ion and
its monosodiated form. Other fragments detected in the negative ion
mode (Fig. 2 A) are consistent with the following
structures:
HexNAc
HexA
Hex
Pent
MU
(SO
H)
( m/z 1092, [M -
H
]
; m/z 1114, [M
- 2H
+
Na
]
; m/z 1136, [M
- 3H
+
2Na
]
),
HexNAc
Pent
MU
(SO
H)
( m/z 1092 [M -
H
]
; m/z 1114, [M
- 2H
+
Na
]
) and
HexNAc
HexA
Hex
Pent
MU
(CH
)
( m/z 1068, [M -
H
+ 2Na
]
). This
indicates that a pentasaccharide with the same primary structure as the
major compound can be substituted with either a monosulfate ester, or a
methyl ester (or ether) in some cases. Very little fragmentation of the
major molecular ion is observed ( m/z 806).
Electrospray MS Analysis of Neutral and Anionic Species
after Glycosidase Digestion
The anionic xyloside mixture (sample
I) was digested with a combination of -glucuronidase and
- N-acetylgalactosaminidase and then separated into
neutral (IA) and anionic (IB) fractions by QAE-Sephadex chromatography
(see ``Experimental Procedures''). Electrospray analyses of
these two fractions are shown in Fig. 2 C (xyloside IA,
positive ion mode), and Fig. 2 D (xyloside IB, negative
ion mode). Since these samples were analyzed after exchanging the
hydroxyl protons with D
O for NMR analysis, and dissolved in
H
O for electrospray analysis, partial deuteration leading
to heterogeneity of individual peaks is observed. The two major ions
seen in the neutral fraction shown in Fig. 2 C at m/z 657 and 331, are consistent with a primary structure containing
Hex
Pent
MU
and
Pent
MU
, respectively, where a proton has been
substituted by sodium. The two corresponding pseudomolecular ions are
observed at m/z 309 and 640.
HexA
Hex
Pent
MU
( m/z 849, minor),
HexA
Pent
MU
( m/z 747),
HexNAc
HexA
Pent
MU
( m/z 687, major),
HexA
Pent
MU
( m/z 615), and
Pent
MU ( m/z 307, major). Fragment ions arising
from the cleavage of the N-acetylhexosamine glycosidic linkage
are also observed at m/z 806 (1011-HexNAc), m/z 645
(849-HexNAc), and 483 (687-HexNAc). The minor novel MU glycans detected
by electrospray mass spectrometry are listed in .
500-MHz
Because
of the small amount of material available (estimated between 10 and 25
µg for the different samples analyzed), one-dimensional and
two-dimensional H NMR Spectroscopy
H NMR analysis were carried out using a
Nano-NMR probe containing 40 µl of the sample solution. The
standard, 7-hydroxy-4-methylcoumarin
-xyloside (Xyl
MU) was
analyzed in the same fashion for comparison (Fig. Z1).
Figure Z1:
Structure 1
Standard 7-Hydroxy-4-methylcoumarin
-Xyloside
H NMR data for
7-hydroxy-4-methylcoumarin is available in the literature
(17) .
The one-dimensional
H NMR of Xyl
MU was recorded in
Me
SO- d
. The signals corresponding to
the methyl and aromatic hydrogens were readily assigned as indicated in
I. The anomeric signal of the
-xyloside appears at
5.045. This represents a downfield shift of 1.132 ppm relative to
the anomeric resonance of a terminal non-reducing
-xylose residue
linked to OH-3 of an underlying mannose residue, recorded in the same
solvent
(18) . However, not all of the expected signals were
found in the one-dimensional spectrum. Only two sharp doublets at
3.364 and 3.770, and two broad signals at
5.37 and 5.10,
corresponding to hydroxyl protons, were observed. These signals were
resolved by a combination of two-dimensional DQFCOSY and TOCSY
experiments (not shown) run at different temperatures (between 22
°C and 65 °C). In this way, the remaining resonances
overlapping the residual HOD peak could be assigned. The DQFCOSY
experiment showed the expected coupling between the aromatic protons
d and e, and between the aromatic proton b and the methyl proton in the coumarin ring. The DQFCOSY spectrum
also located the Xyl H-2 signal at 3.24 ppm, which represents a
downfield shift of 0.2 ppm relative to the value reported in Ref. 2.
The broad doublets at
5.100 and 5.370 were assigned to OH-3 and
OH-2, respectively, on the basis of their coupling with H-1, H-2, and
H-3. From the analysis of the connectivities of these hydroxyl groups
it was also evident that the signal of H-3 is superimposed to that of
H-2 (
3.24). Thus the signal at
3.364 was assigned to Xyl
H-4, and that at
3.770 to Xyl H-5
. H-5
in a
-linked xylose is expected to produce a doublet of
doublets slightly downfield from the H-2 resonance. The TOCSY spectrum
showed a correlation between H-4 and a signal at
3.260 that was
assigned to H-5
. The TOCSY spectrum also showed a
cross-peak between H-4 and a signal overlapped to H-1, this signal most
likely corresponds to OH-4. It has to be pointed out that a downfield
shift of 0.17-0.35 ppm from the values obtained in D
O
was reported when using Me
SO as the solvent
(18) .
The assignments of the proton resonances in the xylose ring (with the
exception of H-1 and H-2, deprotected by the aromatic ring) correspond
very well with the reported assignments for a 4-substituted methyl
xyloside which spectra were also obtained in
Me
SO- d
at 30 °C
(19) .
One-dimensional and Two-dimensional
The one-dimensional
H NMR
Analysis of the Unknown Xyloside I
H NMR spectrum obtained with approximately 25 µg of
xyloside I using a Nano-NMR probe is shown in Fig. 3, and the
H NMR data are summarized in I. This spectrum
is consistent with the electrospray mass spectrometric analysis (see
above), indicating the presence of a mixture. However, considerable
amount of information can be obtained from the spectrum. The signals
expected for the MU aromatic ring, as well as those characteristic
regions of the oligosaccharide ``reporter groups'' are
readily recognized. From the integration of the anomeric region it is
possible to conclude that at least two components (in ratio 2:1) are
present.
Figure 3:
One-dimensional H NMR spectrum
of the
-xyloside I recorded at 500 MHz in D
O at 25
°C. A, complete spectrum; B, expansion of the
anomeric region; C, expansion of the aromatic region; and
D, expansion of the 3.30-4.75 ppm
region.
The first striking feature of this spectrum is the presence
of a pair of -anomeric resonances at
5.480
( J
= 3.91 Hz) and
5.463
( J
= 3.67 Hz), in a ratio 2:1
(Fig. 3 B). This
-anomeric signal is unexpected
because there is no precedent for such a residue in the known sequence
of the GAG chain core. The same 2:1 ratio is observed for the two pairs
of
-anomeric signals at
5.243 ( J
= 7.57 Hz) and
5.254 ( J
= 7.09 Hz), shown enlarged in Fig. 3 B. Two
types of aromatic signals d are also observed
(Fig. 3 C). The signal at
4.598 was assigned to a
-Gal anomeric proton (Fig. 3 D). The remaining
anomeric signals at
4.701 and
4.690 could be tentatively
assigned to another
-Gal residue, and to a
-glucuronic acid
residue on the basis of the known monosaccharide composition, and the
known structure of a GAG chain core region. However, it is not possible
to decide which is which. The signal at
2.496 was assigned to the
MU methyl protons, and that at
2.080 to an N-acetyl
group.
noise
line at 4.9 ppm. The
-anomeric signal at
5.480 presents
cross-peaks with only three other signals. The typical pattern of a
galactose-type ring where the coupling constant between H-4 and H-5 is
very small and cannot produce an observable magnetization transfer.
Furthermore, comparing the spectra acquired with 50 and 100 ms mixing
time (not shown), it is possible to assign the resonance at
4.173
to H-2, the resonance at
3.897 to H-3, and the one at
4.016
to H-4.
Figure 4:
Two-dimensional TOCSY of the
-xyloside I recorded at 500 MHz in D
O at 25 °C.
Expansion of the region between 3.2 and 5.6 ppm showing the
connectivities in each monosaccharide residue. The numbers and
letters in the spectra refer to the corresponding residues in
the structure.
Very few examples of -linked
N-acetylgalactosamine residues have been reported
(20, 21, 22, 23) , and certainly none of
them in GAG chains. Thus, other experiments were carried out to confirm
this assignment (see below). The pattern of cross-peaks for the
anomeric signal at
5.243 is similar to that of the xylose ring on
the standard MU
-xyloside, but with a considerable downfield shift
explained by the different solvent used in both experiments. A detailed
analysis of the two-dimensional TOCSY slices obtained in the f
direction, at 50- and 100-ms mixing times (not shown), allowed
assigning of the different resonances as indicated in I.
The group of cross-peaks correlated to the anomeric signals at
4.6-4.7 is very complex due to the presence of a mixture of
compounds. As shown in Fig. 4, there is considerable overlapping
of signals between the glucuronic acid and galactose residues.
Furthermore, it is clear from the expansion of the two-dimensional
TOCSY spectrum that there are two different uronic acid signals
characterized by the typical triplets of H-2 between 3.1 and 3.3 ppm.
These triplets were also observed in the one-dimensional spectrum (see
Fig. 3A). In fact, the integration of the
one-dimensional spectrum allows us to determine that the uronic acid
with H-2 resonance at higher magnetic field corresponds to the main
component in this mixture. From the expansion of the slices obtained in
the f
direction at 50 and 100 ms (not shown), it was
possible to assign the resonances of Gal-2, Gal-3, and the two GlcA
residues of the major and minor components of the mixture, as indicated
in I. The resonances observed for the two galactose
residues, as well as those for the glucuronic acid residue, correspond
better to the data reported in the literature for serine-linked
oligosaccharides obtained from proteoglycan linkage regions than with
those reported for reduced oligosaccharides
(24, 25, 26, 27) . However, there is
considerable influence of the MU aglycone.
NMR Analysis of Fractions IA and IB
To confirm the
structure of the major component in sample I, approximately 20 µg
of this material was digested with a combination of
- N-acetylgalactosaminidase and
-glucuronidase. This
treatment neutralized 66% of the [
H]galactose
tracer in the preparation and showed that the major component of the
tracer was sensitive to this digestion. The neutralized material was
separated from the remaining anionic material on QAE-Sephadex and
desalted on C-18. Both the neutral component, IA (
15 µg), and
the remaining anionic component, IB (
10 µg), were analyzed by
one-dimensional and two-dimensional
H NMR after
purification.
H NMR,
the neutral product is still a mixture with anomeric signals for
-xylose at
5.260 and 5.290 in a ratio 2:1. However, the
anomeric signal at
5.480 has disappeared, as well as the methyl
signal of the N-acetyl group. This result confirms the
assignment of the resonance at
5.480 to the anomeric proton of a
-GalNAc residue. As expected for this neutral compound, no
resonances that could be attributed to a glucuronic acid residue were
observed. On the contrary, a clear
-galactose anomeric region
shows two resonances at
4.660 and 4.715 in a 1:1 ratio
(Fig. 5 A). Resonances were assigned using the
two-dimensional DQFCOSY and TOCSY experiments shown in Fig. 5,
B and C. The corresponding
H NMR data are
summarized in I. Thus, the
H NMR results
indicate that one of the components in this mixture has the structure,
Gal
(1, 2, 3) Gal
(1, 2, 3, 4) Xyl
(1, 2, 3, 4) MU,
while the other one resembles the starting material
(MU-
-xyloside). These results are in agreement with the mass
spectrometry study described above.
Figure 5:
NMR spectra of the neutral fraction
( IA) of the -xyloside recorded at 500 MHz in
D
O at 30 °C. A, expansion of the carbohydrate
region; B, two-dimensional DQFCOSY spectrum. In the figure,
the assignment pathways for the xylose and galactose residues are
drawn; C, two-dimensional TOCSY spectrum obtained with 100 ms
mixing time. The numbers and letters in the spectra
refer to the corresponding residues in the
structure.
The spectra obtained for the
acidic product showed the presence of a complex mixture of several
compounds in similar ratios, and complete assignment could not be
achieved. However, several pieces of information could be obtained from
these experiments.
-anomeric resonance of N-acetylgalactosamine is observed
in the one-dimensional spectrum in a ratio 0.75:1 with the main
component.
-anomeric resonance (see I). H-5
could
not be detected (data not shown). The higher intensity of this signal
is consistent with the electrospray mas spectrometry data that show the
presence of sequences containing two or three xylose repeats.
-galactose and
-glucuronic acid cross-peaks looks similar to that of the original
mixture. However, in this case all resonances could not be completely
assigned because of lack of sensitivity. It is possible to observe two
galactose patterns, one of which has a superimposed set of signals that
should correspond to the glucuronic acid residue with H-2 resonance at
3.46. A second uronic acid was not found in this product, but a
new set of signals was observed in this region with anomeric resonance
at
4.55 (not shown). This chemical shift is similar to the
reported values for a
-GalNAc residue sulfated at position 6
(26) .
Linkage Analysis of Xyloside I by Two-dimensional
ROESY
The two-dimensional ROESY experiment allowed us to
identify the linkage positions. Interresidue cross-peaks were found
between H-1 of GalNAc and H-4 of GlcA, H-1 of GlcA and H-3 of Gal-3,
between H-1 of Gal-3 and H-3 of Gal-2, and between H-1 of Gal-2 and H-4
of Xyl. Furthermore, the H-5 signal of the GalNAc residue could be
assigned to 3.70 ppm since the closeness in space of the H-4 and H-5
affords a cross-peak in this spectrum (Fig. 6).
Figure 6:
Two-dimensional ROESY spectrum of the
-xyloside I recorded at 500 MHz in D
O at 30 °C,
with a mixing time of 200 ms. Interresidue ROEs are indicated by
arrows. ROESY correlations (phase opposite the diagonal) are
indicated with 10 contour lines; TOCSY artifacts, and the diagonal are
indicated with a single contour line.
-xylosides and metabolically labeled with
[6-
H]Gal make a series of neutral and anionic
labeled di- to tetrasaccharides on the added acceptors
(9) .
These include the expected biosynthetic intermediates of the GAG core
glycan. In the previous study, most of the anionic
[
H]Gal-labeled
-xylosides were structurally
analyzed by enzymatic and chemical degradations, but 10-15% were
resistant to these degradations. Preliminary studies showed that the
unknown molecule(s) contained 4-5 sugars and a single negative
charge. This paper and its companion
(36) focus on the study of
these products.
H]Gal-labeled molecule to
-glucuronidase
digestion suggests that the GlcA residue has modified or not the
terminal residue. When these products were isolated in enough quantity,
and were submitted to monosaccharide compositional analysis, LSIMS, and
electrospray MS analyses, the results were consistent with a
pentasaccharide core typical of chondroitin sulfate. Further analysis
by one-dimensional and two-dimensional NMR showed the expected signals
for a typical core structure except that the terminal GalNAc was in an
rather than
configuration.
-GalNAc
residue at this position
(3) . Heparan sulfate/heparin chains
also share the same core, but have an
-GlcNAc residue at this
position. Addition of either
-GalNAc or
-GlcNAc to the core
is considered the first committed step for building up each type of
chain. The amino acid sequence of the protein itself
(27) or
modifications of the core saccharide may control the choice between
chondroitin sulfate and heparan sulfate
(23, 24, 25, 26, 28) . NMR
analyses of the core region of chondroitin sulfate chains following
chondroitinase digestion have shown that GalNAc at this location is
-linked to the single core GlcA
(24, 25, 26, 27, 29) . In
vitro biosynthetic studies of the core saccharide synthesis using
solubilized membranes also showed that the GalNAc at this location is
totally sensitive to digestion with
-hexosaminidase
(30) .
These studies also showed that the
-GalNAc residue is added to the
core
-GlcA by a different enzyme than that responsible for chain
elongation in chondroitin sulfate.
-GalNAc
residue at this critical location is intriguing because it provides
another, previously unrecognized option for the tetrasaccharide
intermediate. It is possible that
-GalNAc stops further elongation
of the chain. Since glycosyltransferases specifically recognize the
anomeric linkage of the sugar acceptor, it is unlikely that the next
enzyme, presumably a
-glucuronosyltransferase, would recognize
-GalNAc and
-GalNAc residues equally well. The low amount of
-GalNAc terminated xyloside products in these preparations may be
due to their rapid elongation to form larger GAG chains, which are not
retained on the C-18 cartridges used to prepare these samples.
-GalNAc residues are sometimes found in
glycolipids and in blood group A oligosaccharides
(31, 32) , there are no reports of GalNAc
GlcA
-
disaccharide sequences in any glycoprotein in the CarbBank data base.
Furthermore, the melanoma cells that we studied do not make such
glycolipids and are not reactive with anti-A antibodies. Thus, it is
not likely that other
-GalNAc transferases account for the
synthesis of the
-GalNAc-terminated GAG chain cores.
H NMR results allowed us to clearly identify the major
-GalNAc terminated xyloside. However, definitive assignment of all
resonances in the
H NMR spectra of xylosides I and IB was
not possible because of the complexity of these mixtures. Nevertheless,
we gained considerable information from the NMR analysis of the minute
amounts of this complex mixture. These results were achieved with only
20 µg of sample I which represents about one-tenth to one-third of
the minimum amounts used for such analyses. Complete structural
characterization of each product in this mixture of xylosides will
require much more work to perform similar analyses. This is under way
in our laboratories.
MU. The first three types contain one pentose (xylose) and 0,
1, or 2 hexose (galactose) units along with 1-2 HexNAc and HexA.
The fourth type contains 2 pentoses, a HexNAc, and 1-2 HexA. The
fifth type contains 3 pentoses and a HexA. This last type represents
about 10% of sample I. It appears resistant to
-glucuronidase
digestion since it remains anionic (1B) after two rounds of
-glucuronidase digestions, and the corresponding mass for
Xyl
MU is not found in the neutral fraction, 1A.
HexA
Pent
MU ( m/z 818), seen in unfractionated sample I, appears to be susceptible
to
- N-acetylgalactosaminidase digestion. This conclusion
is based in the disappearance of its pseudomolecular ion following
digestion, and the presence of the pseudomolecular ion for the same
molecule minus a HexNAc residue ( m/z 616) in the anionic
xyloside sample IB. On the other hand, this molecule also appears to be
resistant to
-glucuronidase digestion, since an m/z 440
corresponding to the further loss of HexA is not found in the neutral
fraction 1A.
(1, 3) Xyl
(1, 3) Glc
linked to serine
(33) , and Izumi et al. (10) recently found that human fibroblasts make a novel
xyloside, Xyl
(1, 4) Xyl
MU, when incubated with
MU. A xylosyl transferase in the soluble fraction of rat kidney
homogenate glycosylates glycogenin, but this enzyme is not expected to
have access to the xylosides passing through the Golgi
(34, 35) . It is possible that other xylose-containing
sugar chains have been overlooked in the past. On the other hand, the
small amounts of these structures may simply be the result of
glycosyltransferase promiscuity in the presence of high endogenous
concentrations of the artificial acceptors.
-GalNAc cap such an experimental artifact? Although this is
possible, we think this is unlikely, based on the results presented in
the companion paper
(36) . However, it will be necessary to
identify such a structure on a known proteoglycan. These studies are
also in progress.
Table: Recovery of H-Gal radiolabeled anionic xylosides
Table: 1952998688p4in
NA, not applicable.
Table: H chemical shifts of
the constituent monosaccharides of compounds I, IA, and IB and the
reference compound MU b-Xyloside
MU,
4-methylumbelliferyl-
-xyloside; HPAEC-PAD, high pH anion-exchange
chromatography with pulsed amperometric detection; FAB-MS, fast atom
bombardment-mass spectrometry; LSIMS, liquid secondary ion mass
spectrometry; DQFCOSY, double quantum filtered correlation
spectroscopy; TOCSY, totally scalar correlated spectroscopy; MLEV17,
Malcom Levitt-17 mixing sequence; ROESY, rotating frame Overhauser
enhancement spectroscopy; HPLC, high performance liquid chromatography;
pNP, p-nitrophenyl.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.