Lectin-like Characteristics of Recombinant Human Interleukin-1
Recognizing Glycans of the Glycosylphosphatidylinositol Anchor*
(Received for publication, January 14, 1997, and in revised form, February 10, 1997)
Keiko
Fukushima
,
Sayuri
Hara-Kuge
,
Takashi
Ohkura
,
Akira
Seko
,
Hiroko
Ideo
,
Toshiyuki
Inazu
and
Katsuko
Yamashita
§
From the Department of Biochemistry, Sasaki Institute,
Kanda-Surugadai, Chiyoda-ku, Tokyo 101 and the
Noguchi
Institute, Kaga, Itabashi-ku, Tokyo 173, Japan
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES
ABSTRACT
We found that 35S-labeled
recombinant human interleukin-1
(rhIL-1
) binds
phosphatidylinositol-specific phospholipase C-treated human placental
alkaline phosphatase, phosphatidylinositol-specific phospholipase
C-treated trypanosome surface variant glycoproteins, and urinary
uromodulin immobilized on plates or immobilized on CNBr-activated
Sepharose 4B. The interaction between rhIL-1
and these glycoproteins
was lectin-like, since it was inhibited in the presence of specific
saccharides, i.e. mannose 6-phosphate or synthetic
Ac-NH·CH2·CH2·PO4
6Man
1
(±2Man
1
±6Man
1
)propyl
at about 1 µM. On the other hand, a wide variety of
compounds including biantennary sugar chains derived from these
glycoproteins as well as ethanolamine phosphate, inositol phosphate,
mannose 6-sulfate, mannose 1-phosphate, glucose 6-phosphate, and
mannitol 6-phosphate did not show any inhibitory effect at
concentrations up to 1 mM. These results indicate that rhIL-1
interacts with these glycoproteins via the mannose
6-phosphate diester of glycans on the glycosylphosphatidylinositol
(GPI) anchor. Furthermore, when monolayers of polarized
Madin-Darby canine kidney cells on polycarbonate filter membranes were
incubated with 35S-rhIL-1
in either the apical or
basolateral chamber, 35S-interleukin-1
was found to bind
specifically to the apical membranes with a Ka
value of 4.6 × 107 M
1, and
the specific interaction was inhibited by 1 µM mannose
6-phosphate. Since the mannose 6-phosphate diester moiety exists only
in the GPI glycans on plasma membranes, it was evident that
interleukin-1
can directly interact with the mannose 6-phosphate
diester component of the intact glycan of GPI anchors on plasma
membranes.
INTRODUCTION
Until now, over 50 types of cytokines have been reported
that mediate the regulatory network established between lymphoid cells,
hematopoietic cells, and endothelial cells that control their
differentiation and proliferation (1). In this cytokine network, a
single cytokine can act as both a positive signal and a negative one,
depending on the type of target cells. In contrast, in some instances
multiple cytokines act on a single cell and show the same biological
effects. The response of target cells to a given cytokine is determined
by the expression of the cytokine receptor and/or the nature of the
link between the receptor and the signal transduction pathways of the
target cells. We have been interested in the lectin-like character of
cytokines in relation to their role as signal transducers or effectors.
On the basis of their lectin-like character, various cytokines have
been categorized into several groups. Growth factors such as
granulocyte-macrophage colony-stimulating factor (2), heparin-binding
growth factor (3), basic fibroblast growth factor (4, 5), and midkine (6) bind to heparan sulfate, whereas tumor necrosis factor-
(TNF-
)1 (7), lymphotoxin (8),
interleukin-1
and -1
(7, 9), interleukin-2 (10), and
interleukin-3 (11) recognize
N,N
-diacetylchitobiose and/or some oligomannosyl
residues, although the precise carbohydrate binding specificities and
target glycoproteins have not yet been determined. To elucidate the
physiological role of the lectin-like interaction between cytokines and
target molecules, the carbohydrate binding specificities of cytokines
should first be determined. We describe in this paper the lectin-like
interaction between recombinant human IL-1
(rhIL-1
) and target
glycoproteins by measuring the binding of 35S-labeled
rhIL-1
to various glycoproteins immobilized on Sepharose 4B, on
plates coated with phosphatidylinositol-specific phospholipase C
(PI-PLC)-treated human placental alkaline phosphatase (ALP), and on
polarized Madin-Darby canine kidney (MDCK) cells and also by assaying
the inhibition of this interaction by various haptenic compounds.
EXPERIMENTAL PROCEDURES
Materials
Human transferrin, bovine ribonuclease B, mannose
6-phosphate, mannose 1-phosphate, glucose 6-phosphate,
myo-inositol 1-phosphate, mannose 6-sulfate, and
ethanolamine phosphate were purchased from Sigma.
[35S]Methionine (10.27 Ci/mmol) was obtained from ICN
Pharmaceutical Inc. Arthrobacter ureafaciens sialidase and
Bacillus thuringiensis PI-PLC were purchased from Nacalai
Tesque (Kyoto) and Funakoshi Inc. (Tokyo), respectively. Mannitol
6-phosphate was prepared from mannose 6-phosphate by reduction with
NaBH4.
H2N·CH2·CH2·PO4
6(Man
1
2)mannitol
was kindly provided by Dr. S. Iwahara (Kagawa University, Kagawa,
Japan) and
H2N·CH2·CH2· PO4
6mannitol
was prepared from
H2N·CH2·CH2·PO4
6(Man
1
2)mannitol
by Aspergillus saitoi Man
1
2-specific
-mannosidase digestion (12). Gal
1
4GlcNAc
1
2Man
1
6
(Gal
1
4GlcNAc
1
2Man
1
3)Man
1
4GlcNAc (abbreviated
as
Gal2·GlcNAc2·Man3· GlcNAc)
was purified from the urine of patients with
GM1-gangliosidosis (13). ALP was a generous gift from Dr.
M. Ikehara (Fukuoka University, Fukuoka, Japan). The ALP was purified
from human placenta by the method of Ogata et al. (14). MDCK
strain II cells were kindly supplied by Dr. M. Tashiro (National
Institute of Health, Tokyo, Japan).
Synthesis of Phosphate
Derivatives
Ac-NH·CH2·CH2·PO4
6Man
1
OCH2·CH2·CH3,
Ac-NH·CH2·CH2·PO4
6Man
1
2Man
1
OCH2·CH2·CH3,
and
Ac-NH·CH2·CH2·PO4
6Man
1
2Man
1
6Man
1
OCH2·CH2·CH3
were synthesized according to the hydrogen phosphate method as
described by Pannecoucke et al. (15) using van Boom reagent
(16) from N-acetylethanolamine and the corresponding
suitable benzylated allyl mannopyranosides that were prepared by the
dimethylphosphinothioate method (17).
Preparation of Uromodulin
Uromodulin was prepared from
pooled urine (1 liter) from a pregnant donor (in the 12th week of
pregnancy) as follows (18). 0.58 mol of NaCl was added to the urine,
and it was stirred at 4 °C overnight. The precipitate formed was
collected by centrifugation and dialyzed against distilled water.
After centrifugation, the supernatant was freeze-dried and dissolved in
phosphate-buffered saline (PBS).
Preparation of Trypanosome Variant Surface
Glycoproteins
Trypanosomes (Trypanosoma brucei
gambiense) isolated from the blood of an infected mouse according
to the method described by Cross (19) were kindly provided by Dr. Y. Tachibana (Tokai University, Japan). A 100-µl sample of packed
trypanosomes was digested with B. thuringiensis PI-PLC (0.5 units/ml in Tris-buffered saline, pH 7.4, 37 °C, 5 h), and the
solubilized variant surface glycoproteins (VSG) were purified by
Superose 12 (Pharmacia Biotech Inc.) gel permeation chromatography.
Upon analysis by SDS-polyacrylamide gel electrophoresis according to
the method of Laemmli (20) followed by silver staining, the purified
VSG appeared as a broad single band with a molecular mass of about 60 kDa.
Preparation of rhIL-1
cDNA encoding human IL-1
(R&D Systems Europe Ltd., Abingdon, United Kingdom) was used to produce
rhIL-1
in Escherichia coli. Plasmid pET3a (Novagen, Inc.,
Madison, WI) was used as the T7 expression plasmid (21). A
NdeI-BamHI fragment corresponding to the
synthetic human IL-1
gene was inserted between the NdeI and BamHI sites of pET3a to produce the expression plasmid.
The rhIL-1
gene was expressed in E. coli strain BL21(DE3)
under the control of the T7 promoter. The recombinant protein was
released from the cells by osmotic shock, concentrated with an Amicon
PM10 Diaflo membrane, and applied to a Sephacryl S-200 column.
Fractions containing rhIL-1
were pooled and stored at
20 °C
until use.
Preparation of 35S-rhIL-1
The cloned
cDNA encoding IL-1
(R&D Systems Europe Ltd., Abingdon, UK)
inserted downstream from the T7 RNA polymerase promoter in plasmid
pET3a was used as a template for in vitro transcription and
translation in the Single Tube Protein System 2, T7 (Novagen, Inc.) in
the presence of [35S]methionine. The translation products
were separated from free [35S]methionine using a PD-10
column (Pharmacia). One reaction using 2.5 µg of plasmid DNA
templates, 200 µl of lysate, and 200 µCi of
[35S]methionine provided approximately 500 fmol of
35S-rhIL-1
(0.9 × 107 dpm/pmol).
Solid-phase Binding Assay
The binding of
35S-labeled rhIL-1
to various glycoproteins was measured
by a solid-phase binding assay. Enzyme-linked immunosorbent assay
plates (Corning, Inc., Corning, NY) were coated with glycoproteins at
10 µg/ml in 20 mM carbonate buffer, pH 9.6, at 4 °C
overnight. The plates were washed with 0.05% Tween 20 in PBS, pH 7.3, blocked with PBS, 0.05% Tween 20, 1% BSA and then treated with 1 × 105 dpm of 35S-rhIL-1
in PBS, 0.05%
Tween 20, 0.1% BSA at 37 °C for 2 h. After washing with 0.05%
Tween 20 in PBS, the bound 35S-rhIL-1
was released by
treatment with 100 µl of 1 M acetic acid, and the
radioactivity was measured by means of a liquid scintillation
counter.
Preparation of Sepharose 4B-immobilized Glycoproteins
ALP
(1.2 mg) was dissolved in 1 ml of coupling buffer (0.1 M
NaHCO3 (pH 8.3) containing 0.5 M NaCl). 1 ml of
CNBr-activated Sepharose 4B (Pharmacia) in a column (inner diameter 0.7 cm × 5 cm long) was washed with 1 mM HCl (20 ml), and
the HCl was replaced with the coupling buffer. The ALP solution was
quickly added to the CNBr-activated Sepharose 4B resin. After gentle
mixing at 4 °C for 18 h, the gel was washed with 5 gel volumes
of coupling buffer. The gel was shaken in an equal volume of 0.2 M glycine, pH 8.0, at room temperature for 2 h and
washed with 0.1 M acetate buffer containing 1 M
NaCl, pH 4.0, and with coupling buffer (three times each). The amount
of ALP bound to the Sepharose 4B (based on the amount of ALP remaining
in the coupling reaction filtrate) was estimated to be approximately 1 mg/ml gel. Bovine ribonuclease B-Sepharose (10 mg/ml gel), trypanosome
VSG-Sepharose (1 mg/ml gel), uromodulin-Sepharose (1 mg/ml gel), and
human transferrin-Sepharose (10 mg/ml gel) were prepared according to
the same procedures as those employed for ALP-Sepharose.
Fractionation of 35S-rhIL-1
on Sepharose 4B
Column-immobilized Glycoproteins
5000 dpm of
35S-rhIL-1
was applied to a column (inner diameter, 0.7 cm × 5 cm long) of ALP-, VSG-, uromodulin-, transferrin-, or
ribonuclease B-Sepharose (1 ml), which was equilibrated with PBS
containing 0.1% BSA (PBS-BSA) at 4 °C. After standing for 30 min,
the respective columns were washed with 5 column volumes of PBS-BSA and
the bound 35S-rhIL-1
was eluted with PBS-BSA containing
1 mM mannose 6-phosphate.
Cell Culture
MDCK strain II cells were maintained in
minimal essential medium (MEM) supplemented with 10% fetal bovine
serum. For binding experiments, MDCK cells that were plated at
confluent density (approximately 3.5 × 105 cells) in
6.5-mm Transwell filter chambers (Costar) and cultured for 4 days after
plating were used. The polarity of cells was assessed by polarized
uptake of [35S]methionine from the basolateral side
(>10:1, basolateral:apical) (22).
Binding of 35S-rhIL-1
to MDCK Cell
Monolayers
The polarized monolayers on Transwell filters were
washed with ice-cold MEM (200 µl), the same medium was added to both
the apical (0.1-ml) and basolateral (0.5-ml) chambers, and the cells were incubated for 1 h at 4 °C. The medium in both chambers was changed to media with or without 200 µg/ml rhIL-1
, and the cells were incubated for another hour at 4 °C. For assay of apical
binding, the medium in the apical compartment was replaced with 50 µl
of medium containing 0.2 µCi/ml 35S-rhIL-1
with or
without 200 µg/ml rhIL-1
, and the Transwell filters were placed
onto 50 µl of MEM medium on a piece of parafilm. For assay of
basolateral binding, the non-radioactive medium and the radioactive
medium were added to opposite chambers, and the cells were then
incubated for the indicated times at 4 °C. The binding assays were
terminated by rapid washing of each compartment with ice-cold PBS four
times. The cells were lysed in 100 µl of ice-cold Nonidet P-40 lysis
buffer (50 mM Tris-Hcl, pH 8, containing 10 mM
EDTA, 1% Nonidet P-40, and 0.1% SDS) and removed by gentle scraping.
The lysates were centrifuged in a microcentrifuge at 10,000 × g for 10 min to precipitate debris and intact nuclei. Proteins in the supernatants were precipitated with 10%
trichloroacetic acid at 4 °C for 30 min, and the samples were
centrifuged. The pellets were washed with ethyl ether, and
radioactivity was counted using a scintillation counter.
RESULTS
Binding of 35S-rhIL-1
to Glycoproteins Immobilized
on Columns
It has been reported that rhIL-1
exhibits
lectin-like characteristics (7, 9). As the first step to examine the
lectin-like characteristics of IL-1
, the binding of
35S-rhIL-1
to various glycoproteins immobilized on
Sepharose 4B columns was investigated. As shown in Fig.
1, 35S-rhIL-1
bound to PI-PLC-treated
ALP-Sepharose, PI-PLC-treated VSG-Sepharose, or uromodulin-Sepharose (1 mg/ml gel) columns, and the bound 35S-rhIL-1
was eluted
with PBS-BSA containing 1 mM mannose 6-phosphate, which is
a constituent of the glycan portion of the glycosylphosphatidylinositol (GPI) anchor (Fig. 1, A, B, and C). In
contrast, rhIL-1
did not bind to human transferrin-Sepharose or
bovine ribonuclease B-Sepharose (10 mg/ml gel) columns, where the
glycan moiety linked to the protein consists of sialylated biantennary
sugar chains (23) and high mannose-type sugar chains, respectively (24)
(Fig. 1, D and E). The glycoproteins interacting
with rhIL-1
not only have asparagine-linked sugar chains but also
glycans of the GPI anchor at the carboxyl-terminal end. The
structures of the glycan portions of the GPI anchor of ALP (25)
and trypanosome VSG (26) have already been determined, and their side
chain structures differ from each other as shown below.
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Fig. 1.
Binding of 35S-rhIL-1
to
various glycoprotein-Sepharose 4B columns. To each column (0.7 cm
inner diameter × 5 cm long) 5 × 103 dpm of
35S-rhIL-1
was applied at 4 °C. The column was eluted
with PBS-BSA and then with PBS-BSA containing 1 mM mannose
6-phosphate from the position indicated by the black arrows.
A, PI-PLC-treated placental ALP-Sepharose (1 mg/ml gel);
B, PI-PLC-treated trypanosome VSG-Sepharose (1 mg/ml gel);
C, uromodulin-Sepharose (1 mg/ml gel); D, human
transferrin-Sepharose (10 mg/ml gel); E, bovine RNase
B-Sepharose (10 mg/ml gel).
[View Larger Version of this Image (17K GIF file)]
Since the 1-O-alkyl-2-O-acylglycerol moiety
of the GPI anchor is removed by PI-PLC digestion, it was evident
that rhIL-1
recognizes the common structure,
NH2·CH2·CH2·PO4
6Man
1
2Man
1
6Man
1
4GlcNH2,
of the glycans in the GPI anchor.
Conditions for Solid-phase Binding Assay of
35S-rhIL-1
In binding assays using plates coated
with each of the five glycoproteins (10 µg/ml),
35S-rhIL-1
bound to PI-PLC-treated ALP, PI-PLC-treated
VSG, and uromodulin in a dose-dependent manner (data not
shown). For determination of the character of the interaction between
rhIL-1
and these glycoproteins, an assay of inhibition of
solid-phase binding by specific haptens was employed. PI-PLC-treated
human placental ALP was used as the glycoprotein to be immobilized on
plates because the structures of both the N-linked sugar
chain (27) and the glycan portion of the GPI anchor (25) had been
previously determined and are rather simple in comparison with other
glycoproteins. We investigated the inhibitory effects of the
biantennary sugar chain and various simple components of the glycan
portion of the GPI anchor on the interaction between
35S-rhIL-1
and ALP-coated plates. At first, we
investigated the conditions for solid-phase binding of
35S-rhIL-1
to ALP-coated plates. As for the time
dependence, the linearity of the binding activity was maintained for at
least 3 h (Fig. 2A,
), and the
binding activity was inhibited in the presence of excess amounts of
unlabeled rhIL-1
(Fig. 2A,
). These results indicated
that the binding of 35S-rhIL-1
to ALP is specific and is
competitive with unlabeled rhIL-1
. The binding of
35S-rhIL-1
was dependent on the concentration of both
ALP (Fig. 2B) and 35S-rhIL-1
(Fig.
2C,
), and the concentration dependence was linear at
least up to 20 µg/ml (ALP) and 1 × 106 dpm/ml
(35S-rhIL-1
), respectively. The reactivity did not
change in the presence of 1 mM EDTA, 10 mM
HEPES buffer, pH 7.3, 0.1% BSA (Fig. 2C,
) or 1 mM CaCl2, 10 mM HEPES buffer, pH
7.3, 0.1% BSA (Fig. 2C,
) showing that the lectin-like
interaction between rhIL-1
and ALP at least does not require calcium
ions. On the basis of these results, the following inhibitory effects
of various haptens were investigated in the proportional range of the
protein concentration dependence and time dependence.
Fig. 2.
Binding of 35S-rhIL-1
to
ALP-coated plates. Plates were coated with ALP as described under
"Experimental Procedures," and the amount of bound
35S-rhIL-1
was measured using a liquid scintillation
counter after being released with 1 M AcOH. A,
time course. Plates coated with 10 µg/ml ALP were incubated with
1 × 105 dpm of 35S-rhIL-1
alone (
)
or with 1 × 105 dpm of 35S-rhIL-1
and
10 µg/ml unlabeled rhIL-1
(
) for the indicated times at
37 °C; B, ALP concentration dependence. Plates coated with various concentrations of ALP (0-20 µg/ml) were incubated with
1 × 105 dpm of 35S-rhIL-1
for 2 h
at 37 °C; C, 35S-rhIL-1
concentration
dependence. Plates coated with 10 µg/ml ALP were incubated for 2 h with various concentrations of 35S-rhIL-1
(up to
5 × 106 dpm/ml) in PBS-BSA (
), in 10 mM HEPES, 0.1%BSA, pH 7.3, containing 1 mM
EDTA (
), or in 10 mM HEPES, 0.1% BSA, pH 7.3 containing 1 mM CaCl2 (
).
[View Larger Version of this Image (17K GIF file)]
Inhibitory Effects of Various Haptens on the Lectin-like Activity
of rhIL-1
The inhibitory effects of various saccharides listed
in Table I on the lectin-like interaction between
IL-1
and ALP-coated plates were examined, and the inhibition curves
obtained are shown in Fig. 3. The biantennary sugar
chain of ALP, which is an asialo-N-linked sugar chain, did
not show any inhibitory effect at concentrations up to 10 mM (Fig. 3A, Table I). Since the same results
were obtained even when the plates were coated with sialidase-treated
ALP (data not shown), the result is not due to a sialylated biantennary sugar chain of ALP. In contrast, mannose 6-phosphate, which is a
component of the glycan of the GPI anchor, was an effective inhibitor
(Fig. 3B). However, other components of this glycan such as
ethanolamine phosphate and inositol phosphate did not show any
inhibitory effect at 1 mM (see Table I). To elucidate whether mannose 6-phosphate-diester derivatives show inhibitory effects, three synthetic phosphodiester derivatives as shown in Table I
were studied.
Ac-NH·CH2·CH2·PO4
6Man
1
propyl,
Ac-NH·CH2·CH2·PO4
6Man
1
2Man
1
propyl,
and
Ac-NH·CH2·CH2·PO4
6Man
1
2Man
1
6Man
1
propyl
showed the same strong inhibitory effects as mannose 6-phosphate (Fig.
3, C, D, and E). Mannose 6-sulfate,
mannose 1-phosphate, and glucose 6-phosphate did not show any
inhibitory effect at concentrations up to 1 mM (Table I),
indicating that mannose substituted with phosphate at the C-6 position
is necessary for lectin-like binding. Furthermore, mannitol-6-phosphate
and
NH2CH2·CH2·PO4
6mannitol,
which are reduced forms of mannose 6-phosphate and the phosphodiester
derivative, did not show any inhibitory effect at concentrations up to
1 mM. These results showed that a mannopyranoside substituted with phosphate at the C-6 position represents an important locus for the lectin-like interaction between IL-1
and ALP, and IL-1
recognizes the mannose 6-phosphate diester moieties of the glycan of GPI-anchored glycoproteins.
Fig. 3.
Inhibition curves obtained for the binding of
35S-rhIL-1
to ALP-coated plates in the presence of
various saccharides. A, Gal2·GlcNAc2·Man3·GlcNAc;
B, mannose 6-phosphate; C,
Ac-NH·CH2·CH2·PO4
6Man
1
propyl;
D,
Ac-NH·CH2·CH2·PO4
6Man
1
2Man
1
propyl;
E,
Ac-NH·CH2·CH2·PO4
6Man
1
2Man
1
6Man
1
propyl.
[View Larger Version of this Image (17K GIF file)]
Binding of 35S-rhIL-1
to MDCK Cell
Monolayers
All of the results so far described were obtained
using PI-PLC-treated glycoproteins. To determine whether intact
GPI-anchored glycoproteins on plasma membranes interact with
35S-rhIL-1
, the following experiments were performed.
Because it has been reported that GPI-anchored glycoproteins are
distributed on the apical surface of the plasma membrane of polarized
MDCK cells (28), and no IL-1 receptor on the plasma membrane of MDCK cells has been reported, we examined the lectin-like interaction between rhIL-1
and cell surface glycoproteins using polarized MDCK
cells as a model. Polarized MDCK cells on polycarbonate filter membranes were incubated with 0.2 pmol/ml 35S-rhIL-1
in
either the apical or basolateral chamber. The time course of binding at
4 °C is shown in Fig. 4. The amount of
35S-rhIL-1
bound to the cells (Fig. 4,
) was reduced
by the addition of excess unlabeled rhIL-1
(Fig. 4,
), suggesting
that 35S-rhIL-1
and unlabeled rhIL-1
compete for a
limited number of specific binding sites. The specific binding of
rhIL-1
to the apical surface of MDCK cells, which was defined as the
difference between the amounts of radioactivity bound in the absence
and presence of excess unlabeled rhIL-1
, had reached a steady state after incubation for 4 h at 4 °C (Fig. 4,
). In contrast,
the extent of specific binding to the basolateral surface was low throughout the incubation period for up to 3 h (Fig.
4B). The specific binding was not observed in assays of
non-polarized MDCK cells cultured in monolayers (data not shown),
suggesting that rhIL-1
binds to GPI-anchored proteins on the apical
membranes.
Fig. 4.
Time course of 35S-rhIL-1
binding to apical (A) and basolateral (B)
surfaces of MDCK cells at 4 °C. The polarized cell monolayers
were prepared as described under "Experimental Procedures." Either
the apical or basolateral side of cell monolayers was treated with 50 µl of cold MEM containing approximately 0.2 µCi/ml
35S-rhIL-1
with or without excess unlabeled rhIL-1
,
and the cells were incubated for the indicated times at 4 °C. The
cell monolayers were then washed and harvested as described under
"Experimental Procedures."
, total binding;
, specific
binding;
, nonspecific binding.
[View Larger Version of this Image (22K GIF file)]
Scatchard Plot Analysis of 35S-rhIL-1
Binding to
MDCK Cells
Fig. 5 shows Scatchard plots (29) for
35S-rhIL-1
binding to the apical cell surface of
polarized MDCK monolayers, where B is moles of bound
rhIL-1
/dpm as a proportion of the total rhIL-1
/well and
F is moles of unbound rhIL-1
. The plots gave a straight
line, indicating homogeneity with respect to affinity for the
GPI-anchored glycan. The Ka value for binding was
calculated to be 4.6 × 107
M
1. These values were several orders lower
than those determined for both type I and type II IL-1 receptors (30).
Although both types of IL-1 receptors are expressed in several cell
lines, including B lymphoblastoid lines (30), HepG2 (30), and thymoma
lines (31), the expression of IL-1 receptors in renal epithelial cell lines has not been reported. The specific binding shown in Fig. 4
indicated that rhIL-1
binds to neither type I nor type II IL-1 receptors.
Fig. 5.
35S-IL-1
-specific binding to
the apical surface of MDCK cells. The experimental conditions were
the same as those in the legend to Fig. 4, except that the apical
surface of cell monolayers was treated with 50 µl of MEM containing
the indicated concentrations of 35S-IL-1
with or without
excess unlabeled IL-1
, and the cells were incubated for 4 h at
4 °C. A, the amount of specifically bound
35S-IL-1
was determined by subtracting the radioactivity
bound in the presence of unlabeled IL-1
from the radioactivity bound in the absence of unlabeled IL-1
; B,
35S-IL-1
binding to the apical surface was plotted by
the method of Scatchard (29).
[View Larger Version of this Image (16K GIF file)]
Inhibitory Effect of Mannose 6-Phosphate, Mannose 1-Phosphate, or
Mannose 6-Sulfate on 35S-rhIL-1
Binding to MDCK
Cells
To determine whether the ligands required for rhIL-1
binding to polarized MDCK cells are present within the glycan portion of GPI-anchored proteins, the effect of various amounts of mannose 6-phosphate, mannose 1-phosphate, or mannose 6-sulfate on the specific
binding was examined (Fig. 6). The specific binding was dose dependently inhibited by the addition of mannose 6-phosphate (Fig.
6,
), which is the common core structure of the glycan portion; in
contrast, mannose 1-phosphate (
) or mannose 6-sulfate (
) did not
show any inhibitory effect up to 1 mM. These results support the view that the mannose 6-phosphate diester component of the
glycan portions of GPI-anchored proteins in the polarized MDCK cells is
the moiety recognized by rhIL-1
in the lectin-like binding
interaction.
Fig. 6.
Effects of mannose 6-phosphate, mannose
1-phosphate, or mannose 6-sulfate on 35S-IL-1
-specific
binding to the apical surface of MDCK cells. The apical surface of
MDCK cells in monolayers was assayed for 35S-IL-1
binding in the presence of the indicated concentrations of the
components for 4 h at 4 °C. Other experimental conditions were
the same as those in the legend to Fig. 4.
, mannose 6-phosphate;
, mannose 1-phosphate;
, mannose 6-sulfate.
[View Larger Version of this Image (12K GIF file)]
DISCUSSION
This paper demonstrates that rhIL-1
recognizes mannose
6-phosphate diester within the glycans of GPI-anchored proteins
immobilized on plates or immobilized on CNBr-activated Sepharose 4B. It
is also shown that 35S-rhIL-1
binds to intact
GPI-anchored proteins present on the apical membranes of polarized MDCK
cells with the Ka value of 4.6 × 107 M
1, and this binding is
inhibited in the presence of approximately 1 × 10
6
M mannose 6-phosphate.
The lectin-like interaction between rhIL-1
and mannose 6-phosphate
(diester) in the GPI anchor was calcium-independent as shown in Fig.
2C. It is well known that one of two receptors for lysosomal
enzymes trafficking into the lysosome is a Ca2+-independent
mannose 6-phosphate binding lectin with a molecular weight of 275,000 (32). It recognizes mannose 6-phosphate residues in high mannose-type
sugar chains with a Ka value of 7-8 × 106 M
1 (32), but not mannose
6-phosphate diester (33), while IL-1
recognizes both mannose
6-phosphate and its diester equally as described in this study. Since
no homology was observed between the amino acid sequences of the
Ca2+-independent mannose 6-phosphate-binding lectin and
IL-1
using the FASTA method (34), IL-1
may have a different type
of carbohydrate binding domain from that of the
Ca2+-independent mannose 6-phosphate-binding lectin for
lysosomal enzymes.
It has been reported that not only IL-1
but also TNF-
and
lymphotoxin bind to uromodulin immobilized on plates, and in each case
this binding is partially inhibited by high concentrations of
N,N
-diacetylchitobiose (7, 8). Since uromodulin
has not only asparagine-N-linked sugar chains but also a
GPI-anchored glycan at the carboxyl-terminal end (35), it seems
important to ascertain in the near future whether TNF-
and
lymphotoxin have the same carbohydrate binding specificities as
IL-1
.
Although the physiological function of the lectin-like character of
these cytokines remains to be studied, several possible roles may be
considered as follows. It has been reported that TNF-
exerts a
direct trypanolytic activity on salivarian trypanosomes (36). This
activity is not blocked by soluble 55-kDa and 75-kDa TNF receptors, but
is potently inhibited by N,N
-diacetylchitobiose and the hexapeptide of TNF-
, Thr105 to
Glu110, implying the existence of an alternative
recognition domain of TNF-
for constituents of trypanosome parasites
(36). It is of interest to determine whether this recognition region of TNF-
shows binding activity specific for mannose 6-phosphate diester
in the glycan of the GPI anchor, i.e. the same lectin-like specificity as IL-1
. Such a lectin-like activity of TNF-
may exhibit such alternative recognition of microbial constituents.
As another physiological role of the lectin-like interaction between
rhIL-1
and the GPI anchor, the latter may function as a modulator of
IL-1 receptors. Although two types of IL-1 receptors have been
identified (37), it remains to be established whether the cloned IL-1
receptor by itself has the ability to generate signals or whether it is
linked to an unidentified signal transducer or effector. It is clear
only that the two different IL-1 receptors function independently at
the level of ligand binding and do not form a heterodimeric receptor
complex even when they are present on the same cell (37). The GPI
anchor portions that are recognized with low affinity by IL-1
may
serve only to bind ligand and deliver it or enhance binding to the type
I and/or II receptor(s), which would be capable of binding ligands and
delivering the signal into cells, as in the case of the interleukin-2
system (38) or growth factors such as granulocyte-macrophage
colony-stimulating factor (2), heparin-binding growth factor (3), basic
fibroblast growth factor (4, 5), or the midkine-heparan sulfate
interaction (6). The growth factors show low affinity for heparan
sulfate, which behaves as an accessory molecule required for binding of growth factors to the high affinity receptors on the plasma membrane. Such an obligatory relationship of low and high affinity cytokine receptors suggests a physiological role for heparan sulfate and the GPI
anchor as low affinity receptors and constitutes a novel mechanism for
the regulation of cytokine-mediated signal transduction.
It is reported that some IL-1
-specific binding structures with low
affinity are present on the cell wall of virulent bacteria (39) and in
supernatants from vaccinia virus-infected CV-1 cells (40). In these
cases, the role of binding to IL-1
is thought to prohibit or
diminish the inflammatory effects of the cytokine produced by infected
cells. The low affinity binding of the GPI anchor portion to IL-1
may have a similar role in regulating the effects of IL-1
, which are
induced by binding of IL-1
to type I and/or II receptor(s).
The results so far described support the view that these lectin-like
activities of cytokines are fundamental to immunology and bring some
additional information to light concerning the functional role of the
GPI anchor.
FOOTNOTES
*
This work was supported by Grant-in-Aid 06240246 for
Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture, 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.
§
To whom correspondence should be addressed. Tel.: 81-3-3294-3286;
Fax: 81-3-3294-2656.
1
The abbreviations used are: TNF, tumor necrosis
factor; IL-1
, interleukin-1
; rhIL-1
, recombinant human
interleukin-1
; IL-1, interleukin-1; VSG, variant surface
glycoproteins; PI-PLC, phosphatidylinositol-specific phospholipase C;
GPI, glycosylphosphatidylinositol; ALP, human placental alkaline
phosphatase; BSA, bovine serum albumin; PBS, phosphate-buffered saline;
MDCK, Madin-Darby canine kidney cells; MEM, minimal essential
medium.
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