Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary
Soluble Thrombomodulin,
SO4-3GlcA
1-3Gal
1-3(±Sia
2-6)Gal
1-4Xyl*
Hiroyuki
Wakabayashi
§,
Shunji
Natsuka
,
Tomohiro
Mega
,
Naoki
Otsuki
¶,
Mitsuko
Isaji
,
Masaaki
Naotsuka**,
Sadatoshi
Koyama**,
Toshinori
Kanamori**,
Kiyoshi
Sakai**, and
Sumihiro
Hase

From the
Department of Chemistry, Graduate School of
Science, Osaka University, Toyonaka, Osaka 560-0043, ** Pharmaceutical
Laboratory, Mochida Pharmaceutical Co., Ltd., Fujieda, Shizuoka
426-8640, and
Biosciences Research Laboratory, Mochida
Pharmaceutical Co., Ltd., Kita-ku, Tokyo 115-8515, Japan
 |
ABSTRACT |
O-linked sugar chains with xylose as
a reducing end linked to human urinary soluble thrombomodulin were
studied. Sugar chains were liberated by hydrazinolysis followed by
N-acetylation and tagged with 2-aminopyridine. Two
fractions containing pyridylaminated Xyl as a reducing end were
collected. Their structures were determined by partial acid hydrolysis,
two-dimensional sugar mapping combined with exoglycosidase digestions,
methylation analysis, mass spectrometry, and NMR as
SO4-3GlcA
1-3Gal
1-3(±Sia
2-6)Gal
1-4Xyl.
These sugar chains could bind to an HNK-1 monoclonal antibody. This is
believed to be the first example of a proteoglycan linkage
tetrasaccharide with glucuronic acid 3-sulfate and sialic acid.
 |
INTRODUCTION |
Thrombomodulin (TM)1 is
a physiologically important anticoagulant (1) that is present not only
on the endothelial cell surface but also in soluble form in plasma and
urine (2-4). Takahashi et al. (5) have purified the major
active forms of human urinary soluble thrombomodulin (uTM) and
demonstrated that they possess strong cofactor activity for
thrombin-catalyzed protein C activation as well as exhibiting potent
anticoagulant activity in vivo. They have also shown that
uTM improves disseminated intravascular coagulation without excessive
prolongation of the activated partial thromboplastin time (6). The
protein possesses five potential N-linked glycosylation sites (7) as deduced from its amino acid sequence, whereas the
detection of GalNAc suggests the presence of O-linked sugar chains (8). Although uTM does not contain a glycosaminoglycan, recombinant TM and some TMs obtained from cultured human endothelial cells are expressed in both a high molecular weight TM containing chondroitin sulfate and a low molecular weight TM lacking this modification (9, 10). Recombinant TM expressed in Chinese hamster ovary
cells contains chondroitin 4-sulfate (11). TM glycosylation in relation
to biological activity has been discussed in several papers (12-14),
but to understand the structure and biological activities of the
glycoprotein, detailed knowledge of the sugar structures is essential.
Recently, the structures of N-linked sugar chains have been
reported (15). During the course of studies on N- and
O-linked sugar chains of uTM, we have detected new
structures with xylose at the reducing ends. Here, we present a
detailed analysis of the novel sugar structures found in uTM.
 |
EXPERIMENTAL PROCEDURES |
Materials--
uTM was purified from human urine as reported
previously (5).
-Glucuronidase (bovine liver) was obtained from
Sigma. Sialidase (Arthrobacter ureafaciens) was purchased
from Nacalai Tesque (Kyoto),
-galactosidase (Aspergillus
sp.) from Toyobo (Osaka), actinase E from Kaken Pharmaceutical (Tokyo),
chondro-4-sulfatase and chondro-6-sulfatase from Seikagaku Kogyo
(Tokyo), and monoclonal HNK-1 antibody from Cosmo Bio (Tokyo).
Gal
1-4Xyl-PA and GlcA
1-3Gal
1-3Gal
1-4Xyl-PA were
prepared as described (16). Standard partially O-methylated pyridylaminated (PA)-galactoses were prepared by pyridylamination of
the corresponding partially O-methylated galactoses as
reported (17). A Cosmosil 5C18-AR300 column (0.6 × 15 cm) was
purchased from Nacalai Tesque, a Shodex NH2P-50 column
(0.46 × 5 cm) from Showa Denko (Tokyo), a TSKgel sugar AXI column
(0.46 × 7.5 cm) and Toyopearl HW-40F from Tosoh (Tokyo), Sephadex
G-25, Sephadex LH-20, and a Mono Q HR 5/5 column from Amersham
Pharmacia Biotech, a µBondasphere 5C18-300Å column (0.39 × 15 cm) from Nihon Waters (Tokyo), an IonPac AS4A column (0.4 × 25 cm) from Dionex (Sunnyvale, CA), a YMC-Pack Diol-200G column (0.8 × 50 cm) from YMC (Tokyo), DC-Alifolien silica gel 60 plates from
Merck, Advantec 51B filter papers from Toyo Roshi (Tokyo), and
Spectrapor membrane tubing (MWCO: 25,000) from Spectrum Medical
Industries (Los Angeles, CA).
Preparation of Glycopeptides--
uTM (374 mg) was digested with
37 mg of actinase E in 3.7 ml of 0.2 M Tris-HCl buffer, pH
8.2, at 37 °C for 1 h. An additional 3.7 mg of the enzyme was
then added, and the incubation was continued for 24 h. The digest
was applied onto a Toyopearl HW-40F column (2.2 × 100 cm), and
glycopeptides were eluted with 0.2 M ammonium acetate
buffer, pH 6.0.
Preparation of PA Sugar Chains--
PA sugar chains were
prepared and purified according to the reported method (18). A
freeze-dried glycopeptide fraction was heated with 1.6 ml of anhydrous
hydrazine at 60 °C for 50 h followed by
N-acetylation with 3.2 ml of saturated sodium bicarbonate
solution and 128 µl of acetic anhydride. After desalting with Dowex
50 (H+), the solution was freeze-dried, and the residue was
pyridylaminated with 160 µl of 2-aminopyridine reagent and 560 µl
of reducing reagents prepared as reported (18). Excess reagents were
evaporated three times with 50 µl of toluene:methanol (1:1) under a
stream of nitrogen at 60 °C for 10 min.
HPLC of PA Sugar Chains--
HPLC was carried out using the
following elution conditions. PA sugar chains were detected by
fluorescence (excitation wavelength, 320 nm; emission wavelength, 400 nm).
Elution Condition 1 for PA O-linked sugar chains: column,
Cosmosil 5C18-AR300; flow rate, 2 ml/min at 25 °C; eluent, 20 mM ammonium acetate buffer, pH 6.0, containing 0.01%
1-butanol. After injecting a sample, the butanol concentration was
increased linearly from 0.01 to 0.52% in 51 min and then to 1.0% in
12 min.
Elution Condition 2 for methylation analysis: column, µBondasphere
5C18-300Å; flow rate, 1.0 ml/min at 25 °C; eluent, 0.1 M ammonium acetate buffer, pH 6.0. After injecting a
sample, the acetonitrile concentration was increased linearly from 0 to
70% in 45 min.
Size fractionation HPLC: column, Shodex NH2P-50; flow rate,
0.6 ml/min at 25 °C. Two eluents, A and B, were used. Eluent A was
acetonitrile:water:acetic acid (95:5:3, v/v/v) titrated to pH 7.3 with
triethylamine, and Eluent B was water:acetic acid (100:3, v/v) titrated
to pH 7.3 with triethylamine. The column was equilibrated with Eluent
A. Three min after injecting a sample, linear gradient elution was
performed to Eluent A:Eluent B (90:10, v/v) in 2 min and then to Eluent
A:Eluent B (50:50 v/v) in 38 min.
Anion-exchange HPLC: column, TSKgel Sugar AXI; flow rate, 0.3 ml/min at
73 °C. The eluent used was a mixture of 9 parts 0.8 M
boric acid adjusted to pH 9.0 with potassium hydroxide and 1 part acetonitrile.
Mono Q HPLC: column, Mono Q HR 5/5; flow rate 1.0 ml/min at 25 °C.
The eluent used was water titrated to pH 9.0 with aqueous ammonia.
Adsorbed samples were eluted with a linear gradient of ammonium sulfate
from 0 to 1.0 M concentration.
Exoglycosidase Digestion--
A PA sugar chain (100 pmol) was
digested with 100 milliunits of Aspergillus
-galactosidase in 20 µl of 50 mM ammonium acetate buffer, pH 4.5; with 2.5 milliunits of sialidase in 50 µl of 100 mM ammonium acetate buffer, pH 5.0; or with 100 units of
-glucuronidase in 100 µl of 200 mM ammonium acetate
buffer, pH 5.0. Enzymatic reactions were carried out at 37 °C for
16 h and then terminated by heating at 100 °C for 3 min.
Methanolysis--
A PA sugar chain (500 pmol) was methanolyzed
with 50 mM HCl in methanol at 37 °C for 3 h (19).
After evaporation to dryness, the desulfated PA sugar chain was
N-acetylated with 120 µl of water:pyridine:acetic
anhydride (5:25:1) on ice for 1 h. After evaporation of the
solution, a small amount of O-acetyl groups was removed by
heating the solution with 20 µl of 1.0 M aqueous ammonia
at 100 °C for 5 min. The solution was then freeze-dried.
Methylation Analysis--
Freeze-dried X2 (2 nmol) was
permethylated with 0.1 ml of methylsulfinyl carbanion reagent (prepared
from 25 mg of NaH and 0.8 ml of dimethyl sulfoxide) and 0.1 ml of
methyl iodide as reported (20). The reaction mixture was placed on a
Sephadex LH 20 column (0.9 × 24 cm), and the permethylated X2 was
eluted with chloroform. One-ml fractions were collected, and each
fraction was concentrated to dryness by blowing the solvent with
nitrogen. The residue was dissolved in a small amount of chloroform,
and the sample was purified by TLC using a silica gel plate and
methanol:ethyl acetate (2:8, v/v) with one drop of acetic acid as a
solvent. The fluorescent spots revealed under a UV lamp were scraped
and combined, and the permethylated X2 was extracted with 2 ml of
methanol. The extract was evaporated to dryness, and the permethylated
X2 thus purified was then hydrolyzed with 50 µl of 4 M
trifluoroacetic acid at 100 °C for 3 h. The solution was
freeze-dried, and the residue was pyridylaminated with 10 µl of
2-aminopyridine reagent and 35 µl of borane-dimethylamine complex
reagent as reported (21). Excess reagents were evaporated three times
with 50 µl of toluene:methanol (1:1 v/v) under a stream of nitrogen
at 60 °C for 10 min (18). The residue was dissolved in 5 µl of the electrophoresis buffer, and small amounts of contaminating materials were removed by paper electrophoresis. Paper electrophoresis was performed at 30 V/cm at 4 °C using a filter paper (30 cm) and water:acetic acid:pyridine (60:2:3, v/v). The area that migrated like
PA GlcNAc was cut off, and PA derivatives of partially
O-methylated PA monosaccharides were extracted from the
paper 4 times each with 100 µl of water. A part of the solution (10 µl) was analyzed by reversed-phase HPLC using Elution Condition 2.
Mass Spectrometric Analysis--
Mass spectra were
recorded using a Voyager-DE STR BioSpectrometry Workstation, a
matrix-assisted laser desorption ionization time of flight mass
spectrometer (PerSeptive Biosystems, Framingham, MA). For mass
spectrometry, PA sugar chains were dissolved in distilled water (10 pmol/µl). Aliquots of 0.5 µl were applied onto a sample plate.
Subsequently, 0.5 µl of a matrix solution (10 mg/ml 2, 5-dihydroxybenzoic acid in 50% (v/v) acetonitrile) was mixed with the
aliquot and allowed to dry. The analyzer was used in the linear mode.
Nuclear Magnetic Resonance (NMR) Spectroscopy--
PA sugar
chains were exchanged twice with 2H2O at room
temperature with intermediate lyophilization and finally dissolved in 2H2O (99.999 atom % 2H; Isotec,
Miamisburg, OH). Samples were analyzed at 30 or 50 °C on a Varian
Unity Inova 750 MHz NMR spectrometer. Chemical shifts (
) are
expressed by reference to internal acetone (
2.213 ppm at 50 °C,
2.218 ppm at 30 °C).
Equilibrium Dialysis--
A commercial HNK-1 monoclonal antibody
was purified by gel filtration on a YMC-Pack Diol-200G column, and the
IgM fraction was concentrated by ultrafiltration with an Amicon YM-30
membrane. Equilibrium dialysis was performed between 60 µl each of an
antibody solution and an oligosaccharide solution using a Spectrapor
membrane at 2 °C for 48 h (22). Three concentrations of X1
(160, 80, and 40 nM) and one concentration of X2 (80 nM) were used. PA GlcNAc was employed as an internal
standard. After the dialysis, a part of the oligosaccharide fraction
was removed, and the amounts of X1 and X2 were quantified by size
fractionation HPLC. The binding constant was obtained by
double-reciprocal plots of the concentration of the bound
oligosaccharide versus that of the free oligosaccharide, as
reported (23).
Other Analytical Methods--
The structures of the PA sugar
chains were assessed by two-dimensional sugar mapping. A PA sugar chain
was chromatographed by reversed-phase (Elution Condition 1) and size
fractionation HPLC, and its elution position was compared with those of
standard PA sugar chains on a two-dimensional sugar map. The PA sugar
chain was then digested sequentially with exoglycosidases, and the
structure of the product was analyzed on the two-dimensional sugar map
as reported (23).
The reducing ends of PA sugar chains were analyzed according to the
reported method (24). PA sugar chains were hydrolyzed with 100 µl of
4 M trifluoroacetic acid at 100 °C for 3 h in
evacuated sealed tubes. The solution was evaporated to dryness by a
centrifugal concentrator, and the residue was N-acetylated
with saturated sodium bicarbonate solution and acetic anhydride. The PA
monosaccharides obtained were separated and quantified by
anion-exchange HPLC.
Component sugar analysis was done as reported (24). Glycopeptides were
hydrolyzed with 100 µl of 4 M trifluoroacetic acid at
100 °C for 3 h. The solution was freeze-dried, and
monosaccharides liberated were N-acetylated with saturated
sodium bicarbonate solution and acetic anhydride. After desalting with
Dowex 50 (H+), the solution was freeze-dried, and the
monosaccharides were pyridylaminated with 2-aminopyridine and
borane-dimethylamine complex. The excess reagents were removed under a
stream of nitrogen gas with 40 µl of toluene at 40 °C. The residue
was dissolved in a small amount of water, and a part of the solution
was analyzed by anion-exchange HPLC.
Sulfate ion was measured by ion chromatography. A PA sugar chain (2 nmol) was mixed with 20 µl of 20 mM sodium hydroxide. After heating at 250 °C for 30 min, the resulting sulfate ion was
measured with a Dionex 20210i ion chromatography system using an IonPac
AS4A column and 2.8 mM Na2CO3, 2.25 mM NaHCO3 at a flow rate of 1.5 ml/min (25).
Sialic acid was measured by the reported method (26).
 |
RESULTS |
Preparation and Analysis of a PA Sugar Chain Fraction from
uTM--
Glycopeptides were prepared from 374 mg of uTM by digestion
with actinase E, and the digest was purified by gel filtration. After
component analysis of each fraction, the fractions that contained
sugars were combined (data not shown). Sugar chains were liberated from
the combined fraction by hydrazinolysis followed by
N-acetylation, and the reducing ends of the sugar chains
were pyridylaminated. To obtain an overall view of the sugar
structures, the reducing ends of the PA sugar chain fraction were first
analyzed. The acid hydrolysates of the PA sugar chain fraction were
found to contain 0.39 µmol of PA Xyl, 3.0 µmol pf PA GlcNAc, 0.45 µmol of PA GalNAc, and 0.39 µmol of PA Gal (from a by-product
obtained from Gal-GalNAc structures during the preparation (18)),
indicating that a sugar chain containing Xyl at the reducing end
comprised about 8% (mol/mol) of the total sugar chains. Detection of
PA Xyl indicated the presence of proteoglycan-type sugar chains; however, the linking of chondroitin sulfate chains was excluded as
judged from the finding that the uTM bands on SDS-polyacrylamide electrophoresis gels were not changed by digestion with chondroitinase ABC (data not shown). The PA sugar chain fraction was digested with
sialidase and analyzed by Mono Q HPLC, and a pass-through and adsorbed
fractions were obtained. Reducing-end analysis of each fraction
revealed that PA Xyl was present only in the acid hydrolysates of the
adsorbed fraction, indicating that the xylose-containing sugar chains
carried a negative charge(s) in addition to sialic acid (data not shown).
Purification of PA Sugar Chains with Xylose at the Reducing
Ends--
The PA sugar chain fraction was applied onto a TSKgel HW-40F
column, and an aliquot of each fraction was subjected to reducing-end PA monosaccharide analysis (Fig. 1). A
fraction containing PA Xyl (Fraction X) was collected and further
fractionated by reversed-phase HPLC (Fig.
2). Reducing-end analysis of each peak
showed that fractions X1 and X2 contained a PA Xyl residue as a major
component. The other fractions and the fraction eluted between 25 and
60 ml did not contain appreciable amounts of PA Xyl. Other major peaks
contained PA GalNAc, PA Gal, or PA GlcNAc as major components. Small
amounts of contaminating materials were removed by size fractionation
HPLC, and the final fractions were desalted on a Sephadex G-25 column
(1 × 10 cm) using 10 mM ammonium acetate, pH 6.0, as
an eluent. Starting from Fraction X, X1, and X2 were recovered at 17 and 26% (including losses during purification), respectively. X1 and
X2 thus purified showed a single peak when analyzed by reversed-phase
HPLC (data not shown) using Elution Condition 1 and size fractionation
HPLC, indicating that X1 and X2 were pure.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Gel filtration profiles of PA sugar chains
from uTM. PA sugar chains were applied onto a TSKgel HW-40F column
(2 × 80 cm) using 0.2 M ammonium acetate buffer, pH
6.0, and 5-ml fractions were collected. An aliquot of each fraction was
subjected to reducing-end PA sugar analysis. , PA GlcNAc; , PA
GalNAc; , PA Xyl. Fractions indicated by a bar were
pooled as Fraction X.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Subfractionation of Fraction X by
reversed-phase HPLC. Elution Condition 1 was used (see
text).
|
|
Structure Analysis of X1--
The fluorescent fragments obtained
by partial acid hydrolysis (100 °C in 1 M
trifluoroacetic acid for 15 min) of X1 appeared at the elution
positions of GlcA
1-3Gal
1-3Gal
1-4Xyl-PA, Gal
1-4Xyl-PA, and PA Xyl on a two-dimensional sugar map (Fig.
3), indicating that X1 contained the
linkage tetrasaccharide structure of proteoglycans. X1 had 1 mol of
sulfate but no sialic acid (Table I). The
elution position of X1 on the map was not changed by digestion with
-glucuronidase, chondro-4-sulfatase, or chondro-6-sulfatase (data
not shown). When X1 was desulfated by methanolysis, the product
appeared at the position of GlcA
1-3Gal
1-3Gal
1-4Xyl-PA (Fig.
3). X1 was not digested with
-glucuronidase, but desulfated X1
was then susceptible to
-glucuronidase digestion. These results
indicated that the sulfate group was linked to the GlcA-Gal structure.
On further digestion of the
-glucuronidase digest (position
D) with Aspergillus
-galactosidase, a new peak
appeared at PA Xyl. These results suggested that X1 was
SO4-GlcA
1-3Gal
1-3Gal
1-4Xyl-PA.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Characterization of structures of X1 and X2
using a two-dimensional sugar map. The elution positions relative
to that of Gal 1-3GalNAc-PA on reversed-phase HPLC are shown on the
abscissa. Elution Condition 1 was used for the
reversed-phase HPLC. The molecular sizes of the PA sugar chains
measured by size fractionation HPLC are plotted on the
ordinate in terms of glucose units using PA
isomaltooligosaccharides. Arrows with solid lines
indicate changes in coordinate positions caused by chemical
desulfation, and other arrows indicate changes in coordinate
positions caused by digestion with the following exoglycosidases:
dashed lines, -glucuronidase; dot and dash
line; -galactosidase; dotted line,
sialidase. AGGX,
GlcA 1-3Gal 1-3Gal 1-4Xyl-PA; GX,
Gal 1-4Xyl-PA.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Component analysis of Fractions X1 and X2
Reducing end PA Xyl was measured as described under "Experimental
Procedures." Sulfate ion was measured by ion chromatography after
heating with sodium hydroxide at 250 °C for 30 min. Neu5Ac was
quantified by the method of Hara et al. (26). The value of
PA Xyl was taken as 1.00.
|
|
The above findings were confirmed by measuring the molecular weight of
X1 by mass spectrometry (Fig. 4). In the
positive-ion mass spectrum, the peak of the molecule-related ion, [M + Na]+ (m/z 831.2, calculated 831.2),
was observed. Loss of sodium sulfite with hydrogen transfer from the
cation also occurred, resulting in an intense peak at
m/z 729.2 (calculated 729.3). In the negative-ion mass spectrum also, the results verified the proposed structures of X1.
The linkage position of the sulfate group was analyzed by
1H NMR spectroscopy. Reported data on the unsaturated
linkage hexasaccharide of proteoglycans
(
GlcA
1-3GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xylol) (27), the synthetic trisaccharide, YM677
(SO4-3GlcA
1-3Gal
1-4GlcNAc) (28), and an oviducal
mucin oligosaccharide
(SO4-3GlcA
1-3Gal
1-4Gal
1-3GalNAcol) (29)
provided the necessary reference data for assignment of the proton
signals of X1 (Table II). The proton
signal at 4.605 ppm was assigned to H1 of Gal-2 and that at 4.668 ppm,
to H1 of Gal-3 as compared with those of the unsaturated linkage
hexasaccharide (R1). Analysis of the two-dimensional NMR spectra of the
SO4-GlcA residues in X1 (Fig.
5) revealed identical spin patterns to
the SO4-3GlcA residues in the the synthetic trisaccharide,
YM677 (R2) and oviducal mucin oligosaccharide (R3). Therefore, the
proton signals at 4.757 and 4.317 ppm were, respectively, assigned to H1 and H3 of SO4-3GlcA, confirming that the HNK-1
disaccharide element was present in X1. Taking these results together
with those given above, the structure of X1 was determined to be
SO4-3GlcA
1-3Gal
1-3Gal
1-4Xyl-PA.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Matrix-assisted laser desorption
ionization mass spectra of Fraction X1 and X2. a and
b, X1; c and d, X2. a and
c, positive-ion mode; b and d,
negative-ion mode.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
1H chemical shifts of structural-reporter groups of constituent
monosaccharides of X1, X2, and reference compounds
X2,
SO4-3GlcA[4] 1-3Gal[3] 1-3(Neu5Ac 2-6)Gal[2] 1-4Xyl-PA[1];
X1,
SO4-3GlcA[4] 1-3Gal[3] 1-3Gal[2] 1-4Xyl-PA[1];
R1,
GlcA 1-3GalNAc 1-4GlcA[4] 1-3Gal[3] 1-3Gal[2] 1-4Xylol
(27); R2, SO4-3GlcA[4] 1-3Gal[3] 1-4GlcNAc (28); R3,
SO4-3GlcA[4] 1-3Gal[3] 1-4Gal 1-3GalNAcol (29).
Code numbers of sugar residues are indicated in brackets as described
above. X1 was measured at 50 °C, X2 at 50 and 30 °C, R1 at
15 °C, R2 at 22 °C, and R3 at 27 °C.
|
|

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 5.
Analysis of sugar structures of Fraction X1
and X2 by total correlated spectroscopy. Parts of the total
correlated spectroscopy spectra of X1 (A) and X2
(B) are shown. Specific spin patterns observed for GlcA are
shown by dashed lines.
|
|
Structure Analysis of X2--
Partial acid hydrolysis of X2 gave
the same fluorescent fragments as found for X1, indicating that X2 also
contained the linkage tetrasaccharide structure. X2 had 1 mol each of a
sulfate and Neu5Ac (Table I). The sialidase digest of X2 was eluted at
the position of X1 on the two-dimensional sugar map (Fig. 3),
indicating that X2 was a sialylated form of X1. The elution position of
X2 on the map was also not changed by digestion with
-glucuronidase, chondro-4-sulfatase, or chondro-6-sulfatase (data not shown). When X2
was desulfated by methanolysis, the product appeared at position A
(Fig. 3). Desulfated X2 was then susceptible to
-glucuronidase digestion, and the digest was eluted at position B. These results indicated that the sulfate group was linked to the GlcA-Gal structure. On further digestion of the
-glucuronidase digest with
Aspergillus
-galactosidase, a new peak with a molecular
size 0.9 glucose units smaller appeared at position C. The sialidase
digest of the
-galactosidase digest was eluted at the position of
Gal
1-4Xyl-PA, indicating that Neu5Ac was linked to the Gal-Xyl
structure (Fig. 3). Methylation analysis of X2 indicated
3,6-disubstituted and 3-substituted Gal structures (Fig.
6). These results suggested that X2 was
SO4-GlcA
1-3Gal
1-3(Neu5Ac
2-6)Gal
1-4Xyl-PA.
The structure was confirmed by measuring the molecular weight of X2 by
mass spectrometry (Fig. 4). In the positive-ion mass spectrum, peaks of
the molecule-related ions, [M + Na]+
(m/z 1122.2, calculated 1122.3) and [M + K]+ (m/z 1138.5, calculated 1138.3),
were observed. Loss of sodium sulfite with hydrogen transfer from these
cations also occurred, resulting in an intense peak at
m/z 1020.2 (calculated 1020.4). In the
negative-ion mass spectrum also, the results verified the proposed
structure of X2. The linkage position of the sulfate group was analyzed
by 1H NMR in the same manner as for X1. Analysis of the
two-dimensional NMR spectra of the SO4-GlcA residue in X2
(Fig. 5) revealed identical spin patterns to the SO4-3GlcA
residues in X1. Therefore, the proton signal at 4.756 ppm and that at
4.320 ppm were, respectively, assigned to H1 and H3 of
SO4-3GlcA, confirming that the HNK-1 disaccharide element
was also present in X2. Three typical signals (H3e 2.688, H3a 1.634, and NAc 2.012 ppm) of the Neu5Ac residue were observed. The NMR spectra
of X2 were almost the same as those of X1 besides Neu5Ac and H4 of
Gal-2. The H4 signal of Gal-2 in X1 was shifted, indicating that Neu5Ac
was linked to C6 of Gal-2. Taking these results together with those
given above, the structure of X2 is
SO4-3GlcA
1-3Gal
1-3(Neu5Ac
2-6)Gal
1-4Xyl-PA.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 6.
Reversed-phase HPLC of partially
O-methylated PA sugars obtained by methylation
analysis of Fraction X2. Arrows indicate the elution positions of
the derivatives of PA galactose: 1, 3,4-di-O-methyl; 2, 2,3-di-O-methyl and 2,6-di-O-methyl; 3, 3,6-di-O-methyl; 4, 4,6-di-O-methyl; 5, 2,4-di-O-methyl; 6, 3,4,6-tri-O-methyl; 7, 2,3,6-tri-O-methyl; 8, 2,3,4-tri-O-methyl; 9, 2,4,6-tri-O-methyl; 10, 2,3,4,5-tetra-O-methyl.
Arrow A indicates the elution position of the derivative obtained from
the reducing-end PA xylose.
|
|
Binding Analysis of HNK-1 Antibody to X1 and
X2--
Double-reciprocal plots of the concentrations of the bound
oligosaccharide (X1) versus those of the free
oligosaccharide in equilibrium dialysis gave the binding constant of
HNK-1 antibody to X1. The apparent association constant was 2.5 × 106 M
1. The binding constant of
X2, 1 × 106 M
1, was smaller
than that of X1. These analyses showed that Neu5Ac in X2 did not
contribute to the binding to the antibody used.
 |
DISCUSSION |
Analysis of sugar chains from human uTM revealed the presence of
oligosaccharides with xylose at the reducing end. On the basis of the
results obtained with X1 and X2, the structures were determined to be
linkage tetrasaccharides of proteoglycans with SO4-3GlcA at
the nonreducing end and with partial substitution of sialic acid. These
are novel structures and to our knowledge are the first reported
instance of a proteoglycan linkage tetrasaccharide with
SO4-3GlcA and sialic acid residues solely linked to a
glycoprotein. Because X1 and X2 were liberated and their structures
determined by chemical means, the possibility of another chemically
labile substituent(s) cannot be excluded. uTM does not contain
chondroitin sulfate (2-4), although TM produced in cultured human
endothelial cells and recombinant TMs were expressed in both cases with
a high molecular weight TM containing chondroitin sulfate and a low
molecular weight TM lacking this modification (10). Platelet factor 4 binds to the glycanated form of TM but not to the chondroitin sulfate-lacking TM (12). Acceleration of the inhibition of thrombin by
antithrombin III by TM is dependent upon the presence of chondroitin sulfate linked to TM (10). Hence, the binding of chondroitin sulfate
seems to be important for several of the versatile functions of TM and
to affect its cell-surface anticoagulant potential. Chondroitin sulfate
is linked to Ser-474 in the high molecular weight TM of recombinant TM,
but Ser-474 is also modified with a small substituent in the low
molecular weight TM (9, 10). By way of explanation of the fact that TM
is expressed in two distinct forms, Gerlitz et al. (9)
postulated a model involving glycosyltransferase competition between
xylosyltransferase and N-acetylgalactosaminyltransferase for
Ser-474, whereas Lin et al. (10) suggested that occupation
of the adjacent O-linked glycosylation may be important
because of the steric hindrance for xylosyltransferase of the cell line.
If the new structures determined in the present paper link to a similar
position, the following possibilities arise. The key step in producing
the low molecular weight TM is the first GalNAc transfer reaction to
the proteoglycan linkage tetrasaccharide regulated by sulfation at 3C
of GlcA, which leads to regulation of the cell-surface anticoagulant
potential. This is compatible with the finding that GalNAc transferase
catalyzing chondroitin chain elongation cannot transfer GalNAc to
3-O-sulfated GlcA (30). This sulfation may be accomplished
either with a similar sulfotransferase, as reported for the detection
of SO4-3GlcA
1-4Xyl
-4-methylumbelliferone from the
substrate, 4-methylumbelliferyl
-Xyl (31), or with the
sulfotransferase involved in the biosynthesis of the HNK-1 carbohydrate
epitope (32, 33). These results suggest that glycosaminoglycan chain
elongation of human TM seems to be abolished at the linkage
tetrasaccharide core structure by addition of a sulfate group,
indicating that 3-O-sulfation is a stop signal leading to TM
lacking chondroitin sulfate.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. K. Lee (Osaka
University, Graduate School of Science) for operation of the Varian
750-MHz NMR spectrometer of the Venture Business Laboratory, Osaka
University and Koutarou Hoshida for operation of the mass spectrometer.
 |
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.
§
Present address: Pharmaceutical Laboratory, Mocida Pharmaceutical
Co., Ltd., Fujieda, Shizuoka 426-8640, Japan.
¶
Present address: Pharmaceutical Development Laboratory, Kirin
Brewery Co., Takasaki, Gunma 370, Japan.

To whom correspondence should be addressed.: Tel:
+81-6-6850-5380; Fax: +81-6-6850-5383.
 |
ABBREVIATIONS |
The abbreviations used are:
TM, thrombomodulin;
uTM, urinary soluble TM;
PA, pyridylaminated;
HPLC, high performance
liquid chromatography.
 |
REFERENCES |
-
Esmon, C. T.
(1995)
FASEB J.
9,
946-955[Abstract/Free Full Text]
-
Yamamoto, S.,
Mizoguchi, T.,
Tamaki, T.,
Ohkuchi, M.,
Kimura, S.,
and Aoki, N.
(1993)
J. Biochem. (Tokyo)
113,
433-440[Abstract]
-
Jackson, D. E.,
Tetaz, T. J.,
Salem, H. H.,
and Mitchell, C. A.
(1994)
Eur. J. Biochem.
221,
1079-1087[Abstract]
-
Nakano, M.,
Furutani, M.,
Hiraishi, S.,
and Ishii, H.
(1998)
Thromb. Haemostasis
79,
331-337[Medline]
[Order article via Infotrieve]
-
Takahashi, Y.,
Hosaka, Y.,
Niina, H.,
Nagasawa, K.,
Naotsuka, M.,
Sakai, K.,
and Uemura, A.
(1995)
Thromb. Haemostasis
73,
805-811[Medline]
[Order article via Infotrieve]
-
Takahashi, Y.,
Hosaka, Y.,
Imada, K.,
Adachi, T.,
Niina, H.,
Watanabe, M.,
and Mochizuki, H.
(1997)
Thromb. Haemostasis
77,
789-795[Medline]
[Order article via Infotrieve]
-
Suzuki, K.,
Kusumoto, H.,
Deyashiki, Y.,
Nishioka, J.,
Maruyama, I.,
Zushi, M.,
Kawahara, S.,
Honda, G.,
Yamamoto, S.,
and Horiguchi, S.
(1987)
EMBO J.
6,
1891-1897[Abstract]
-
Parkinson, J. F.,
Vlahos, C. J.,
Yan, S. C. B.,
and Bang, N. U.
(1992)
Biochem. J.
283,
151-157[Medline]
[Order article via Infotrieve]
-
Gerlitz, B.,
Hassell, T.,
Vlahos, C. J.,
Parkinson, J. F.,
Bang, N. U.,
and Grinnell, B. W.
(1993)
Biochem. J.
295,
131-140[Medline]
[Order article via Infotrieve]
-
Lin, J-H.,
McLean, K.,
Morser, J.,
Young, T. A.,
Wydro, R. M.,
Andrews, W. H.,
and Light, D. R.
(1994)
J. Biol. Chem.
269,
25021-25030[Abstract/Free Full Text]
-
Nawa, K.,
Sakano, K.,
Fujiwara, H.,
Sato, Y.,
Sugiyama, N.,
Teruuchi, T.,
Iwamoto, M.,
and Marumoto, Y.
(1990)
Biochem. Biophys. Res. Commun.
171,
729-737[Medline]
[Order article via Infotrieve]
-
Dudek, A. Z.,
Pennell, C. A.,
Decker, T. D.,
Young, T. A.,
Key, N. S.,
and Slungaard, A.
(1997)
J. Biol. Chem.
272,
31785-31792[Abstract/Free Full Text]
-
Gysin, J.,
Pouvelle, B.,
Tonqueze, M. L.,
Edelman, L.,
and Boffa, M-C.
(1997)
Mol. Biochem. Parasitol.
88,
267-271[CrossRef][Medline]
[Order article via Infotrieve]
-
Rogerson, S. J.,
Novakovic, S.,
Cooke, B. M.,
and Brown, G. V.
(1997)
Exp. Parasitol.
86,
8-18[CrossRef][Medline]
[Order article via Infotrieve]
-
Edano, T.,
Kumai, N.,
Mizoguchi, T.,
and Ohkuchi, M.
(1998)
Int. J. Biochem. Cell Biol.
30,
77-88[CrossRef][Medline]
[Order article via Infotrieve]
-
Makino, Y.,
Kuraya, N.,
Omichi, K.,
and Hase, S.
(1996)
Anal. Biochem.
238,
54-59[CrossRef][Medline]
[Order article via Infotrieve]
-
Hase, S.,
Ikenaka, T.,
and Matsushima, Y.
(1978)
Biochem. Biophys. Res. Commun.
85,
257-263[Medline]
[Order article via Infotrieve]
-
Kuraya, N.,
and Hase, S.
(1992)
J. Biochem.
112,
122-126[Abstract]
-
Slomiany, A.,
Kojima, K.,
Banas-Gruszka, Z.,
and Slomiany, B. L.
(1981)
Biochem. Biophys. Res. Commun.
100,
778-784[Medline]
[Order article via Infotrieve]
-
Hakomori, S.
(1964)
J. Biochem. (Tokyo)
55,
205-208[Medline]
[Order article via Infotrieve]
-
Hase, S.
(1994)
Methods Enzymol.
230,
225-237[Medline]
[Order article via Infotrieve]
-
Mega, T.,
and Hase, S.
(1991)
J. Biochem. (Tokyo)
109,
600-603[Abstract]
-
Mega, T.,
Oku, H.,
and Hase, S.
(1992)
J. Biochem. (Tokyo)
111,
396-400[Abstract]
-
Hase, S.,
Hatanaka, K.,
Ochiai, K.,
and Shimizu, H.
(1992)
Biosci. Biotechnol. Biochem.
56,
1676-1677
-
Sugahara, K.,
Masuda, M.,
Yamada, S.,
Harada, T.,
Yoshida, K.,
and Yamashita, I.
(1989)
in
Proceedings of the 10th International Symposium on Glycoconjugates, Sept. 10, 1989 (Sharon, N., Lis, H., Duskin, D., and Kahane, I., eds), p. 322, Jerusalem, Israel
-
Hara, S.,
Takemori, Y.,
Yamaguchi, M.,
Nakamura, M.,
and Ohkura, Y.
(1987)
Anal. Biochem.
164,
138-145[Medline]
[Order article via Infotrieve]
-
Sugahara, K.,
Ohi, Y.,
Harada, T.,
de Waard, P.,
and Vliegenthart, J. F. G.
(1992)
J. Biol. Chem.
267,
6027-6035[Abstract/Free Full Text]
-
Voshol, H.,
van Zuylen, C. W. E. M.,
Orberger, G.,
Vliegenthart, J. F. G.,
and Schachner, M.
(1996)
J. Biol. Chem.
271,
22957-22960[Abstract/Free Full Text]
-
Florea, D.,
Maes, E.,
and Strecker, G.
(1997)
Carbohydr. Res.
302,
179-189[CrossRef][Medline]
[Order article via Infotrieve]
-
Kitagawa, H.,
Tsutsumi, K.,
Ujikawa, M.,
Goto, F.,
Tamura, J.,
Neumann, K. W.,
Ogawa, T.,
and Sugahara, K.
(1997)
Glycobiology
7,
531-537[Abstract]
-
Takagaki, K.,
Tazawa, T.,
Munakata, H.,
Nakamura, T.,
and Endo, M.
(1997)
J. Biochem. (Tokyo)
122,
1129-1132[Abstract]
-
Bakker, H.,
Friedmann, I.,
Oka, S.,
Kawasaki, T.,
Nifant'ev, N.,
Schachner, M.,
and Mantei, N.
(1997)
J. Biol. Chem.
272,
29942-29946[Abstract/Free Full Text]
-
Ong, E.,
Yeh, J-C.,
Ding, Y.,
Hindsgaul, O.,
and Fukuda, M.
(1998)
J. Biol. Chem.
273,
5190-5195[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.