From the Department of Biochemistry, Sasaki Institute, 2-2 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 and CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation, 2-3 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 Japan
Received for publication, September 26, 2000, and in revised form, November 10, 2000
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ABSTRACT |
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Interleukin-2 (IL-2) specifically recognizes
high-mannose type glycans with five or six mannosyl residues. To
determine whether the carbohydrate recognition activity of IL-2
contributes to its physiological activity, the inhibitory effects of
high-mannose type glycans on IL-2-dependent CTLL-2 cell
proliferation were investigated.
Man5GlcNAc2Asn added to CTLL-2 cell
cultures inhibited not only phosphorylation of tyrosine kinases but
also IL-2-dependent cell proliferation. We found that a
complex of IL-2, IL-2 receptor Interleukin-2 (IL-2)1 is
a cytokine synthesized by activated T cells (1). IL-2 promotes the
proliferation of IL-2-dependent T cells and functions as an
immunomodulator of activated B cells, macrophages, and natural killer
cells (2). IL-2 expresses its physiological functions through
interaction with its receptor complex, which consists of three receptor
subunits, Although research on the carbohydrate recognition of IL-2 has a long
history, its physiological function has not been clearly determined.
Sherblom et al. (11) and Zanetta et al. (12)
reported that IL-2 recognizes high-mannose type glycans with five or
six mannosyl residues as determined by the plate method. Later, Najjam et al. (13) found that rhIL-2 binds to heparin specifically. However, since the addition of heparin did not show any inhibitory effect on IL-2-dependent cell proliferation, it was
suggested that the interaction between IL-2 and heparin is not related
to such activity. Zanetta et al. (12) presented a
cross-linking model in which it was hypothesized that, in the case of
human peripheral lymphocytes, IL-2 binds to not only the IL-2 receptor via the IL-2 receptor-binding sites but also the TCR complex containing glycosylated CD3 (12). This tentative model was proposed on the basis
of the results of analysis of immunoprecipitates obtained using
IL-2R In this paper, we report that addition of high-mannose type glycans
inhibits not only IL-2-dependent CTLL-2 cell proliferation but also the phosphorylation of the related tyrosine kinases including Jak1, Jak3, Lck, and Lyn. Furthermore, a high affinity complex including IL-2R Materials and
Chemicals--
Endo- Preparation of RhIL-2--
cDNA encoding human IL-2 (RandD
Systems Europe Ltd., Abingdon, UK) was used to produce rhIL-2 in
Escherichia coli. Plasmid pET3a (Novagen Inc., Madison, WI)
containing a T7 promoter was used as the rhIL-2 expression plasmid. A
NdeI-HindIII fragment corresponding to a
synthetic human IL-2 gene was inserted between the
NdeI and HindIII sites of pET3a to produce the
expression plasmid. The rhIL-2 gene was expressed in E. coli strain BL21(DE3) under the control of the T7 promoter. A
15-ml culture of E. coli BL21(DE3) cells containing the IL-2
plasmid was incubated overnight until the cells reached the stationary
phase of growth and this culture was used to inoculate 500 ml of L
broth containing 100 µg/ml ampicillin. After incubation for 2.5 h at 37 °C, IL-2 production was induced by addition of 0.5 mM isopropyl Cell Culture--
Mouse T cell line CTLL-2 (RCB0637) was
obtained from the RIKEN Cell Bank (Ibaraki, Japan). CTLL-2 cells were
maintained in complete RPMI 1640 medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum and 100 units/ml rhIL-2 at
37 °C under a 5% CO2 atmosphere. The cells were
cultured until the cell density reached 1.5 × 106
cells/ml and the culture was then split.
Bioassay of rhIL-2--
For the bioassay, 2 days after the last
addition of rhIL-2, the cells were washed three times in RPMI 1640 medium. The cells were then resuspended in complete medium at a cell
density of 1 × 105 cells/ml and plated out in
microtiter plates, 100 µl/well. Then 100 µl of rhIL-2 at various
concentrations, diluted in complete RPMI 1640 medium, was added. The
cells were incubated at 37 °C in a 5% CO2 atmosphere
for 2 days, then 20 µl of Cell Titer 96TM Aqueous one
solution reagent was added to each well. After incubating the mixture
for 2 h, the absorbance at 525 nm was read using a dual wavelength
flying spot scanning densitometer CS-9300PC (Shimazu Corp. Kyoto,
Japan). Cell Titer 96TM Aqueous one solution reagent used
to measure cell proliferation activity was obtained from Promega Corp.
The solution is composed of a novel tetrazolium compound and an
electron coupling reagent, phenazine ethosulfate in Dulbecco's
phosphate-buffered saline (pH 6.0).
Oligosaccharides--
Man Cell Lysis and Immunoprecipitation--
To investigate the
phosphorylation of kinases in the presence and absence of
Man5GlcNAc2Asn, the following experiments were performed. CTLL-2 cells were washed twice with RPMI 1640 medium containing 10% fetal calf serum, suspended at 2 × 106 cells/ml and incubated for 6 h at 37 °C in the
absence of IL-2. The cells were then stimulated with rhIL-2 (5 units/ml) for 20 min at 37 °C in the presence or absence of 10 µM Man5GlcNAc2Asn, followed by
centrifugation at 3000 rpm for 5 min at 4 °C. Cells (1 × 107 cells/lane) were lysed for 60 min on ice by adding 1 ml
of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS,
50 mM sodium fluoride, 1 mM sodium
orthovanadate, 10 µM pepstatin A, 1 µg/ml leupeptin,
100 kallikrein units/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride. Cell lysates were cleared by
centrifugation for 15 min at 1.5 × 104 rpm and used
for the immunoprecipitation. The tyrosine kinases were individually
immunoprecipitated from cell lysates with anti-Jak1, anti-Jak3 (Upstate
Biotechnology, NY), anti-Lyn, or anti-Lck antibody (Santa Cruz
Biotechnology, Inc., CA) according to the manufacturer's protocol.
After fractionation of the immunoprecipitates by SDS-PAGE, the proteins
were transferred to a nitrocellulose membrane. The blots were then
probed with anti-phosphotyrosine monoclonal antibody (4G10, UBI) and
with the appropriate second antibody and visualized by means of the ECL
system (Amersham Pharmacia Biotech). The blots were stripped with 62.5 mM Tris/HCl (pH 6.7) containing 2% SDS and 100 mM
To detect any glycoprotein with high-mannose type glycans among the
constituents of the IL-2 receptor complex, the following experiments
were performed. Cells (1 × 107 cells/lane) which had
been cultured continuously in the presence of IL-2 were lysed by adding
1 ml of lysis buffer containing 50 mM Tris-HCl (pH 8.0),
150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1%
SDS, 10 µM pepstatin A, 1 µg/ml leupeptin, 100 kallikrein units/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride, and 1 mM mannolactone (Sigma-Aldrich) and
incubating the mixture for 60 min on ice. Cell lysates were cleared by
centrifugation for 15 min at 1.5 × 104 rpm and used
for the immunoprecipitation. The supernatants of the cell lysates were
treated with rabbit anti-IL-2R Preparation of Biotinylated G. nivalis Agglutinin--
G.
nivalis agglutinin was obtained from Sigma-Aldrich Co. and
sulfo-NHS-biotin was obtained from Pierce. 1 mg of GNA was dissolved in
500 µl of phosphate-buffered saline and 1 mg of sulfo-NHS-biotin was
dissolved in 1 ml of distilled water. The GNA solution was mixed with
30 µl of sulfo-NHS-biotin solution and left to stand on ice for
2 h. After dialysis against phosphate-buffered saline, the
biotinylated GNA was used as a probe.
The IL-2 Lectin Activity Is Required to Induce
IL-2-dependent Cell Proliferation--
It is known that
IL-2 specifically recognizes high-mannose type glycans with 5 or 6 mannosyl residues (11, 12). We studied whether the lectin activity is
indispensable for induction of IL-2-dependent cell
proliferation. As the first step, we investigated whether this process
is inhibited by addition of high-mannose type glycans. It is known that
CTLL-2 cells, a mouse T-cell line, proliferate in a manner dependent on
IL-2. Upon incubating the cells (1 × 104/well) in the
presence of rhIL-2 at 5 units/ml for 48 h, the cells showed a
proliferative response which was dependent on the concentration of
rhIL-2 (Fig. 1A). The extent
of cell proliferation was determined colorimetrically (see
"Experimental Procedures"). Since the concentration of rhIL-2
required to stimulate maximum IL-2-dependent cell
proliferation was found to be 5 units/ml, the following experiments
were performed at this concentration.
Mixtures were prepared containing 5 units/ml rhIL-2 and high-mannose
type glycans at various concentrations and, after being left standing
for 2 h at 37 °C, the mixtures were added to the wells
containing the cultured cells. In this experiment,
Man5GlcNAc2Asn and
Man6GlcNAc2Asn were found to dose dependently
inhibit the proliferative response of these cells to rhIL-2 in
vitro, whereas Man7GlcNAc2Asn,
Man8GlcNAc2Asn,
Man9GlcNAc2Asn, and
Man3GlcNAc2 did not show any inhibitory effect
(Fig. 1B). These results suggested that the lectin activity
of IL-2 is required for stimulation of IL-2-dependent
T-cell proliferation.
Inhibitory Effects of Man5GlcNAc2Asn on
Phosphorylation of Tyrosine Kinases Activated by IL-2--
It has been
reported that, in the case of IL-2-induced proliferation of CTLL-2
cells, signal transduction occurs via tyrosine kinases including Lck
(4), Jak1, and Jak3 (5-7). In preliminary experiments, we found that
Lyn is also phosphorylated as a result of IL-2 stimulation in CTLL-2
cells. Although Lyn was originally reported to be phosphorylated as a
result of IL-2 stimulation in a B-cell line, whether the association
site is IL-2R GNA Staining of the IL-2 Receptor Complex Including Tyrosine
Kinases--
The results described above indicated that the
carbohydrate recognition function of IL-2 was involved in the cellular
signaling system. Although it is known that IL-2 induces the formation
of an IL-2·IL-2 receptor complex which includes the three receptor subunits
In view of these results, after the immunoprecipitates obtained with
anti-IL-2R
Since the nonglycosylated rhIL-2R Our findings presented in this paper clearly demonstrate
that the dual recognition by IL-2 of a specific peptide sequence and a
carbohydrate epitope in the IL-2R,
,
subunits, and tyrosine
kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the
components of this complex, only the IL-2 receptor
subunit was
stained with Galanthus nivalis agglutinin which
specifically recognizes high-mannose type glycans. This staining was
diminished after digestion of the glycans with
endo-
-N-acetylglucosaminidase H or D, suggesting that at
least a N-glycan containing
Man5GlcNAc2 is linked to the extracellular
portion of the IL-2 receptor
subunit. Our findings indicate that
IL-2 binds the IL-2 receptor
subunit through
Man5GlcNAc2 and a specific peptide sequence on
the surface of CTLL-2 cells. When IL-2 binds to the IL-2R
subunit,
this may trigger formation of the high affinity complex of
IL-2-IL-2R
, -
, and -
subunits, leading to cellular signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
(IL-2R
, -
, and -
) (3). Although
none of the receptor subunits has intrinsic tyrosine kinase activity,
intracellular portions of the IL-2R
and -
subunits associate with
intracellular tyrosine kinases including Lck (4), Jak1 and Jak3 (5-7)
in IL-2-stimulated T-cells, and cellular signaling occurs through
tyrosine phosphorylation of several proteins (3). From these
observations, it is suggested that a complex consisting of at least
IL-2, IL-2R
, IL-2R
, IL-2R
, and tyrosine kinases including Lck,
Jak1, and Jak3 might be formed in CTLL-2 cells stimulated by IL-2.
However, it has been reported that each IL-2 receptor subunit alone
shows only weak binding to IL-2. IL-2R
binds IL-2 with low affinity
(Kd ~10 nM), IL-2R
binds IL-2 with
very low affinity (Kd ~100 nM), and
IL-2R
has no measurable affinity for IL-2 (8-10). Accordingly, the
mechanism by which IL-2 stimulates the formation of a high affinity
IL-2-IL-2R
, -
, or -
complex remains unclear.
antibody. However, they did not directly show that phosphorylation of Lck kinase co-immunoprecipitated with IL-2R
subunit occurs, or that high-mannose type glycan has an inhibitory effect on IL-2-dependent cell proliferation. Accordingly,
whether the carbohydrate recognition activity of IL-2 contributes to
the physiological function of IL-2 still remains unclear. Moreover, it
has been reported recently that the catalytic activation of Jak1 and
Jak3 kinases is induced within minutes after formation of a IL-2·IL-2
receptor high-affinity complex (5-7).
, -
, -
subunits, and Jak1, Jak3, Lck, Lyn
tyrosine kinases is formed in IL-2-stimulated CTLL-2 cells. Among the
co-immunoprecipitated components of the complex, only the IL-2R
subunit was stained with Galanthus nivalis agglutinin (GNA)
which specifically recognizes high-mannose type glycans (14) and the
staining was diminished after digestion of the glycans with
Man5GlcNAc2-specific
endo-
-N-acetylglucosaminidase D (Endo D) (15). Our
findings suggest that dual binding of IL-2 to both a
Man5GlcNAc2 moiety and a specific peptide
sequence in the IL-2 receptor
subunit serves to trigger the
formation of a high-affinity complex of IL-2- IL-2R
, -
, and -
subunits, leading to cellular signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-N-acetylglucosaminidase H (Endo H)
and Endo D were obtained from Seikagaku Kogyo Co. (Tokyo,
Japan). Prestained protein markers used as molecular weight markers for
SDS-PAGE were obtained from BioLabs Inc. (Hertfordshire, United
Kingdom). Arthrobacter protophormiae
endo-
-N-acetylglucosaminidase (16) was kindly provided by
Dr. K. Takegawa, Faculty of Agriculture, Kagawa University, Japan.
Fmoc-conjugated Asn-GlcNAc (17) was kindly provided by Dr. T. Inazu,
Noguchi Institute, Tokyo, Japan.
-thiogalactoside, and the cells were grown
for 2.5 h. IL-2 was produced mainly in inclusion bodies. The
inclusion bodies were solubilized and IL-2 was refolded by a method
described previously (18), with slight modification, as follows. The
cells were collected by centrifugation and homogenized by lysozyme
treatment and sonication at 4 °C. The lysate was centrifuged at
10,000 rpm for 10 min, and the precipitate was collected. The pellet
was dissolved in 20 mM Tris-HCl buffer (pH 8.3) containing
10 mM EDTA and 6 M guanidine hydrochloride. Then, the solution was treated with 10 mM reduced
glutathione and 1 mM oxidized glutathione in the presence
of 2 M guanidine hydrochloride at pH 8.0. The solution was
kept for 16 h at room temperature, then it was dialyzed against
phosphate-buffered saline. An aliquot of the dialysate was
subjected to SDS-PAGE using a 15% acrylamide gel to check the purity
of the rhIL-2. The biological activity of the recovered soluble rhIL-2
protein was determined in a proliferation assay using CTLL-2 cells.
Human IL-2 purchased from Sigma-Aldrich Co. was used as the standard
for the units of activity. Protein concentration was estimated using
the Bio-Rad Protein Assay dye reagent with bovine serum albumin as the
standard. The amount of activity displayed by the rhIL-2 used in this
study was 1-10 units/ng.
1
6(Man
1
3)Man
1
6(Man
1
3)
Man
1
4GlcNAc
1
4GlcNAc-Asn(Man5GlcNAc2Asn)
and
Man
1
6(Man
1
3)Man
1
6(Man
1
2Man
1
3)Man
1
4GlcNAc
1
4GlcNAc-Asn (Man6GlcNAc2Asn) were prepared by
exhaustive Pronase digestion of ovalbumin followed by Dowex
50 × 2 (H+ form) column chromatography (200-400
mesh, 1.5 × 150 cm) according to the method described by Tai
et al. (15).
(Man
1
2)2-4[Man
1
6- (Man
1
3)Man
1
6(Man
1
3)]Man
1
4GlcNAc
1
4GlcNAc
(Man7GlcNAc2, Man8GlcNAc2,
Man9GlcNAc2) and
Man
1
6 (Man
1
3)Man
1
4GlcNAc
1
4GlcNAc (Man3GlcNAc2) were prepared from 3 g of
porcine thyroglobulin glycopeptides by hydrazinolysis followed by
re-N-acetylation, and Man7-9GlcNAc2
were each isolated by Bio-Gel P-4 (under 400 mesh, 2.0 × 100 cm)
column chromatography. Each oligosaccharide was converted to the
asparaginyl oligosaccharide from
Man7-9GlcNAc2 and Fmoc-conjugated Asn-GlcNAc
by treatment with A. protophormiae endo-
-N-acetylglucosaminidase according to the method
described by Kuge et al. (19). The structures of these
different glycoasparagines and oligosaccharides were determined through
a combination of methylation analysis (20),
-mannosidase digestion,
partial acetolysis (21), and matrix-assisted laser desorption-time of flight mass spectrometry (Shimadzu Corp., Kyoto, Japan).
-mercaptoethanol at 50 °C for 30 min and reprobed with anti-Jak1, anti-Jak3, anti-Lyn, or anti-Lck antibody to evaluate the amount of the corresponding tyrosine kinase.
, anti-IL-2R
, or anti-IL-2R
antibody (Santa Cruz Biotechnology, Inc., CA) according to the
manufacturer's protocol and the immunoprecipitates were fractionated
by SDS-PAGE. The immunoprecipitates were then probed with
anti-IL-2R
, -
, -
, anti-Lck, anti-Jak1, anti-Jak3, or anti-Lyn antibody and with the appropriate second antibody and visualized by
means of the ECL system (Amersham Pharmacia Biotech). Otherwise, membranes were incubated at 37 °C for 18 h in the presence or absence of Endo H (10 milliunits/100 µl of citrate-phosphate buffer (pH 6.5)/cm2) or Endo D (10 milliunits/100 µl of
citrate-phosphate buffer (pH 6.5)/cm2) and stained with
biotinylated GNA (80 µg/ml), followed by treatment with avidin
peroxidase, and visualized by means of the ECL system. Then, the blot
was reprobed with anti-IL-2R
subunit antibody and with the
appropriate second antibody and visualized.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
rhIL-2-dependent
proliferation of CTLL-2 cells (A) and the effects of
high-mannose type glycans on the proliferative response
(B). A, rhIL-2 at increasing
concentrations was added to 5 × 104 cells/well. After
2 days, the extent of cell proliferation was determined using Cell
Titer 96TM Aqueous one solution reagent. B,
rhIL-2 (5 units/ml) and various high-mannose type glycans were mixed,
and each mixture was kept at 37 °C for 2 h before being added
to the culture of CTLL-2 cells. The inhibition curves obtained
using Man7GlcNAc2Asn or
Man8GlcNAc2Asn were the same as that obtained
using Man3GlcNAc2 (data not shown). Results are
means of three experiments (standard deviations were less than 5%).
M5, M6, M9, and M3 indicate
Man5GlcNAc2Asn,
Man6GlcNAc2Asn,
Man9GlcNAc2Asn, and
Man3 GlcNAc2, respectively.
or -
remains to be determined (22). To further
confirm whether the lectin activity of IL-2 modulates the cellular
signal transduction mechanism, phosphorylation of Jak1, Jak3, Lck, and
Lyn were comparatively studied in the presence and absence of
Man5GlcNAc2Asn in the medium. After culturing
the cells in the absence of IL-2 for 6 h, CTLL-2 cells in
G0 phase were stimulated with rhIL2 (10 units/ml) at 37 °C for 30 min in the presence or absence of
Man5GlcNAc2Asn (10 µM). Then, the
cells (1 × 107 cells/lane) were solubilized and
proteins in the lysates were immunoprecipitated with anti-Jak1,
anti-Jak3, anti-Lck, or anti-Lyn antibody. Tyrosine-phosphorylated
proteins were identified by immunoblotting with an antiphosphotyrosine
monoclonal antibody, 4G10 (anti-Tyr(P)). As shown in Fig.
2, proteins in lysates of CTLL-2
cells in G0 phase (lanes 1, 4, 7, and
10), lysates of IL-2-treated cells (lanes 3, 6, 9, and 12), and lysates of cells incubated with IL-2 in
the presence of Man5GlcNAc2Asn (lanes 2, 5, 8, and 11) were immunoprecipitated with anti-Jak1
(lanes 1-3), anti-Jak3 (lanes 4-6), anti-Lck
(lanes 7-9), and anti-Lyn antibody (lanes 10-12). The levels of phosphorylated Jak1, Jak3, Lck, and Lyn, which were detected by the phosphotyrosine-specific 4G10 antibody, were
increased in IL-2-induced cells (lanes 3, 6, 9, and
12) as compared with cells in G0 phase
(lanes 1, 4, 7, and 10). In contrast, phosphorylation of these tyrosine kinases in cells stimulated with IL-2
in the presence of Man5GlcNAc2Asn was
exclusively reduced (lanes 2, 5, 8, and 11).
These results indicate that the carbohydrate recognition function of
IL-2 modulates signal transduction through Jak1, Jak3, Lck, and Lyn
linked to IL-2 receptor subunits
and
.
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Fig. 2.
The inhibitory effect of
Man5GlcNAc2Asn on the phosphorylation of Jak1,
Jak3, Lck, and Lyn kinases. CTLL-2 cells were incubated in the
absence of IL-2 for 6 h and then further incubated without
stimulation (lanes 1, 4, 7, 10), or stimulated
with 10 units/ml IL-2 (lanes 3, 6, 9, and 12) or
10 units/ml IL-2 in the presence of 10 µM
Man5GlcNAc2Asn (lanes 2, 5, 8, and
11) for 20 min. Immunoprecipitation was performed using anti-Jak1
antibody (lanes 1-3), anti-Jak3 antibody (lanes
4-6), anti-Lck antibody (lanes 7-9), and anti-Lyn
antibody (lanes 10-12). Following SDS-PAGE and transfer to
a nitrocellulose membrane, the immunoblot was probed with the
antiphosphotyrosine (Tyr(P)) monoclonal antibody 4G10. The blot was
stripped and reprobed with anti-Jak1 antibody (lanes 1-3),
anti-Jak3 antibody (lanes 4-6), anti-Lck antibody
(lanes 7-9), and anti-Lyn antibody (lanes
10-12). M5 indicates
Man5GlcNAc2Asn.
,
, and
(IL-2R
, -
, and -
) (3), the soluble IL-2R
, -
, and -
independently show low affinity binding to IL-2. That is, the
-subunit binds IL-2 with low affinity
(Kd ~ 10 nM), the
-subunit binds
IL-2 with very low affinity (Kd ~ 100 nM), and the
-subunit has no measurable affinity for
IL-2 (8-10). However, as soon as IL-2 forms the high affinity complex with the IL-2R
, -
, and -
subunits, cellular signaling is
triggered. If a lectin-like interaction between IL-2 and a specific
glycoprotein is the trigger for formation of the high-affinity receptor
complex, a specific glycoprotein having
Man5-6GlcNAc2 should be co-immunoprecipitated with the IL-2 receptor complex in the lysates of IL-2-stimulated CTLL-2
cells using antibody against the IL-2R
, -
, or -
subunit. To
detect such a glycoprotein containing
Man5-6GlcNAc2 in these immunoprecipitates,
we used G. nivalis agglutinin which specifically recognizes
high-mannose type glycans (14). CTLL-2 cells (1 × 107
cells/lane) which had been continuously cultured in the presence of
IL-2 were solubilized with the lysis buffer containing 0.1% SDS, 0.5%
deoxycholate, and 1% Nonidet P-40, the proteins were immunoprecipitated with anti-IL-2R
, anti-IL-2R
, or anti-IL-2R
antibody, and each of the immunoprecipitates was fractionated by
polyacrylamide gel electrophoresis on a 10% acrylamide gel and blotted
onto nitrocellulose membranes. The membranes were then treated with
anti-IL-2R
, anti-IL-2R
, anti-IL-2R
, anti-Lck, anti-Lyn,
anti-Jak1, or anti-Jak3 antibody. Although antibody against each
subunit of IL-2R was used for immunoprecipitation, all
immunoprecipitates showed a 55-kDa band corresponding to IL-2R
upon
staining with anti-IL-2R
, a 75-kDa band corresponding to IL-2R
upon staining with anti-IL-2R
, a 64-kDa band corresponding to
IL-2R
upon staining with anti-IL-2R
, a 56-kDa band corresponding to Lck upon staining with anti-Lck, a 115-kDa band corresponding to
Jak1 upon staining with anti-Jak1 kinase, a 115-kDa band corresponding to Jak3 upon staining with anti-Jak3, and a 56-kDa band corresponding to Lyn upon staining with anti-Lyn antibody (3) (Fig.
3A). These results indicated
that all of the immunoprecipitates obtained with anti-IL-2R
, -
,
or -
antibody in analysis of CTLL-2 cells exposed to IL-2 consisted
of the IL-2R complex which at least included IL-2R
, -
, and -
,
and the kinases Lck, Lyn, Jak1, and Jak3. In contrast, this complex
could not be observed in CTLL-2 cells incubated in the absence of IL-2
(data not shown).
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Fig. 3.
GNA staining of the IL-2R complex.
CTLL-2 cells (1 × 107 cells/lane) were solubilized
and proteins were immunoprecipitated with anti-IL-2R subunit
antibody (lanes 1, 4, 7, and 10-12),
anti-IL-2R
subunit antibody (lanes 2, 5, and
8) or anti-IL-2R
subunit antibody (lanes 3, 6,
and 9). Each immunoprecipitate was fractionated by
polyacrylamide gel electrophoresis on a 10% acrylamide gel, and
blotted onto a nitrocellulose membrane. A, each immunoblot
was treated with antibodies against IL-2R
, IL-2R
, IL-2R
, Jak1,
Jak3, Lyn, or Lck kinase. B, each immunoblot was stained
with biotinylated GNA (lanes 4-6) and reprobed with
anti-IL-2R
subunit antibody (lanes 7-9). C,
immunoblots treated with anti-IL-2R
subunit antibody digested with
Endo H (lane 11), Endo D (lane 12), or
nondigested (lane 10) were stained with biotinylated
GNA.
, -
, and -
had been fractionated by polyacrylamide gel electrophoresis on a 10% acrylamide gel and blotted onto
nitrocellulose membranes, the membranes were stained with biotinylated
GNA which rather specifically recognizes
Man5GlcNAc2Asn (14), to detect the constituent
to which IL-2 can bind through its carbohydrate recognition site. Since
only a single 55-kDa band corresponding to the IL-2R
subunit was
stained in each instance, the membranes were reprobed with
anti-IL-2R
subunit antibody. As shown in Fig. 3B, a
protein band in the same position as the GNA-stained protein band was
positively stained with anti-IL-2R
subunit antibody. Furthermore,
although only the IL-2R
subunit was immunoprecipitated with
anti-IL-2R
subunit antibody in the case of CTLL-2 cells incubated in
the absence of IL-2, the same constituent of the immunoprecipitate was
positively stained with GNA (data not shown). When each blot was
treated with Endo H (Fig. 3, lane 11) or Endo D (Fig. 3,
lane 12), the band positively stained with GNA (Fig. 3,
lane 10) was diminished to 3% (Endo H) or 15% (Endo D),
calculated on the basis of the intensity of chemiluminescence (Fig.
3C). Since Endo H hydrolyzes high-mannose type glycans
including Man4-9GlcNAc2 and hybrid-type
glycans (23), whereas Endo D hydrolyzes
Man3-5GlcNAc2 (15, 24) and since
IL-2-dependent proliferation of CTLL-2 cells was inhibited
by the addition of Man5GlcNAc2Asn or
Man6GlcNAc2Asn, the carbohydrate structure of
IL-2R
to which IL-2 binds appears to include
Man5GlcNAc2. These results suggest that only
the IL-2R
subunit has the high-mannose type glycan with
Man5GlcNAc2, among the components of the IL-2R
complex in CTLL-2 cells, and that IL-2 bifunctionally binds a
high-mannose type glycan and a specific peptide sequence of IL-2R
,
although all of the subunits of IL-2R have several potential
N-glycosylation sites (25-27).
subunit recognizes
Lys35, Lys38, Thr42, and
Lys43 residues in IL-2 (28), another peptide sequence of
IL-2 may bind a high-mannose type glycan with
Man5GlcNAc2 which is linked to
Asn33, Asn43, or Asn200 of mouse
IL-2R
(25). As soon as IL-2 bifunctionally binds to the IL-2R
subunit, formation of the IL-2·IL-2R
complex might occur resulting
in a change in conformation of IL-2 which increases the accessibility
to the IL-2R
and IL-2R
subunits. This high-affinity complex of
IL-2R
, IL-2R
, and IL-2R
subunits may stimulate cellular signaling through tyrosine kinases including Jak1, Jak3, Lck, and Lyn.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
molecule is required to trigger
the formation of a high-affinity complex of IL-2-IL-2R
, -
, -
,
and that Man5GlcNAc2Asn or
Man6GlcNAc2Asn in the medium inhibits not only
IL-2-dependent CTLL-2 proliferation but also tyrosine
phosphorylation of Jak1, Jak3, Lck, and Lyn. Furthermore, the
IL-2-IL-2R
, -
, and -
complex immunoprecipitated with
anti-IL-2R
, -
, or -
antibody contains GNA-stainable IL-2R
,
suggesting that bifunctional binding of IL-2 to
Man5GlcNAc2 and a specific peptide sequence in
the IL-2R
molecule immediately leads to formation of the
high-affinity complex of IL-2-IL-2R
, -
, and -
, which subsequently induces tyrosine phosphorylation of IL-2R
and
linked to Jak1, Jak3, Lck, and Lyn. Our results indicate that IL-2R
is a candidate glycoprotein for IL-2 lectin-like binding in
vivo, and as soon as the tetramer including IL-2-IL-2R
, -
, and -
is tightly formed, the tyrosine kinases linked to
intracellular domains of IL-2R
and -
are immediately
phosphorylated and induce cellular signaling. On the basis of the
results described, we propose a tentative schematic model as shown in
Fig. 4, although the binding site of Lyn
has not been determined to be the IL-2R
or -
subunit.
View larger version (30K):
[in a new window]
Fig. 4.
A tentative schematic model of the formation
of the high-affinity IL-2·IL-2R ,
-
, or -
complex
triggered by recognition of Man5GlcNAc2 in the
IL-2R
subunit.
In our investigation of the inhibitory effects of
Man5GlcNAc2Asn and
Man6GlcNAc2Asn on IL-2-dependent
cell proliferation, the 2-h preincubation time before addition of the
mixture to the cells was found to be critical and exogenous
Man5GlcNAc2Asn added to the mixture could not
replace the glycan bound to IL-2. As soon as the high-mannose type
glycan linked to the extracellular domain of the IL-2R subunit binds
to IL-2, it seems that IL-2 binds a specific region of the IL-2R
subunit and this dual recognition is too strong to be replaced by
exogenous Man5GlcNAc2Asn. On the basis of the
experimental results, we speculate that the conformation of
carbohydrate-bound IL-2 may immediately change to fit with a specific
peptide sequence in IL-2R
and formation of the IL-2·IL-2R
complex may be a trigger to form the high-affinity complex which consists of all constituents required for the cell signaling to occur.
This may be the reason why large amounts of exogenous
Man5GlcNAc2Asn cannot substitute for the
endogenous glycoprotein, and why inhibitory effects of oligomannosides
on the T-cell proliferative response to IL-2 have not been reported
until today. To our knowledge, this is the first report to directly
demonstrate that carbohydrate recognition activity is essential for
stimulation of IL-2-dependent T-cell proliferation and
cellular signaling. Anyway, it indicates that IL-2 bifunctionally
recognizes both a high-mannose type glycan and a specific peptide
sequence in IL-2R
, and the sequential binding to IL-2R
and -
subunits is necessary for expression of IL-2-induced cellular signal transduction.
Similar dual recognition of protein and carbohydrate epitopes has
been reported in the case of several proteins. P-selectin is one of the
members of the selectin family which can mediate the initial
rolling interaction between leukocytes and vascular endothelium. As
all members of the selectin family can bind to related fucosylated or
sialylated tetrasaccharide structures, such as
sialyl-Lewisx or sialyl-Lewisa, P-selectin can
bind to P-selectin glycoprotein ligand 1 which has sialyl
Lewisx-type structures on the O-linked glycan
(29). Additionally, P-selectin glycoprotein ligand 1-P-selectin binding
requires the sulfotyrosine residues located within the region
consisting of the first 19 amino acids, although E-selectin can bind to
P-selectin glycoprotein ligand 1 without the sulfotyrosine residues
(30, 31). Specific glycosyltransferases need to bind not only
carbohydrate epitopes but also a specific peptide sequence, as follows.
For example, UDP-GlcNAc:lysosomal enzyme
N-acetylglucosamine-1-phosphotransferase is indispensable
for the biosynthesis of phosphomannosyl residues on lysosomal enzymes
which mediate their binding to mannose 6-phosphate receptors and which
mediate targeting to an endosomal compartment where the hydrolases are
subsequently packaged into lysosomes. Selective transfer of
N-acetylglucosamine-1-phosphate to mannose residues on
lysosomal enzymes by this enzyme involves the dual recognition of
mannosyl residues and the carboxyl lobe of the lysosomal hydrolase
cathepsin D which is shared among lysosomal hydrolases (32). GalNAc
transferase, responsible for the formation of
SO4-GalNAc1,4GlcNAc
1,2Man on the glycoprotein
hormones lutropin and thyrotropin, etc., recognizes both
N-acetylglucosaminyl residues and the peptide motif
Pro-Xaa-(Arg/Lys) present in each of these glycoproteins (33). These
reports suggest that these are members of an emerging family of binding
proteins with specificity for both protein and carbohydrate epitopes.
On the basis of the mechanisms of carbohydrate recognition
involved, cytokines have been grouped into three types to date. It has
been determined that growth factors including granulocyte-macrophage colony-stimulating factor (34) and bovine fibroblast growth factor (35) recognize glycosaminoglycans, interleukin-1 binds the mannose 6-phosphodiester in
glycosylphosphatidylinositol-anchored glycoprotein (36), and
IL-2 recognizes high-mannose type glycans. However, whether other
cytokines have strict carbohydrate recognition ability should be more
precisely investigated. Cytokines have not been reported to have a
common carbohydrate recognition domain. However, IL-2 has a limited
degree of sequence homology in the amino-terminal portion compared with
the COOH-terminal domains of three C-type mannose binding lectins (11).
In preliminary experiments, since we found that at least one of the
conserved amino acid residues was involved in carbohydrate recognition
by IL-2, the further confirmation will be required in the near future.
Zanetta et al. (12) previously reported that IL-2 binds a
glycosylated CD3 of TCR which is linked to the Lck kinase in human peripheral lymphocytes. However, we could not find any TCR subunit when we immunostained the IL-2-IL-2R
, -
, -
, Lck, Lyn, Jak1, and Jak3 complex, which was immunoprecipitated with
anti-IL-2R
, -
, or -
antibody in lysates of CTLL-2 cells
exposed to IL-2, using anti-TCR
subunit antibody (data not
shown). In contrast, the immunoprecipitates obtained with anti-TCR
subunit antibody from lysates of CTLL-2 cells exposed to IL-2
did not include any IL-2R
, -
, or -
as determined by
immunostaining (data not shown). These results also support the view
that the IL-2R
subunit itself is a glycoprotein containing the
carbohydrate recognition site of IL-2 in murine CTLL-2 cells. Which of
the three N-glycosylation sites in murine IL-2R
has the
carbohydrate to which IL-2 binds will be determined in the near future.
We are further investigating whether IL-2 has another carbohydrate
recognition mechanism as proposed by Zanetta et al. (12) in
the case of human peripheral lymphocytes and whether the human IL-2R
subunit has high-mannose type N-glycans to which IL-2 can bind.
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ACKNOWLEDGEMENTS |
---|
We thank H. Ideo and Y. Kanaya for technical assistance.
![]() |
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.
To whom correspondence should be addressed: Dept. of Biochemistry,
Sasaki Institute, 2-2 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel.: 81-3-3294-3286; Fax: 81-3-3294-2656; E-mail: yamashita@sasaki.or.jp.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M008781200
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ABBREVIATIONS |
---|
The abbreviations used are:
IL-2, interleukin-2;
Man, D-mannose;
GlcNAc, D-N-acetylglucosamine;
Asn, asparagine;
IL-2R, IL-2 receptor;
Endo H, endo--N-acetylglucosaminidase H;
Endo D, endo-
-N-acetylglucosaminidase D;
GNA, Galanthus nivalis agglutinin;
rhIL-2, recombinant human
interleukin-2;
PAGE, polyacrylamide gel electrophoresis;
TCR, T-cell receptor;
Fmoc, N-(9-fluorenyl)methoxycarbonyl.
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