From the Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-0071, Japan
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ABSTRACT |
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Tamm-Horsfall glycoprotein (THGP) and the
oligosaccharide fraction liberated from THGP by hydrazinolysis
inhibited tetanus toxoid-induced T cell proliferation. Intact THGP
showed approximately 100-fold more inhibitory activity than the
free oligosaccharides. After fractionating the oligosaccharides by
anion-exchange column chromatography, the inhibitory activity could be
detected in a sialidase-resistant acidic oligosaccharide fraction
(fraction AR). The inhibitory activity of fraction AR was not observed
when the fraction was added to the T cell culture medium 24 h
after the addition of tetanus toxoid. Increased concentration of
interleukin (IL) 1 Most eukaryotic proteins occur as glycoproteins. The
carbohydrate moieties of glycoproteins play important roles not only in
modulation of protein properties such as stability and biological activities but also in various molecular recognition processes, including initial reaction in bacterial and viral infections, cell adhesion in inflammation and metastasis, differentiation, development, regulation, and many other intercellular communication events (1-3). Understanding the molecular mechanisms of carbohydrate recognition is therefore important for the biology of multicellular organisms. Carbohydrate-binding proteins (lectins) are widely distributed in eukaryotic cells and mediate in many specific biological functions including intercellular recognition, protein trafficking, and
primitive defense reactions (4). We will describe here the novel
lectin-like property of a cytokine.
Tamm-Horsfall glycoprotein
(THGP)1 is a glycoprotein
produced by kidney and contains approximately 30% carbohydrate.
Serafini-Cessi's group (5-7) reported that THGP works as a potential
suppressive agent of both the lymphocyte proliferation induced by
phytohemagglutinin-L4 treatment and the mixed lymphocyte
reaction. They also suggested that the inhibitory activity resides in
the sugar moiety of the glycoprotein (6, 7). Muchmore and co-workers
(8-10) found an immunosuppressive glycoprotein in the urine of
pregnant women, and named it uromodulin. It inhibited T cell
proliferation induced by tetanus toxoid as well as T lymphocyte
proliferation induced by interleukin 1 (IL-1). They also suggested that
the carbohydrate portion of uromodulin played a fundamental role in its
inhibitory activity (11). That uromodulin is identical to THGP was
confirmed by amino acid sequencing (12). Muchmore and Decker (13)
proposed that uromodulin inhibits T cell proliferation via binding to
IL-1 We found that the oligosaccharide fraction, obtained by hydrazinolysis
of THGP followed by N-acetylation, inhibits the T cell proliferation induced by tetanus toxoid. This paper reports the partial
structural characterization of the oligosaccharides with this
inhibitory activity.
Reagents--
NaB3H4 (360 mCi/mmol) and
[3H]thymidine were purchased from NEN Life Science
Products. Cell Lines--
IL-2-dependent mouse T cell line,
CTLL cells, was obtained from Dr. Takiguchi (Institute of Medical
Science, University of Tokyo) and cultured in RPMI 1640 medium (ICN
Biomedicals Ltd.) containing 10% fetal calf serum (FCS) (Cell Culture
Laboratories, Cleveland, OH) and 2 units/ml human rIL-2 (kindly
provided by Shionogi Co., Osaka, Japan) in 5% CO2 at
37 °C. IL-1 Analytical Methods--
Anion-exchange column chromatography was
performed using a fast protein liquid chromatography apparatus
(Pharmacia Biotech) equipped with a Mono-Q HR5/5 column. Elution was
programmed as follows: 5 mM sodium acetate, pH 4.0, for 10 min and then 500 mM sodium acetate, pH 4.0, for 20 min, at
a flow rate of 1 ml/min at room temperature. Bio-Gel P-2 column
chromatography using distilled water was performed to remove the salt
or the monosaccharide from oligosaccharide fraction. Descending paper
chromatography was performed using a solvent
(1-butanol:ethanol:water = 4:1:1, v/v). Affinity chromatography on
W. floribunda agglutinin-agarose was performed as follows.
The sample dissolved in 100 µl of 10 mM phosphate-buffered saline (PBS), pH 7.4, was applied to a W. floribunda agglutinin-agarose column (2 ml) and eluted with 15 ml
of the same buffer. Oligosaccharides bound to the column were eluted with 8 ml of the buffer containing 100 mM
N-acetylgalactosamine. After elution, the oligosaccharide
fraction was freed from N-acetylgalactosamine by passing
through a Bio-Gel P-2 column. Mild methanolysis was performed according
to the procedure described previously (19).
Glycosidase Digestion--
Oligosaccharides were incubated with
the following enzyme solutions: (i) A. ureafaciens sialidase
(200 milliunits) in 50 µl of 0.1 M acetate buffer, pH
5.0; (ii) jack bean Preparation of THGP and Release of Its N-Linked Sugar Chains as
Oligosaccharides--
THGP was isolated from urine of a healthy adult
male according to the procedure described previously (20). The THGP,
thus obtained, gave a single band under both reducing and nonreducing conditions stained with Coomassie Brilliant Blue after being subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (data not shown). For the inhibitory assay, purified THGP solution was
filtered to free from bacteria. Thoroughly dried THGP (50 mg), thus
obtained, was subjected to 10 h of hydrazinolysis followed by
N-acetylation as described previously (21). One-twentieth of
the oligosaccharide fraction thus obtained was reduced with NaB3H4 (400 µCi) in 100 µl of 0.05 N NaOH at 30 °C for 4 h, and the resulting
radioactive oligosaccharide was purified as described previously (21).
Detection of Sulfate Ion Using Anion Chromatography--
One
nmol of oligosaccharides in fraction AR was heated in 6 N
HCl at 110 °C for 24 h followed by evaporation with methanol to
remove HCl. The residue was dissolved in 100 µl of water, and the
two-fifths of the solution was applied to HPLC TSKgel
IC-Anion-PWXL (4.6 mm inner diameter × 3.5 cm, Tosoh
Corp., Tokyo) to detect sulfate ion. Elution was performed with 1.5 mM potassium gluconate, 1.5 mM sodium borate,
5.8 mM boric acid, 4% acetonitrile, 3% 1-butanol, and
0.5% glycerol. The column temperature was maintained at 35 °C at a
flow rate of 1 ml/min.
Tetanus Toxoid-induced T Cell Proliferation--
Tetanus
toxoid-induced proliferation of normal human peripheral blood
mononuclear cells was performed by using slight modifications of the
previously described methods (22). Briefly, 10 ml of normal heparinized
human blood was diluted with an equal volume of RPMI 1640 medium, and
placed above 20 ml of Ficoll-Hypaque solution, and centrifuged at
800 × g for 15 min at room temperature. Monocyte-enriched population located between upper and lower layers was
carefully collected. The cells were washed twice with RPMI 1640 medium
containing 10% autologous plasma by alternate suspension and
centrifugation. The viable cells (2 × 105) in 10%
autologous plasma were incubated in 96-well microtiter plates with
tetanus toxoid and various amounts of each oligosaccharide in a final
volume of 200 µl. After incubation for 6 days, the cultures were
pulsed with 0.5 µCi of [3H]thymidine for 6 h, and
the amount of [3H]thymidine incorporated into the cells
was determined.
Measurement of the Amount of Cytokine in the Culture Medium of T
Cells Proliferated by Tetanus Toxoid--
The amount of IL-1 Growth Inhibition of IL-1 Binding of IL-1 Dot Blot Analysis--
One or 10 µg of each glycoprotein (THGP
and bovine TSH) was spotted onto a nitrocellulose filter and air-dried
for several hours. The filter was washed three times with PBS, soaked
in 5% BSA/PBS, and incubated at room temperature for 4 h. After
being washed with PBS, the filter was soaked in IL-1 Binding of IL-1 N-Linked Sugar Chains Obtained from THGP Inhibited Tetanus
Toxoid-induced T Cell Proliferation--
The oligosaccharide fractions
obtained from THGP by hydrazinolysis followed by
N-acetylation were added to the culture medium of tetanus
toxoid-specific T cell proliferation. The effect of these
oligosaccharides on the proliferation reaction is shown in Fig.
1. The concentration required for 50%
inhibition of tetanus toxoid-induced proliferation was around 10 µM. At this concentration, neither the oligosaccharides
liberated from
The oligosaccharide mixture was subjected to anion-exchange column
chromatography with a Mono-Q HR5/5 column. As shown in Fig.
2A, oligosaccharides were
separated into a neutral fraction (N) that eluted with 5 mM
sodium acetate, pH 4.0, and an acidic fraction (A) that eluted with 500 mM sodium acetate, pH 4.0. When fraction A was exhaustively
incubated with A. ureafaciens sialidase, 67% of it was
converted to neutral components (named fraction AN), and the remainder
(named fraction AR) was resistant to this enzymatic treatment (Fig.
2B). Approximately 80% of oligosaccharides in fraction AR
were converted to neutral components (named fraction ARN) by mild
methanolysis (Fig. 2C). These results suggest that the
acidic nature of the majority of the oligosaccharides in fraction AR
may be due to the presence of sulfate residues as it was previously reported that sulfate residues are removed from sugar moieties under
these conditions (19). In order to confirm the presence of sulfate
residues in fraction AR, this fraction was treated with 6 N
HCl at 110 °C for 24 h followed by anion chromatography using a
TSK gel IC-Anion-PWXL column. The sulfate ion in the above reaction mixture was detected as shown in Fig.
3. This result indicates that the acidic
nature of sialidase-resistant oligosaccharides is due to the sulfate
residue. Based on the analytical data, about 3 nmol of sulfate ion was
released from 1 nmol of oligosaccharide mixture in fraction AR under
these experimental conditions. Remaining acidic oligosaccharides in
fraction ARR in Fig. 2C were converted to the neutral
components by heating in 0.1 N CF3COOH at
80 °C for 2 h. However, the acidic nature of oligosaccharides
in fraction ARR could not be determined due to the limited amounts of
sample. Based on the radioactivities, the molar ratio of
oligosaccharides in each fraction was calculated as follows: fraction N
(10%), fraction A (90%), fraction AN (60%), fraction AR (30%),
fraction ARN (25%), and fraction ARR (5%).
On the other hand, when fraction A was directly subjected to mild
methanolysis in order to determine the ratio of oligosaccharides carrying only the sulfate residue, a very small part of the fraction was converted to a neutral oligosaccharide mixture (named fraction AMN), and most of the oligosaccharides remained acidic as indicated by
the fraction AMR peak in Fig. 2D. The molar ratio of the
oligosaccharides in the two fractions was calculated on the basis of
their radioactivities, fraction AMN (3%) and fraction AMR (87%).
These results indicate that 60% of the THGP oligosaccharides contain
only sialic acid residues, 3% contain only sulfate residues, and 27%
contain both sialic acid and sulfate residues as their acidic components.
Inhibition Activities of the Oligosaccharide Fractions--
The
inhibitory activity of each oligosaccharide fraction thus obtained on
the T cell proliferation was examined, and the results are summarized
in Fig. 4. Oligosaccharides in fraction A
and fraction AR showed strong inhibitory activity. On the other hand,
oligosaccharides in fraction ARN, which was converted to a neutral
fraction by mild methanolysis treatment as described above, showed no
inhibitory activity. These results indicate that the sulfate residue of
oligosaccharides in fraction AR was essential for the expression of
inhibitory activities. This interpretation is supported by the fact
that fractions ARR, AMN, and AMR showed no inhibitory activity (Fig. 4). It is noteworthy that fraction AR showed only slightly more potent
inhibitory activity than fraction A, whereas fraction A was separated
into bioactive fraction AR and inactive fraction AN after sialidase
treatment as described above. The reason for this has not yet been
determined, although a possible explanation is that the presence of the
sialic acid residues, which is not essential for
proliferation-inhibitory activity, on the sugar chains in fraction A
may enhance the inhibitory activity of the sulfated oligosaccharides.
As compared with other inactive fractions, the neutral fraction N,
which was reported to contain only high mannose-type oligosaccharides
(24, 25), had a slight inhibitory effect on the T cell proliferation at
a concentration of ~100 µM.
Inhibitory Mechanism of Oligosaccharides on T Cell
Proliferation--
To clarify the mechanisms of inhibitory activity of
oligosaccharides in fraction AR, we changed the time of oligosaccharide addition. When added 24 h or more after the addition of tetanus toxoid, oligosaccharides in the fraction AR showed no inhibitory activity at all (Fig. 5). This result
suggests that sulfated oligosaccharides in fraction AR acted at an
early stage of antigen-specific T cell proliferation.
Therefore, we quantified the amount of IL-1
In order to find out whether the oligosaccharides in fraction AR can
directly act on the IL-1 Binding of IL-1
In order to elucidate the structure of the IL-1 Binding of Glycoproteins and Glycosphingolipids with
IL-1 Muchmore's group (25, 31) reported that the glycopeptides
containing high mannose type sugar chains derived from THGP inhibited
antigen-specific T cell proliferation by 50% in the concentration
range 0.2-2 µM. Dall'Olio et al. (7)
reported that glycopeptides containing the complex type or high mannose type sugar chains obtained by Pronase digestion of THGP inhibited the
lymphocyte proliferation induced by mixed lymphocyte reaction. Conflicting evidence is that the glycopeptides obtained from ovalbumin by Pronase digestion, which should contain a series of high mannose type sugar chains together with a series of hybrid type sugar chains
(2), did not show any inhibitory activity (7). Our data demonstrate,
however, that oligosaccharides in fraction N, which contains high
mannose type sugar chains (24, 25), showed very little inhibitory
activity even at the concentration of 100 µM (Fig. 4). In
contrast, oligosaccharides in fraction AR inhibited T cell
proliferation by 50% at the concentration of 2 µM.
We propose in this report that the immunosuppressive properties of THGP
are expressed by the interaction of its carbohydrate moieties with
IL-1 THGP has been found to contain more than 150 N-linked sugar
chains, and the structures of only 30 oligosaccharides have been determined (35, 36). Among them, two types of terminal sulfated elements were found, 4-O-sulfated GalNAc and
3-O-sulfated Gal (36). None of these are considered to be
the actual ligand of IL-1 and decreased concentration of IL-2 were observed
in the T cell culture medium after the addition of fraction AR. The
oligosaccharides in fraction AR also inhibited the growth of an
IL-1-dependent cell line, D10-G4. These results strongly
suggested that the oligosaccharides in fraction AR bind to IL-1
and
suppress its cytokine activity. IL-1
actually bound to the fraction
AR immobilized on an amino-bonded thin layer plate. Fractionation of
the oligosaccharides indicated that only oligosaccharides containing an
N-acetylgalactosamine residue and a sulfate residue bound
specifically to IL-1
. Removal of either the sulfate residue or the
N-acetylgalactosamine residue from the oligosaccharides
abolished both the proliferation-inhibition and IL-1
binding
activities. Since IL-1
did not bind to thyroid-stimulating hormone,
which has the sulfate group at C-4 of the
N-acetylgalactosamine residue in its N-linked
sugar chains, the binding of IL-1
toward oligosaccharides in
fraction AR was considered to be highly specific.
INTRODUCTION
Top
Abstract
Introduction
References
through its carbohydrate moieties and inactivates a mediator. However, Moonen et al. (14) rejected this explanation,
because uromodulin interacted only with the denatured IL-1
adsorbed
to the plastic plate but not with the native soluble IL-1
.
Therefore, the precise immunosuppressive mechanism of uromodulin has
not yet been clarified.
MATERIALS AND METHODS
-N-Acetylhexosaminidase was purified from jack
bean meal by the methods of Li and Li (15). Sialidase from
Arthrobacter ureafaciens and 4-chloro-1-naphthol were
purchased from Nacalai Tesque, Kyoto. Concanavalin A (Con A) and
Ficoll-Hypaque were purchased from Pharmacia Biotech (Uppsala, Sweden).
Human recombinant interleukin 1
(rIL-1
) and recombinant
interleukin 2 (rIL-2) were purchased from Genzyme (Cambridge, MA).
Anti-human IL-1
was from Collaborative Research Inc. (Bedford, MA).
Biotinylated anti-rabbit immunoglobulins (G+M) antibody and
Wisteria floribunda agglutinin-agarose were from E-Y
Laboratories Inc. (San Mateo, CA). 125I-Labeled anti-rabbit
immunoglobulins (G+M) was from Amersham Corp. (Tokyo, Japan).
Avidin-biotin-peroxidase reagent (ABC reagent, Vectastain ABC Kit) was
purchased from Vector Laboratories, Inc. (Burlingame, CA). Tetanus
toxoid (400 limit of flocculation/ml) was kindly donated by
Kagaku-Kessei Therapeutic Research Laboratories (Kumamoto, Japan).
1-Acid glycoprotein (
1AGP), bovine
ribonuclease B (RNase B), fetuin, and porcine thyroglobulin were
purchased from Sigma. Bovine thyroid-stimulating hormone (TSH) was
purchased from UCB-Bioproducts S.A. (Belgium). GM2 and sulfatide were
purchased from DIA-IATRON (Tokyo). SM2 from rat kidney (16) was kindly donated by Professor Ineo Ishizuka, Teikyo University School of Medicine. An amino-bonded high performance silica gel plate (HPTLC Fertigplatten NH2F254) was obtained from Merck
(Darmstadt, Germany).
-dependent T cell clone, D10-G4 (17, 18),
was obtained from Dr. Kakiuchi (Institute of Medical Science,
University of Tokyo) and cultured in RPMI 1640 medium containing 10%
FCS and 5% Con A supernatant, which was derived from the culture
medium of rat splenic cells treated with Con A.
-N-acetylhexosaminidase (4 units) in
50 µl of 0.3 M citrate phosphate buffer, pH 5.0. One drop
of toluene was added to the reaction mixtures to inhibit bacterial
growth during incubation. After being incubated for 48 h at
37 °C, enzymes were inactivated by heating the reaction mixture in a
boiling water bath for 3 min.
1AGP, RNase B, porcine thyroglobulin, and fetuin were
also subjected to hydrazinolysis followed by N-acetylation according to the procedures described above.
in the
culture medium was measured at day 3 using an enzyme-linked
immunosorbent assay kit for IL-1
(Otsuka Bioassay Research,
Tokushima, Japan). The amount of IL-2 in the culture medium was
measured at day 4 by counting the [3H]thymidine
incorporated into CTLL cells, whose growth is dependent on IL-2.
Briefly, 5 × 103 cells were incubated with 100 µl
of the culture medium of RPMI 1640 containing 10% FCS at 37 °C for
18 h. The cultures were then pulsed with 0.5 µCi of
[3H]thymidine overnight, and the amount of
[3H]thymidine incorporated was determined as described
above. For the quantitation, known amounts of rIL-2 were added to
aliquots of the above assay mixture as standards.
Responsive T Cell Clone, D10-G4 by
Oligosaccharides--
In each well of a 96-well plate, 2 × 105 of D10-G4 cells/50 µl of RPMI 1640 containing 10%
FCS were incubated with 50 µl of 8 µg/ml Con A (final concentration
of 2 µg/ml) and 50 µl of 4 units/ml IL-1
(final concentration of
1 unit/ml). To this mixture, various concentrations of oligosaccharides
were added and incubated at 37 °C for another 2 days. The cultures
were then pulsed with 0.5 µCi of [3H]thymidine
overnight, and the amount of [3H]thymidine incorporated
was determined as described above.
to Oligosaccharides Immobilized on an
NH2 HPTLC Plate--
Binding assays of IL-1
to
oligosaccharides were performed using a slight modification of the
previously described methods (23). An amino-bonded high performance
silica gel plate (HPTLC Fertigplatten NH2F254)
was soaked in NaBH3CN solution and air-dried. Each
oligosaccharide (1 nmol) was spotted onto the plate and left at room
temperature (20-25 °C) for an appropriate length of time. The plate
was washed three times with distilled water for 1 min and then soaked
in saturated sodium bicarbonate solution containing 5% acetic
anhydride to block the remaining active amino groups on the plate by
N-acetylation. The plate was then overlaid with 5% bovine
serum albumin (BSA) in 10 mM Tris-HCl, pH 7.8, containing 0.15 M NaCl and incubated at room temperature for 4 h.
After being washed with PBS, the plate was overlaid with IL-1
solution in 1% BSA/PBS and incubated at 4 °C for 16 h. After
being washed five times with PBS, the plate was overlaid with rabbit
anti-human IL-1
antibodies and incubated at 4 °C for a further
2 h. The plate was then washed with PBS, overlaid with
125I-labeled goat anti-rabbit immunoglobulin (G+M)
antibodies, and incubated at room temperature for 2 h. The plate
was then washed with PBS, dried, and exposed to XAR-5 x-ray film
(Eastman Kodak Co.) in the dark at
80 °C for 2-5 days.
solution in 1% BSA/PBS and incubated at 4 °C for 16 h. After being washed
three times with PBS, the filter was soaked in the solution containing rabbit anti-human IL-1
antibodies and incubated at 4 °C for
2 h. After a further wash with PBS, the filter was soaked in a
solution containing biotinylated goat anti-rabbit immunoglobulins (G+M) antibodies and incubated at room temperature for 2 h. The filter was then washed with PBS, incubated with ABC reagent at room
temperature for 30 min, and washed with PBS again. The filter was
incubated with a substrate solution (10 mM Tris-HCl buffer,
pH 7.2, 0.3% 4-chloro-1-naphthol in methanol, 30%
H2O2 aqueous (5:1:0.01, v/v)) at room
temperature for an appropriate length of time.
of Glycosphingolipids on an HPTLC
Plate--
SM2, GM2, and sulfatide (1 nmol each) were spotted onto a
silica polygram Sil G plate (Nagel, Germany). In order to prevent nonspecific binding, the plate was treated with 1%
polyvinylpyrrolidone for 1 min and then soaked in PBS containing 1%
BSA for 2 h at room temperature. The plate was incubated with
IL-1
solution (0.1 µg/ml) at 4 °C for 18 h with gentle
shaking. After washing three times with PBS, the plate was then
incubated with rabbit anti-human IL-1
antibody solution at 4 °C
for 2 h. After washing three times with PBS, the plate was
incubated with biotinylated goat anti-rabbit Ig(G+M) antibody at room
temperature for 1 h. Bound biotinylated antibody was detected as
described above.
RESULTS
1AGP nor those from RNase B inhibited
tetanus toxoid-induced T cell proliferation (data not shown). In order
to characterize the oligosaccharide showing the inhibitory activity,
the oligosaccharide mixture obtained from THGP was fractionated. To
facilitate detection of the oligosaccharides in further fractionation
procedures, a small amount of tritium-labeled oligosaccharide mixture
from THGP was added.
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Fig. 1.
Effect of oligosaccharide fraction, obtained
from THGP by hydrazinolysis, on tetanus toxoid-induced T cell
proliferation. Values, expressed as counts/min of
[3H]thymidine incorporated, are means of three separate
experiments. The bars indicate the standard
deviations.
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Fig. 2.
Fractionation of oligosaccharides from THGP
by an anion-exchange column. Anion-exchange column chromatography
was carried out using a fast protein liquid chromatography apparatus as
described under "Materials and Methods." In order to facilitate
detection of the oligosaccharides, a trace tritium-labeled
oligosaccharide mixture (5 × 106 cpm) from THGP was
added. A, oligosaccharides liberated from THGP by
hydrazinolysis; B, fraction A in A digested
exhaustively with A. ureafaciens sialidase; C,
fraction AR in B subjected to mild methanolysis treatment;
D, fraction A in A subjected to mild methanolysis
treatment. The arrows indicate the positions where the
elution buffer was switched to 500 mM sodium acetate.
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Fig. 3.
Detection of sulfate anion by ion
chromatography. Detailed procedures are described under
"Materials and Methods." The triangles indicate the
elution positions of standard anions. a, chloride anion (3.1 min); b, phosphate anion (6.8 min); c, sulfate
anion (8.7 min). Peaks other than sulfate anion were also detected in
fraction AR not subjected to acid hydrolysis.
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Fig. 4.
Inhibition of tetanus toxoid-induced T cell
proliferation by various oligosaccharide fractions. The name of
each oligosaccharide fraction corresponds to that in Fig. 2. Values
expressed as counts/min of [3H]thymidine incorporation
are the means of three separate experiments, and the bars
indicate the standard deviations.
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Fig. 5.
Time dependence of the inhibitory effect of
fraction AR. Fraction AR was added to the tetanus toxoid-induced
proliferation assay 0, 24, or 48 h after the incubation with
tetanus toxoid was initiated. DNA synthesis was determined at day 6 by
measuring the amount of [3H]thymidine incorporated.
Values are means of three separate experiments, and the bars
indicate the standard deviations.
and IL-2 molecules in
the culture medium of tetanus toxoid-activated T cells under the effect
of various amounts of fraction AR, since these soluble mediators are
known to play important roles at the early stage of antigen-specific T
cell proliferation. As shown in Fig. 6,
the amount of IL-1
in the T cell culture medium increased in
parallel with the amount of fraction AR added. In contrast, the amount
of IL-2 decreased on increasing the concentration of oligosaccharides.
A possible explanation for these interesting results is that fraction
AR inhibited the binding of IL-1
to its receptor on the surface of T
cells. As a result, the decline of signal transduction induced by
IL-1
in T cells would reduce IL-2 production. This inhibition can be
induced by binding of the oligosaccharides to either IL-1
molecules
or IL-1 receptors.
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Fig. 6.
Determination of the amounts of
IL-1 and IL-2 in the T cell culture medium,
which were activated by tetanus toxoid.
, amounts of IL-1
in
the culture medium were determined using an enzyme-linked immunosorbent
assay kit.
, amounts of IL-2 in the culture medium were measured
using CTLL cells. The detailed procedures are described under
"Materials and Methods." Inhibition of tetanus toxoid-induced T
cell proliferation by fraction AR was performed as described in Fig. 4.
Values expressed as counts/min of [3H]thymidine
incorporation are the means of three separate experiments, and the
bars indicate the standard deviations.
molecule or not, we examined the effect of
fraction AR on the culture of an IL-1-responsive D10-G4 cell line (17,
18). As shown in Fig. 7, both total oligosaccharides liberated from THGP (W in the figure) and
fraction AR inhibited the proliferation of D10-G4 cells. However,
oligosaccharides derived from other glycoproteins,
1AGP
and RNase B, did not show any effect at all (Fig. 7). These results
suggest that the inhibition of antigen-specific T cell proliferation
might be due to interaction of the unique oligosaccharides of THGP with
IL-1
.
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Fig. 7.
Effects of additions of oligosaccharide
fractions on the proliferation of D10-G4 cells. D10-G4 cells
(2 × 104) were incubated with 2 µg/ml Con A and 1 unit/ml human rIL-1 at 37 °C. Each oligosaccharide fraction
obtained from THGP (W, total oligosaccharides mixture;
AR, oligosaccharides mixture in fraction AR), from
1AGP, or from RNase B by hydrazinolysis followed by
N-acetylation was added at the beginning of the incubation.
Incubation was continued at 37 °C for another 48 h, and then
[3H]thymidine incorporation was determined. Values are
the means of the data obtained from three separate experiments. The
bars indicate the standard deviations.
to Immobilized Oligosaccharides on an
NH2 HPTLC Plate--
We then examined whether IL-1
binds directly to the oligosaccharides of THGP by a thin layer overlay
method as described previously (23). As shown in Fig.
8A, IL-1
bound to
oligosaccharides in fractions A and AR but not to those in fractions
AN, ARN, ARR, AMR, and AMN. These results are consistent with the data
that only fractions AR and A could inhibit T cell proliferation (see Fig. 4). IL-1
also did not bind to the oligosaccharide fractions from thyroglobulin and
1AGP, which did not inhibit the T
cell proliferation (Fig. 8A). The radioactivities retained
by immobilized oligosaccharides in Fig. 8A were quantified
densitometrically, and the data are shown in Fig. 8B. The
data clearly indicated that IL-1
bound only to the oligosaccharides
in fractions A and AR derived from THGP. It must be stressed here that
IL-1
very weakly bound to the porcine thyroglobulin
oligosaccharides, which included sulfated biantennary sugar chains
(26). The results indicate that oligosaccharides with not only sulfate
residues but also additional structural features are recognized by the IL-1
molecule.
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Fig. 8.
Binding of IL-1 to
various oligosaccharide fractions immobilized on an NH2
HPTLC plate. One nanomole each of the oligosaccharides was fixed
on the plate. A, fractions A, AN, AR, ARN, ARR,
AMR and AMN are the same as shown in Fig. 2;
thyro, the oligosaccharide fraction obtained from porcine
thyroglobulin;
1AGP, that from
1-acidic
glycoprotein. B, quantification of the binding of IL-1
to
oligosaccharide fractions immobilized on an NH2 HPTLC
plate. The amounts of IL-1
bound to the oligosaccharide fractions on
the plate were determined by densitometry. Detailed procedures are
described under "Materials and Methods."
ligand, fraction AR
was applied to a W. floribunda agglutinin-agarose column, which is known to recognize a peripheral
-linked
N-acetylgalactosamine residue (27), and separated into an
unbound fraction (fraction I in Fig.
9A) and a bound fraction
subsequently eluted with 100 mM
N-acetylgalactosamine (fraction II in Fig.
9A). The percent molar ratio of these two fractions was 53 and 47%, respectively. After removing salt and
N-acetylgalactosamine by passing through a Bio-Gel P-2
column, both the inhibitory activity on T cell proliferation and the
binding ability to IL-1
were examined for the two fractions. As
shown in Fig. 10, A and
B, both activities resided exclusively in fraction II, and
fraction II showed more potent inhibitory activity than fraction AR.
Furthermore, removal of terminal N-acetylgalactosamine residues from fraction II by jack bean
-N-acetylhexosaminidase digestion (fraction III in Fig.
9B) abolished the binding ability to IL-1
completely
(Fig. 9C). These data indicate that at least two elements, a
sulfate residue and a
-N-acetylgalactosamine residue at
the nonreducing termini of the oligosaccharides, are necessary for
their binding to IL-1
.
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Fig. 9.
Chromatography of oligosaccharides in
fraction AR on a W. floribunda agglutinin-agarose
column. A, fraction AR was subjected to a W. floribunda agglutinin-agarose column as described under
"Materials and Methods." Arrows indicate the positions
where the elution buffer was switched to that containing 100 mM N-acetylgalactosamine; B,
rechromatography of oligosaccharides in fraction II in A
after digestion with jack bean -N-acetylhexosaminidase;
C, binding of IL-1
to the oligosaccharides separated by
the W. floribunda agglutinin column chromatography.
Fractions I and II in A, fraction III in B, and
fraction AR were immobilized on an NH2 HPTLC plate. One
nanomole of each oligosaccharide was spotted onto the plate. Detailed
procedures for staining are described under "Materials and
Methods."
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Fig. 10.
Coexistence of the inhibitory activity and
IL-1 binding activity in fraction AR and
fraction II obtained by W. floribunda agglutinin
column chromatography. A, effect of addition of the
oligosaccharides fractionated by the W. floribunda
agglutinin-agarose column on tetanus toxoid-induced lymphocyte
proliferation. Fractions I and II are the same as in Fig.
9A. B, binding of IL-1
to each oligosaccharide
fraction in A. The amount of IL-1
bound to each
oligosaccharide fraction was determined by densitometry of the
NH2 HPTLC plate as described in Fig. 8. Detailed procedures
are described under "Materials and Methods."
--
In an attempt to clarify the relationship between the
sulfate and N-acetylgalactosamine residues, we examined the
binding of IL-1
to TSH because the SO4-4GalNAc group was
present in the sugar chains of TSH (28). When we studied the
interaction of THGP and TSH with IL-1
, only THGP could be stained
with IL-1
under the experimental conditions used (Fig.
11A). This suggests that
IL-1
could not bind the SO4-4GalNAc group. Next, we
examined the reactivity of IL-1
with various glycolipids containing
sulfate and
-N-acetylgalactosamine residues. As shown in
Fig. 11B, IL-1
did not bind to sulfatide at all. This is
consistent with the results that the oligosaccharides of thyroglobulin
did not react with IL-1
as shown in Fig. 8, because sulfatide
contains only the SO4-3Gal group similar to thyroglobulin
(26, 29). On the other hand, it is quite interesting that IL-1
can
bind weakly to SM2 but not to GM2 at all, although SM2 and GM2 have
similar peripheral structures: the GalNAc
1
4(R-3)Gal
1
, in
which R is SO4 for SM2 and is sialic acid for GM2 (30).
Therefore, the GalNAc
1
4(SO4-3)Gal
1
group could
be at least a part of the ligand for IL-1
molecule.
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Fig. 11.
Binding of IL-1 to
glycoproteins (A) and glycosphingolipids
(B). One nanomole of each glycosphingolipid was
spotted onto the plate. Detailed procedures are described under
"Materials and Methods."
DISCUSSION
. Our results demonstrate that oligosaccharides in fraction AR
inhibited the proliferation of D10-G4 cells, which shows
IL-1-dependent growth (17, 18), and that IL-1
interacted specifically with the oligosaccharides in fraction AR immobilized on
the NH2 HPTLC plate. By further fractionation of the
oligosaccharides in fraction AR, it was found that the oligosaccharides
containing
-N-acetylgalactosamine and sulfate residues
specifically bind to IL-1
. Two conflicting data were reported
relating to our findings. Muchmore and Decker (13) reported that the
N-linked sugar chains of uromodulin interacted with IL-1,
although Moonen et al. (14) reported that THGP did not
interact with soluble native cytokines and could only bind to denatured
cytokines at low pH. Additionally, Fukushima et al. (32)
demonstrated that IL-1
interacted with the
glycosylphosphatidylinositol anchor. We recently found that IL-1
molecules radiolabeled with 125I by Bolton-Hunter reagent,
which reacted with N-terminal amino acid and lysine residues of the
peptide portion (33), could no longer bind to THGP
oligosaccharides.2 This
result suggests that N-terminal amino acid and/or lysine residues of
IL-1
may play a role as the carbohydrate-binding site. In contrast,
it was reported that even after the 125I-labeling IL-1
molecules can bind to their receptors on the cell surface (34).
Therefore, care must be taken when using an 125I-labeled
IL-1
as a probe to investigate the lectin-like activity of IL-1
.
The possibility that the inhibitory oligosaccharides bind to tetanus
toxoid and consequently inhibit the T cell proliferation will be ruled
out because toxoid-independent T cell proliferation induced by
combination between phytohemagglutinin-L4 and IL-1
was
also inhibited by oligosaccharides in fraction AR (data not shown).
from our current study. These results
indicate that not only the presence of a sulfate residue and a
-N-acetylgalactosamine residue but also a specific
linkage(s) between the two groups is required for the ligand of
IL-1
. Based on the reactivity with glycosphingolipids, the
GalNAc
1
4(SO4
-3)Gal
1
group
could be considered as a part of an epitope for binding to the to
IL-1
molecule. Therefore, structural determination of the remaining
oligosaccharides of THGP, especially sulfated and
-N-acetylgalactosamine-containing N-linked
sugar chains, will be required in order to elucidate the inhibitory
mechanism of the IL-1
molecule and the physiological roles of the
lectin-like property.
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ACKNOWLEDGEMENTS |
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We thank Professor Jiro Tatsuno, National Defense Medical College, for encouraging this work and Professor Ineo Ishizuka, Teikyo University School of Medicine, for the kind gift of SM2.
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FOOTNOTES |
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* This work was supported by a grant from The Naito Foundation and Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.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: Dept. of Physiology, National Defense Medical
College, 3-2 Namiki, Tokorozawa-shi, Saitama 359-8513, Japan.
§ Present address and to whom correspondence should be addressed: Dept. of Glycobiology, Tokyo Metropolitan Institute of Gerontology, Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan. Tel.: 81-3-3964-3241 (ext. 3080); Fax: 81-3-3579-4776; E-mail: endo{at}tmig.or.jp.
¶ Present address: Director's Office, Tokyo Metropolitan Institute of Gerontology, Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan.
The abbreviations used are:
THGP, Tamm-Horsfall
glycoprotein; IL-1, interleukin 1; IL-1, interleukin 1
; IL-2, interleukin 2; rIL, recombinant interleukin; BSA, bovine serum albumin; Con A, concanavalin A;
1AGP,
1-acid
glycoprotein; FCS, fetal calf serum; TSH, thyroid-stimulating hormone; PBS, phosphate-buffered saline; HPTLC, high performance thin layer chromatography.
2 M. Tandai-Hiruma, unpublished results.
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REFERENCES |
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