From the Laboratoire de Chimie Biologique Université des Sciences et Technologies de Lille, CNRS Unité Mixte de Recherche 8576 Glycobiologie Structurale et Fonctionnelle, 59655 Villeneuve d'Ascq cedex, France
Received for publication, September 21, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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A method was developed for the determination of
putative lectin activities of cytokines. It involved the immunoblotting
measurement of the quantity of these cytokines unbound to a series of
different immobilized glycoconjugates and displacement of the bound
cytokines with oligosaccharides of known structures. This method allows demonstrating that the following interleukins specifically recognize different oligosaccharide structures in a calcium-independent mechanism: interleukin-1 Cytokines are modulators of the activity of the immune system,
their mechanism of action remaining, for the most part, not decrypted. As a general rule, the action of a cytokine results from its
binding to membrane receptors, a series of molecules coupled to
signaling systems involving kinases and/or phosphatases (1-5). The
binding of a cytokine to its receptor(s) generally results in the
phosphorylation/dephosphorylation of the intracytoplasmic domain of the
receptor(s), the first step of the signaling. In general, the
phosphorylation/dephosphorylation mechanism is cell type-specific, the
kinases/phosphatases involved in these processes being quite
specifically associated with surface molecular complexes different from
the cytokine receptor complex (3, 6). Even when two different cytokines
use the same receptor, the signal transduction pathways may be specific
of the cytokines (7).
We made the hypothesis that the specific association of interleukin
receptors with other surface complexes could be due to carbohydrate-binding properties of these cytokines, a property already
suggested in the literature (8-14). The lectin activity of
interleukin-2 (IL-2)1 for
specific oligomannosides (15) appeared to be essential, because IL-2
behaves as a bifunctional molecule able to extracellularly associate
its Biochemicals--
Recombinant human cytokines (produced in
bacteria) and their respective polyclonal rabbit antibodies were from
Chemicon International Inc. (Temecula, CA).
Alkaline-phosphatase-labeled anti-rabbit IgG and normal goat serum were
from Sigma. Ovalbumin, ribonuclease B (from bovine pancreas),
fetuin, and glycosaminoglycans (chondroitin sulfate A, B, and C,
heparan sulfate, heparin, dermatan sulfate, and hyaluronic acid) were
from Sigma. Bovine lactotransferrin was from Euromedex
(Souffelweyersheim, France). Equine and ovine submaxillary mucins were
prepared according to Tettamanti and Pigman (18), and Bufo
bufo egg mucins were prepared according to Morelle and Strecker
(19). Gangliosides were extracted from young rat cerebella as
previously described (20). Neutral glycolipids were extracted from
human meconium by Folch extraction and Folch partition followed by
separation by silica gel chromatography (21). Asialo-GM1
was prepared by limited acid hydrolysis (aqueous formic acid, pH 2.0, during 30 min at 50 °C) followed by a Folch partition. Cerebrosides
and sulfatides were isolated from the rat cerebellum (phospholipids
were eliminated using mild alkaline methanolysis). Sciatic nerve
extracts were obtained by homogenization of freshly dissected sciatic
nerves from adult Wistar albino rats in 1% SDS at the concentration of
10 mg of protein/ml. Myelin-associated glycoprotein (MAG) was purified
from adult rat brain myelin (22) according to Quarles et al.
(23). The P0 glycoprotein was prepared from rat sciatic
nerve myelin according to Kitamura et al. (24). Oligosaccharides were isolated from different sources, and their structures were determined by NMR as previously described (19, 25-28).
Glycopeptides were obtained from different glycoproteins by extensive
Pronase digestion followed by Biogel P2 chromatography (29).
Nitrocellulose (0.45-µm pore size) was from Schleicher & Schüll. GC/MS analyses of monosaccharide, long-chain bases, and
fatty acid derivatives liberated by acid methanolysis were performed as
heptafluorobutyrate derivatives as previously described (30). Sialic
acids liberated after mild acid hydrolysis (2 M acetic
acid, 105 min, 80 °C) were methyl-esterified using diazomethane followed by formation of heptafluorobutyrate
derivatives.2 Volatile
compounds were analyzed using a Carlo Erba GC 8000 apparatus coupled to
a Finnigan Automass II MS apparatus. Analyses were performed routinely
in the electron impact mode or, when necessary, in the chemical
ionization mode in the presence of NH3 gas and detection of
positive or negative ions as previously described (30-32).2
Immobilization of Glycoconjugates on Plastic
Microwells--
In routine screening experiments, the following
classes of glycoconjugates were immobilized to plastic microwells: 1)
fetuin, a glycoprotein containing N- and
O-glycans but presenting a large microheterogeneity
concerned with the degree of sialylation of its N-glycans
and its different O-glycans, differently bound sialic acid
residues, and minor glycans with unknown structures (33); 2) a mixture
of bovine RNase B and of bovine lactotransferrin (bLTF)
containing oligomannosidic N-glycans (with 5 and 6 mannose residues as major glycans for RNase B (34) and with 8 and 9 mannose
residues for bovine lactotransferrin, but for the latter, minor
biantennary complex-type N-glycans (35)); 3) ovalbumin, a
glycoprotein presenting mostly oligomannosidic N-glycans and hybrid-type N-glycans with a very large structural
microheterogeneity (36); 4) a mixture of mucins, the ovine and the
equine submaxillary mucins (rich in different sialic acid residues,
including O-acylated sialic acids (37)), and the mucins from
the eggs of B. bufo (rich in fucosylated
oligosaccharides (19, 27)); 5) a glycosaminoglycan mixture containing
chondroitin sulfate A (4-sulfate), C (6-sulfate), and B (dermatan
sulfate), keratan sulfate, heparan sulfate, heparin, and hyaluronic
acid; 6) a sciatic nerve SDS extract, this material contains special
glycolipids (cerebrosides and sulfatides and glycolipids bearing the
HNK-1 epitope (glucuronic acid-3-sulfate (38, 39))), glycoproteins
possessing different glycans including those having the HNK-1 epitope
(40, 41), and different proteoglycans; 7) a total mixture of
gangliosides isolated from young rat cerebella in mild conditions
(absence of alkaline treatment), this material contains 28 different
sialylated glycolipids corresponding essentially, but not exclusively,
to compounds of the ganglio series (20); and 8) a mixture of neutral
lipids from the human meconium (containing especially globosides and
blood group substance glycolipids (42)), supplemented with
galactosylceramides, sulfatides, and asialo-GM1 (see above).
Glycoproteins and glycosaminoglycans were dissolved in PBS (25 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl), each at the concentration of 1 mg/ml. Sciatic
nerve extract was dissolved (1 mg of protein/ml) in PBS containing
0.1% SDS. Gangliosides and glycolipids were dissolved in methanol (10 µg of hexose/ml of each compound). 50 µl of the previous mixtures
were added to the wells and put in an oven at 50 °C until dried.
After immobilization, the microwells were washed thrice with 300 µl
of PBS and then saturated thrice during 2 h with periodate-treated
BSA (pBSA). As reported by Glass et al. (43), the periodate
treatment followed by elimination of excess of periodate with glycerol
of BSA, reduced by a factor 100 the quantity of lectin necessary to
reveal specific ligands because of the absence of binding to
oligosaccharide impurities of BSA (44). Wells were washed with PBS and
then water and dried at room temperature. The quantity of bound
glycoconjugates was calculated from the amount of hexose equivalents
determined directly in the microwells using the resorcinol sulfuric
acid micromethod (45) relative to dilutions of a galactose solution
treated in parallel. Measurements of the absorbance were performed
using a Bio-Rad model 550 microplate reader at 430 nm and applying the coefficient of relative absorbance as reported by the previous authors
(45). A mean value of 0.5 µg galactose equivalent was found for
fetuin, ovalbumin, OSM, gangliosides, neutral glycolipids, and RNase B,
1.2 µg for equine submaxillary mucin and the mucin of B. bufo, and 1.5 µg for the mixture of glycosaminoglycans. These
quantities of bound glycans remained the same after incubations with
the cytokines as determined in the wells after recovery of the supernatant.
Detection of the Binding of Cytokines to Immobilized
Glycoconjugates--
The wells containing the immobilized
glycoconjugates were saturated once as above, washed thrice in PBS, and
then supplemented with 50 µl of PBS containing 0.3% pBSA and 5 mM EDTA. Cytokines (0.1-0.5 µg in 5 µl of PBS) were
added, and the plates were incubated for 2 h at room temperature
with a rotatory agitation. The supernatants were carefully recovered
and diluted with 25 µl of the Laemmli dissociating buffer (46).
Samples were placed for 15 min in boiling water. Identical volume
aliquots (10-25 µl) corresponding to the same cytokine were
submitted to polyacrylamide gel electrophoresis (13% acrylamide) in
the presence of SDS (46) followed by blotting onto nitrocellulose (47).
After saturation with 3% BSA in PBS containing a 1/20 dilution of
normal goat serum, blots were revealed using the proper polyclonal
anti-cytokine antibody followed by the alkaline phosphatase-labeled
second antibody and then staining with the nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (48). The
binding of a cytokine to one or several glycoconjugate mixtures was
evidenced by its decreased amount on blots. For quantitation, blots
were scanned using a Scanjet 4c (Hewlett Packard), and integrations were performed using the Quantiscan 32 software. Because for most cytokines the polyclonal antibodies were allowed to detect down to 10 ng of cytokines, optimal results (difference in intensity between
samples for which a binding was observed and the others) were obtained
using 50 to 100 ng of cytokines in each incubation. All determinations
were performed in triplicate. Incubations in the presence of 0.3% pBSA
also reduced nonspecific binding. However, the very high amount of pBSA
produced specific electrophoretic artifacts; these were frequently
curving of the electrophoretic front and presence of a double band for
a single compound. These artifacts could be substantially reduced but
not eliminated by decreasing the electric power of the electrophoresis
during the concentration step of the sample.
Determination of the Specificity of the Cytokine-Glycoconjugate
Interactions--
Once a binding of a cytokine to a specific mixture
was observed, a refinement of the nature of the interaction was
performed. The first step consisted in studying the binding of the
cytokine to individual glycoconjugates present in the mixture (for
example separated RNase B and bLTF for IL-1 The rationale of the method used for studying the lectin
activities of interleukins was to detect the cytokines unbound to different immobilized glycoconjugates with known glycan structures using an immunoblotting technique as schematized in Fig.
1. The reasons for choosing such a method
were as follows: 1) because cytokines could loose their
carbohydrate-binding properties upon chemical or radiochemical labeling
(49), we used unlabeled recombinant human cytokines having a preserved
biological activity; 2) because no precise carbohydrate-binding
properties of most cytokines were found in the literature, we studied
the binding of cytokines to a large variety of glycoconjugates or
mixtures of glycoconjugates, the structure of their glycans being in
large part determined; and 3) because the presence of calcium (above 1 mM in Tris-buffered saline) induced a nonspecific fixation
of all interleukins to all immobilized glycoconjugates, the experiments
were performed in the presence of 5 mM EDTA. Consequently,
the data reported in this manuscript were concerned only with
Ca2+-independent carbohydrate-binding properties.
binds to the biantennary disialylated N-glycan completed with two Neu5Ac
2-3 residues;
interleukin-1
to a GM4 sialylated glycolipid
Neu5Ac
2-3Gal
1-Cer having very long and unusual long-chain bases;
interleukin-4 to the 1,7 intramolecular lactone of
N-acetyl-neuraminic acid; interleukin-6 to compounds having
N-linked and O-linked HNK-1-like
epitopes; and interleukin-7 to the sialyl-Tn antigen. Because the
glycan ligands are rare structures in human circulating cells, it is
suggested that such activities could be essential for providing
specific signaling systems to cells having both the receptors and the
oligosaccharide ligands of the interleukin at their cell surface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor (IL-2R
) to other surface receptor complexes bearing
N-glycans recognized by IL-2. This is the case for the CD3·TCR complex in which a N-glycosylated form of
CD3 is an IL-2 ligand. This specific extracellular association is
responsible for the specific phosphorylation of the IL-2R
by the
CD3·TCR-associated kinase p56lck (15), considered as a
first step in the antigen-specific activation process of
CD4+ T cells. As a consequence of this carbohydrate-binding
property, it was suggested that oligomannosides accumulated in specific diseases or bound to specific microorganisms could alter this essential
role of IL-2 (16, 17). Because several groups reported lectin-like
activities for cytokines (8, 10-13), we undertook a study of such
properties for recombinant human interleukins. This work demonstrates
that IL-1
, IL-1
, IL-4, IL-6, and IL-7 recognize very specific
oligosaccharide ligands in a calcium-independent mechanism. The
implications of these findings are discussed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or individual mucins for IL-4 and IL-7 or purified glycoproteins for IL-6 or purified
gangliosides for IL-1
). When a specific binding to one of these
compounds was observed, attempts were made to inhibit this binding
first with mixtures of oligosaccharides isolated from the
glycoconjugate ligand and then with separated individual
oligosaccharides. In crude inhibition assays, oligosaccharides were
added to the wells at a concentration of 10 µg/ml in PBS for
mixtures, or 1 µg/ml in PBS for purified compounds, prior to addition
of the cytokine. For testing the efficiency of the different inhibitory
glycans, serial 10-fold dilutions of these compounds in PBS were performed.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Illustration of the methodology used for the
screening of the lectin activities of the interleukins. As the
initial step, various glycoconjugates are immobilized on plastic
microwells. After saturation with pBSA, cytokines are incubated in
wells in the presence of pBSA. Supernatants are carefully recovered and
submitted to SDS polyacrylamide gel electrophoresis and blotting onto
nitrocellulose (a). Note the large amounts of pBSA used for
eliminating nonspecific interactions with glycoconjugates that could in
some cases perturb electrophoresis. Blots are then revealed using the
proper anti-cytokine primary antibody followed by alkaline
phosphatase-labeled anti-rabbit secondary antibody and then staining
with the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate
reagent (b). For example in b, compare lane
1 (control) and lane 4 (coincubation with a high
affinity ligand of the cytokine). Note an increased amount of the
cytokine in the supernatant, thus indicating an inhibition of the
fixation of the cytokine to the glycoconjugate.
Interleukin 1 Is a Lectin Specific for Disialylated Biantennary
N-Glycans with Two
2,3-Linked Sialic Acid Residues--
As shown in
Fig. 2a, IL-1
binds
significantly to fetuin and to the mixture of RNase B and bLTF, whereas
no binding was observed to the other immobilized glycoconjugates. These
data were surprising, because the major glycans of RNase B and bLTF
were of the oligomannosidic type, in contrast with fetuin, which
contains essentially sialylated O- and N-glycans.
When incubations were performed separately on RNase B and bLTF, the
binding of IL-1
was observed only to the latter. From the quantity
of IL-1
bound to bLTF, it could be calculated that IL-1
binding
sites were present on about 7% of the bLTF molecules, a result that
indicated that the ligands of IL-1
corresponded to minor glycans of
this molecule, as they were minor glycans of fetuin. In fact, the
O-glycan fraction obtained from fetuin after reductive
-elimination and Biogel P2 chromatography, as well as the two major
O-glycans of fetuin, were devoid of inhibitory activity, in
contrast with the N-glycan-enriched fraction obtained by
extensive Pronase digestion of fetuin. However, the purified major
triantennary N-glycan of fetuin and the products obtained after its partial or complete desialylation did not show any inhibitory activity. Because bLTF possessed minor biantennary N-glycans
with the Neu5Ac
2,6GalNAc
1,4GlcNAc sequence (35), we tested the inhibitory activity of this compound. As for the previous ones, no
significant inhibitory activity was obtained at the 10
4
M concentration range. We therefore tested a variety of
biantennary oligosaccharides isolated from the urine of patients with
sialidoses (25, 26). The one containing two
2,6-linked Neu5Ac
residues was ineffective at 10
4 M, whereas
the binding of IL-1
was completely inhibited using 10
6
M of the biantennary N-glycan containing two
2,3-linked Neu5Ac residues (Fig. 2, b and c).
A quasi-equimolar mixture of the two isomers of biantennary
N-glycans containing
2,3- and
2,6-linked Neu5Ac
residues showed a weak inhibition only at the 10
4
M concentration range. Therefore, it was concluded that
IL-1
is a calcium-independent lectin endowed with a higher affinity for the biantennary N-glycan having the
Neu5Ac
2,3Gal
1, 4GlcNAc
1,2 sequence on its two branches (Fig.
2d).
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Previous authors (8) reported that IL-1 was able to bind to
uromodulin, a urine glycoprotein rich in sialylated
N-glycans, the binding being inhibited by the bulk of
glycopeptides isolated by Pronase digestion of fetuin. These authors
suggested that IL-1
was a lectin specific for the major glycan of
fetuin, the trisialylated triantennary N-glycan. Our
experiments demonstrated that the inhibitor of the interaction was not
the previous glycan but the biantennary N-glycan with two
2,3-linked Neu5Ac residues. 50% inhibition of the binding to fetuin
was obtained using 0.5-1 µM of this compound, i.e. 100-200-fold higher efficiency than the
total N-glycans of fetuin. The reason why the biantennary
N-glycan was a high affinity ligand, whereas
2,3-sialyl-lactose or the mixture of the linear monoantennary
2,3-sialylated glycan isolated from patients with sialidosis (25,
26) was ineffective, remained speculative. This might be related to the
tendency of IL-1
to associate into dimers, with each subunit being
able to bind one of the branches of the biantennary
N-glycan. Such a specifically increased affinity upon
formation of oligomers of lectins was previously demonstrated for the
asialo-fetuin receptor (50).
IL-1 Is Likely a Lectin Specific for the GM4
Glycolipid--
IL-1
binds only significantly to the fraction
containing the mixture of gangliosides isolated from the rat cerebellum
(Fig. 3a). Because these
gangliosides were previously fractionated according to their charges by
ion-exchange chromatography (20), we examined the binding of IL-1
to
the different classes of mono-, di-, tri-, and tetrasialo-gangliosides,
respectively. Only the monosialo-ganglioside fraction showed a binding
of IL-1
(Fig. 3b). The different constituents of the
fraction of monosialo-gangliosides isolated by preparative TLC (20)
were immobilized individually. As shown in Fig. 3c, IL-1
binds to a single fraction, previously identified as the sialylated
galactosylceramide GM4 (20).
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This binding was surprising, because GM4 is a very simple
sialylated glycolipid, Neu5Ac2,3Gal
1-Cer. It was therefore
expected that the binding could be inhibited by small oligosaccharides with similar structures. In fact, sialyl-lactoses (with
2,3- or
2,6-linked Neu5Ac) were without inhibitory effects at the concentration of 10
3 M. Because it was
described that IL-1
binds to glycolipids of GPI anchors (13), we
tested the possibility that the GM4 sample used in this
study was contaminated by GPIs. This possibility was ruled out using
GC/MS analysis of this GM4 sample (30, 31). Using a new
procedure allowing the complete liberation of all GPI
constituents,3 the analysis
showed the total absence of D-mannose, inositol, and GlcN,
which are essential constituents of GPIs. To further document this
point, the binding of IL-1
was tested to an immobilized glycosylphosphotydylinositol-ceramide isolated from
Tulamen (kindly provided by Drs. J. and L. Previato). No
binding was observed. Furthermore, the binding of IL-1
to
GM4 was not inhibited by Man-6-P at the concentration of
10
3 M, indicating that the binding to
GM4 observed here was unrelated to the binding to GPI
oligosaccharides (13).
The question remained to know how a compound with a simple carbohydrate
composition could be a specific ligand of a cytokine. Based on the
observations of Karlsson (51) on the influence of the lipid moiety on
the binding of microbe lectins, we made the hypothesis that part of the
specificity could be due to the lipid portion of the rat cerebellum
GM4. This question was answered using GC/MS analysis (30).
Although the fatty acid composition was classical (C16:0 and C18:0 in
equivalent amounts representing more than 98% of total fatty acids),
the long-chain base composition was unusual. Indeed, the linear C22:0
phytosphingosine (60%) and the linear C22:1 sphingenine (30%) were
the major constituents. A minor constituent (10%) was identified as
18-O-ethyl-C18:1 sphingenine. Although it was not
demonstrated that the long-chain base composition of the
GM4 was important for the binding of IL-1, this remained a stimulating possibility. Because of the lack of an inhibitor, the
lectin activity of IL-1
could not be ascertained as such. Nevertheless, the completely different behavior as lectins of IL-1
and IL-1
could explain why these interleukins, endowed with a common
receptor, have different biological functions (see "Discussion").
IL-4 Is a Lectin Specific for the 1,7 Intramolecular Lactone of
N-Acetyl Neuraminic Acid (Neu5Ac-1,7L)--
IL-4 binds strongly to the
mucin mixture (Fig. 4a). When
the binding was studied for the three different isolated mucins, IL-4
binds strongly to the mucin of the eggs of B. bufo and at a
lesser extend to OSM (Fig. 4b). The binding was not
inhibited using the mixtures of the acidic or neutral oligosaccharide
alditols isolated from the mucin by reductive -elimination (see Ref.
27 and Fig. 4c). This suggested two possibilities; either
the interaction was carbohydrate-independent, or an essential
determinant was lost during the alkaline treatment of the
-elimination procedure. In fact, the binding of IL-4 to the B. bufo mucin was eliminated when the mucin was first exposed to
NH3 gas for 72 h at room temperature, indicating that
an important determinant for the binding was an O-acyl
group, possibly located on a sialic acid residue of this mucin. This
was actually the case, because the binding of IL-4 to the mucin of the
eggs of B. bufo was inhibited using the sialic acids
liberated from the mucin by mild acid hydrolysis (Fig. 4c, arrow). Previous studies (27) showed that the sialic acids
detected in this mucin after the procedure of
-elimination were
Neu5Ac and Neu5Gc in equivalent amounts. Because the commercial
compounds were not inhibitory, it was concluded that the ligand of IL-4 was an O-acylated derivative of one of these two
compounds.
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When GC/MS analyses of the sialic acids present in this mucin were
performed,2 four major peaks were detected (Fig.
5). Two of them corresponded to Neu5Ac
and Neu5Gc, respectively, but Neu5Ac was a very minor constituent of
the mucin. In contrast, the sialic acids derived from the
oligosaccharide-alditols obtained by reductive -elimination showed
an equivalent ratio of Neu5Ac and Neu5Gc (27). Classical O-acetylated and O-lactylated sialic acids were
absent from this mucin. In contrast, the major peak corresponded to a
compound with a mass of 879 (as detected in the chemical ionization
mode, i.e. a deficit of mass of 228 relative to
Neu5Ac) suggesting that the major compound corresponded to a lactone
derived from Neu5Ac. Based on the fine fragmentation pattern of the
compound in the electron impact mode of ionization, it was concluded
that this compound was the 1,7 lactone of Neu5Ac (Fig. 4d).
The fourth peak corresponded to the 1,7 lactone of Neu5Gc. The
existence of such intramolecular lactones of sialic acids was
previously suggested in the literature but never obtained in sufficient
amounts to be identified with security.
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The problem remained to know whether the ligand of IL-4 in the B. bufo mucins was the 1,7 lactone of Neu5Ac or that of Neu5Gc or both. Because OSM, showing only a weak binding for IL-4, contains a significant amount of the 1,7 lactone of Neu5Ac but no trace of the corresponding lactone of Neu5Gc, it was concluded that the ligand of IL-4 in the B. bufo mucins could not be the 1,7 lactone of Neu5Gc but was the 1,7 lactone of Neu5Ac.
IL-6 Is a Lectin Specific for Specific Compounds with a Glucuronic
Acid-3-sulfate (HNK-1) Group--
As shown in Fig.
6a, IL-6 binds only to the
sciatic nerve extract, suggesting the presence of a unique ligand in
this heterogeneous fraction. We made the hypothesis that a possible
ligand was the HNK-1 epitope (SO3H (3-)GlcA) present both
on glycolipids and glycoproteins found in this tissue (52). IL-6 also
binds to the mucins isolated from the eggs of Rana
temporaria, also rich in glycans bearing this epitope (see Ref. 28
and Fig. 6b). Because the two major glycoproteins of rat
peripheral nervous system myelin (MAG and overall
P0) possessed this epitope, we immobilized these two
isolated glycoproteins on plastic and measured the binding of IL-6
relative to the other immobilized glycoconjugates. IL-6 actually binds
to these two isolated glycoproteins. The binding of IL-6 was not
inhibited by glycopeptides isolated from RNase B or from fetuin. In
contrast, the binding was inhibited using 104
M of a glycopeptide fraction obtained from the rat brain
synaptosomal fraction (53) in which the HNK-1 epitope was present as
minor constituents based on matrix-assisted laser desorption
ionization/time of flight analysis.4 Because a
series of reduced oligosaccharides possessing the SO3H (3-)GlcA motive were isolated from the mucins of the eggs of R. temporaria (28), we tested these compounds for their inhibitory activity of the binding of IL-6 to the rat P0 glycoprotein.
Interestingly, the best inhibitor was a compound comprising a Fuc
residue (see Ref. 28 and Fig. 6, c-e), whereas compounds
lacking the Fuc residue were not inhibitory at 10
4
M. The presence of an additional Gal residue also abolished
the inhibitory potency of the oligosaccharides (Fig. 6,
c-e). Because none of the other oligosaccharides lacking
the SO3H (3-)GlcA group were inhibitory (data not shown),
it was concluded that, although the SO3H (3-)GlcA motive
was necessary for the binding to IL-6, it was not sufficient.
Considering the structure already determined of the
N-glycans possessing the HNK-1 epitope (41) and the
structure of the O-glycans inhibiting the binding of IL-6 to
these N-glycans, it was suggested that the recognition
domain of IL-6 consisted in the sequence SO3H
(3-)GlcA
1,3Gal
1,4 followed by a monosaccharide sequence
possessing a methyl group in the vicinity of this structure (issued
either from the acetamido group in GlcNAc in N-glycans or
from the methyl group in the Fuc residue in R. temporaria
oligosaccharide alditols).
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IL-7 Binds Specifically to a Glycopeptide of the Ovine Submaxillary
Mucin--
As shown in Fig.
7a, IL-7 binds to fetuin and
the mixture of mucins and not to the glycosaminoglycan mixture, as
contrasted from the data of previous authors (12). When the analysis
was subsequently performed on the individual mucins, IL-7 binds only to
the ovine submaxillary mucin (Fig. 7b). The binding was not changed when the OSM was submitted to partial or total deacylation with
NH3 gas, indicating that O-acylation of sialic
acid residues was not important for the binding. In contrast this
binding was inhibited when the mucin was desialylated by mild acid
hydrolysis, indicating that a sialic acid residue was important for the
binding. However, this binding could not be inhibited using
104 M of purified Neu5Ac (the major sialic
acid present in OSM) or purified Neu5Gc or by the mixture of sialic
acids isolated from OSM by mild acid hydrolysis. Furthermore,
O-glycans isolated from OSM by a reductive
-elimination
procedure did not show any inhibitory activity. This suggested that the
presence of a GalNAc-OH instead of GalNAc residue abolished the binding
or that the binding of IL-7 involved a portion of the polypeptide
chain. These assumptions were sustained by the observation that complex
glycopeptide mixtures obtained by prolonged Pronase digestion of OSM
were inhibitory of the interaction of IL-7 with OSM. As shown in Fig.
7c, an active inhibitory compound was found as a low
Mr fraction isolated by Biogel P2
chromatography. This fraction contained a single resorcinol-positive spot migrating faster than the major monosialo-glycoserine of fetuin
Neu5Ac
2,3Gal
1,3GalNAc
1-Ser and containing one Ser, one GalNAc,
and one Neu5Ac residue (Fig. 7d). Consequently, these data
were compatible with the hypothesis that the IL-7 ligand was the
sialyl-Tn antigen, an onco-fetal antigen relatively abundant on the
ovine submaxillary mucin and comprising both an oligosaccharide and
peptide determinant (54).
|
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DISCUSSION |
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This paper focuses on a new methodology allowing the discovery of
lectin activities of cytokines. Because for most of cytokines no data
exist on the exact nature of the carbohydrate ligand, we undertook a
systematic screening of such carbohydrate-binding properties for
several interleukins including IL-1, IL-1
, IL-4, IL-6, and IL-7.
Similar studies have been undertaken for other cytokines or chemokines.
However, lectin activity has not been demonstrated for some of them,
possibly because we lack the specific ligand. The case of IL-4 is very
relevant to this view, because its lectin activity would have been
missed in the absence of the B. bufo mucin as a ligand.
Several authors have demonstrated that cytokines could loose their
lectin activity upon chemical or radiochemical labeling. Indeed, as
demonstrated for IL-2, the labeling of the cytokine by iodine or biotin
inhibited its biological function, although the binding to the receptor
was not affected (49). Thus, the principal interest of this technique
is to allow studies with proteins having a fully preserved biological
activity. The discovery of the high affinity ligand will permit the
labeling of the cytokine in the presence of the carbohydrate epitope to protect the carbohydrate recognition domain and leads to further investigations, like quantitative studies and identification of the
endogenous ligands of cytokines on cells or on blots.
We demonstrate that except for IL-1 (for which a soluble high
affinity oligosaccharide ligand was not yet isolated), the other
interleukins have high affinity oligosaccharide or glycopeptide ligands
inhibiting the binding of the cytokine to the immobilized ligands at a
concentration range lower than 10
6 M.
Although the Kd of the interaction could not be
determined using the present methodology, the identified ligands were
of high affinity. For example, the ligand of IL-6 from R. temporaria was able to inhibit the binding of IL-6 to MAG at
concentration as low as 10
10 M. This
suggested that if endogenous ligands are present on cells of the immune
system, the lectin activity of the human interleukins may provide
another targeting system different from the classical interleukin
receptors. Although high affinity ligands of these interleukins were
identified, the endogenous ligands of the different cytokines are still
not identified. They may be structurally different from (or at least
not identical to) the endogenous ligands but should share at least a
common determinant. For example, IL-6 binds to N-linked
HNK-1 epitope found on MAG and P0 glycoproteins, the
structure being HSO3 (3-)GlcA
1,3Gal
1,4GlcNAc
1,2
(44). The ligands found in R. temporaria had a significantly
different structure, HSO3
(3-)GlcA
1,3Gal
1,4[Fuc
1,2]Gal
1,3, whereas the linear oligosaccharide lacking the Fuc residue was inactive at 10
4 M. This suggested that, besides a site
for the SO3H (3-)GlcA determinant, the carbohydrate
recognition domain of IL-6 contains a domain interacting with a methyl
group provided either by the 2-acetamido group of GlcNAc or the methyl
group of Fuc, the HSO3 (3-)GlcA
1,3Gal
1,4 being not
sufficient for a high affinity interaction. For IL-4, the ligand, the
1,7 lactone of Neu5Ac, is a compound actually present in glycoproteins
of the human lymphocyte membrane.5
The question of the function of such lectin activities of cytokines
remains largely unanswered. As suggested by the function of the IL-2
lectin activity (15), an essential role in cytokine signaling is
expected, consisting in association of the cytokine receptor complex
with specific glycoprotein or glycolipid ligands of another surface
complex. As a consequence of this hypothesis, only cells having both
the receptor and the ligand of the cytokine could respond to the
cytokine. This could explain why different cytokines having the same
receptor can stimulate specifically certain cell types and not the
others. As an example, IL-1 and IL-1
have the same receptors but
have different signaling profiles in different cells. IL-1
, but not
IL-1
, is able to stimulate human astrocytes, a mechanism responsible
for the nervous regulation of fever (55). It is noteworthy considering
that astrocytes are the only cells of the central nervous system
producing IL-1
and possessing both IL-1 receptors (56) and the
GM4 glycolipid (57).
The experimental approaches to answer the question of the biological
function of the lectin activity of interleukins appear relatively
simple, because the binding of a cytokine to its receptor induces
specific changes in phosphorylation/dephosphorylation, as observed for
IL-2 (15). Indeed, the high-affinity oligosaccharide ligand should
inhibit the changes of phosphorylation/dephosphorylation induced by the
cytokine. This is actually the case for the IL-6 ligand from R. temporaria, because this compound at a 1010
concentration range inhibits the dephosphorylation of a few
tyrosine-phosphorylated proteins induced by IL-6 in a whole population
of resting human lymphocytes.6
Therefore, this study provides new concepts and tools
for understanding the mechanisms of the regulation of the human immune system and suggests that fine modulations of the immune system could be
obtained using specific oligosaccharides or glycans isolated in
consistent amounts from natural sources. These substances may be an
alternative to therapies using antibodies against interleukins or
interleukin receptors in different pathologies.
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ACKNOWLEDGEMENTS |
---|
We thank L. Poissonnier, C. Alonso, and V. Jactat for assistance and B. Coddeville for providing the biantennary bLTF N-glycan.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant 9925 of the French Association pour la Recherche sur le Cancer.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: Laboratoire de Chimie
Biologique USTL, CNRS UMR 8576, 59655 Villeneuve d'Ascq cedex, France.
Tel.: 33-03-20-43-40-10; Fax: 33-03-20-43-65-55; E-mail:
Jean-Pierre.Zanetta@univ-lille1.fr.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M008662200
2 J.-P. Zanetta, A. Pons, M. Iwersen, C. Mariller, Y. Leroy, P. Timmerman, and R. Schauer, submitted for publication.
3 A. Pons, J. Préviato, L. Préviato-Mendoza, P. Timmerman, Y. Leroy, and J.-P. Zanetta, manuscript in preparation.
6 C. Cebo, V. Durier, P. Lagant, E. Maes, G. Vergoten, and J.-P. Zanetta, manuscript in preparation.
7 J.-P. Zanetta, unpublished data.
4 J.-P. Zanetta, unpublished data.
5 C. Mariller, A. Pous, and J.-P. Zanetta, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: IL, interleukin; GPI, glycosyl-phosphatidylinositol; PBS, phosphate-buffered saline; BSA, bovine serum albumin; pBSA, periodate-treated BSA; bLTF, bovine lactotransferrin; OSM, ovine submaxillary mucin; MAG, myelin-associated glycoprotein; GC/MS, gas chromatography-mass spectrometry.
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