The Heparan Sulfate Binding Sequence of Interferon-
Increased the On Rate of the Interferon-
-Interferon-
Receptor
Complex Formation*
Rabia
Sadir
§,
Eric
Forest¶, and
Hugues
Lortat-Jacob
**
From the
Institut Pasteur de Lyon, CNRS URA 1459, ¶ Laboratoire de Spectrométrie de Masse, and
Laboratoire de Biophysique Moléculaire, Institut de
Biologie Structurale, CNRS UPR 9015, Avenue des Martyrs,
38027 Grenoble Cedex 01, France
 |
ABSTRACT |
Interferon-
(IFN
), in common
with a number of growth factors, binds both to heparan sulfate or
heparin-related molecules and to a specific high affinity receptor
(IFN
R). Using surface plasmon resonance technology, kinetic analysis
of the IFN
·IFN
R complex formation was performed with the
extracellular part of IFN
R immobilized on a sensor chip. At the
sensor chip surface, IFN
was bound by two IFN
R molecules with an
affinity in the nanomolar range (0.68 nM). This binding was
characterized by an important on rate, kon = 7.3 × 106
M
1·s
1, and an off rate,
koff = 5 × 10
3·s
1. This binding assay was used to
investigate a possible role of heparin in the IFN
·IFN
R complex
formation. In contrast to growth factors for which binding to heparin
is usually required for high affinity receptor interaction, we found in
this study that IFN
bound to heparin displayed a strongly reduced
affinity for its receptor. This is consistent with the fact that a
cluster of basic amino acids (KTGKRKR, called the C1 domain) in the
carboxyl-terminal sequence of the cytokine was involved both in heparin
and receptor recognition. To understand how a single domain of IFN
could be implicated in two discrete functions (i.e. binding
to heparin and to IFN
R), we also analyzed in a detailed manner the
role of the IFN
carboxyl-terminal sequence in receptor binding.
Using forms of IFN
, with carboxyl terminus truncations of defined
regions of the heparin binding sequence, we found that the C1 domain
functioned by increasing the on rate of the IFN
·IFN
R binding
reaction but was not otherwise required for the stability of the
complex. Interactions between the IFN
carboxyl-terminal domain and
IFN
R could increased the association rate of the reaction either by
increasing the number of encounters between the two molecules or by
favoring productive collisions. The mechanisms by which heparan sulfate regulates IFN
activity may thus include both control of selective protease cleavage events, which directly affect the cytokine activity, and also an ability to modulate the interaction of IFN
with the IFN
R via competitive binding to the C1 domain.
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INTRODUCTION |
Interferon-
(IFN
)1
is a highly pleiotropic protein secreted by activated T-lymphocytes and
natural killer cells (1). The active form of this cytokine is a
homodimer consisting of two intertwined 143-amino acid polypeptides.
Each monomer consists of six helices (denoted A-F), linked by loops,
and an unfolded sequence at the carboxyl-terminal side (downstream to
the F helix, amino acids 124-143), which extends away from the
molecule (2-4). IFN
mediates its pleiotropic activities through a
specific transmembrane receptor (IFN
R) expressed at the surface of
almost all cells (5). This receptor is composed of a ligand-binding
subunit, or
-chain (IFN
R
) and an accessory factor, or
-chain which is required for signal transduction (6, 7). The
IFN
·IFN
R
complex consists of two receptors bound to an
IFN
dimer, a stoichiometry consistent with the symmetry of the
ligand (8, 9). IFN
residues involved in receptor binding are
situated in two areas of the molecule (10, 11): the loop connecting the
A and B helices (residues 18-26) and the helix F (residues 108-124).
The carboxyl-terminal sequence (residues 124-143) is highly flexible and/or adopts multiple conformations (3, 4). It contains two small
clusters of basic amino acids (C1, residues 125-131, KTGKRKR; C2,
residues 137-140, RGRR), which confer on the molecule a important
sensitivity to a variety of proteases (12-15). As a consequence,
native IFN
is usually a mixture of carboxyl-truncated molecules
lacking up to 16 amino acids and therefore may end anywhere from
Gln143 (full-length molecule) to Gly127. Since
deletion of less than 10 amino acid increases the bioactivity of
IFN
, while more extended cleavages of the carboxyl-terminal domain
have the opposite effect, it has been speculated that the cytokine
activity can be modulated by limited proteolysis. The portion of the
carboxyl terminus that appeared to be the most important in this
process is the basic sequence KRKR of the C1 domain, since the
beginning of its removal correlated with loss of activity, while
cleavages downstream of this sequence resulted in the observed
increased activity (13). However, as revealed by the crystal structure
of the IFN
·IFN
R
complex, specific interactions between this
basic sequence of IFN
and IFN
R have not been identified, and the
carboxyl-terminal tail of the cytokine did not appear to be involved in
receptor binding (11). Furthermore, while both antibodies directed
against amino or carboxyl terminus block biological activity, only
synthetic peptides comprising the amino-terminal domain of the cytokine
block binding to its receptor in a competitive manner (15). In
addition, it has been shown that if a positive charge localized in the
carboxyl-terminal part of the cytokine is important for activity, it
can be contributed by a number of different combinations of positions.
Therefore, this part of the molecule did not appear to function in a
specific manner, and its function remains unclear (16). The
carboxyl-terminal domain of IFN
also confers on the molecule a high
affinity (Kd = 1.5 nM) for heparan
sulfate (17) or heparin-related molecules (18). A fragment of heparan
sulfate that displays high affinity for IFN
has been isolated. It
consists of two small sulfated heparin-like sequences linked together
by an extended internal N-acetyl-rich domain (19). In the
IFN
·heparan sulfate complex, the two carboxyl termini of an IFN
dimer interacted with the two heparin-like sequences of the heparan
sulfate fragment, mainly through the basic C1 and C2 domains (20).
In this study, we investigated whether or not IFN
bound to heparin
could also interact with its high affinity cell surface receptor. Using
the Biacore system, we analyzed kinetic aspects of the IFN
·IFN
R
complex formation and, in particular, the importance of the heparin
binding sequence of IFN
for this interaction. We found that IFN
binding to heparin and to IFN
R are mutually exclusive and that the
C1 sequence increased the association rate of the IFN
·IFN
R
binding reaction.
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MATERIALS AND METHODS |
Equipment and Reagents--
An upgraded Biacore system,
certified CM5 sensor chips, and HBS buffer (10 mM HEPES,
150 mM NaCl, 3.4 mM EDTA, 0.05% surfactant P20, pH 7.4) were from Biacore AB. Biotin-LC-hydrazide was from Pierce,
and streptavidin was from Sigma. Proteolytic enzymes (activated factor
X, carboxypeptidase Y, endoproteinase Arg-C) and enzyme inhibitor
(pefablock) were from Boehringer. Recombinant IFN
(IFN
143, batch number L 405), IFN
lacking the last 19 amino acids on the carboxyl-terminal side (IFN
124), and
the two monoclonal antibodies (293-4-45 and 13-16-2) used in this study
were kind gifts of Roussel Uclaf company. The extracellular part of the
human IFN
receptor (sIFN
R)
-chain, produced in Sf9
cells, was a kind gift of Dr. L. Ozmen (Hoffmann La Roche), and
fractionated heparin was from Sanofi Recherche.
Preparation of IFN
Lacking Carboxyl-terminal Amino Acids and
the IFN
Assay--
Full-length IFN
(IFN
143) was
digested with either activated factor X (1 unit/ml in 50 mM
Tris, 150 mM NaCl, pH 8, buffer for 1 h at 25 °C),
carboxypeptidase Y (20), or endoproteinase Arg-C (2.5 units/ml in 50 mM Tris, 150 mM NaCl, pH 8, buffer for 4 h
at 25 °C). Enzymatic reactions were stopped with 1 mM
pefablock and analyzed by 15% SDS-polyacrylamide gel electrophoresis
and by electrospray ionization mass spectrometry. For that purpose, digested samples were made 0.4 mg/ml in 10 mM ammonium
acetate buffer and analyzed using a Perkin-Elmer Sciex API
III+ triple quadrupole mass spectrometer equipped with a
nebulizer-assisted electrospray (ionspray) source. Samples were
directly infused into the source using a syringe pump at a flow rate of
5 µl/min and an injector equipped with a 1-µl internal loop or were
desalted using a reversed phase high pressure liquid chromatography
column coupled to the electrospray source. The ionspray probe tip was held at 5000 V, and spectra were recorded in the 900-1800 range of
mass-to-charge (m/z) ratios in steps of 0.4 m/z, with a 1.7-ms dwell time. The signal was
averaged over four scans. Specific antiviral activity of the IFN
samples were determined with a microtiter inhibition of cytopathic
effect assay using Wish cells against vesicular stomatitis virus
(21).
Biotinylation of the sIFN
R--
The purified sIFN
R (22)
was prepared at 0.27 mg/ml (8 µM) in 20 mM
phosphate buffer, pH 6, and reacted for 20 min in the dark and at
4 °C with 10 mM sodium periodate to oxidize the glycan parts of the molecule. The reaction was quenched with 15 mM
glycerol, and the sample was dialyzed against the 20 mM
phosphate buffer. Biotin-hydrazide (including a 2.5-nm spacer arm
between the biotin and hydrazide groups) was then added to a
concentration of 5 mM, and the mixture was incubated for
6 h at 4 °C. The sample was made to 100 mM
ethanolamine, and then extensively dialyzed against phosphate-buffered
saline, pH 7.2. Biotinylation was checked by Western blot analysis, and
samples were aliquoted and stored at
80 °C.
Preparation of the Biacore Binding Surface--
The flow rate of
the running buffer (HBS) was maintained at 5 µl/min, and the
temperature was maintained at 25 °C. Two flow cells of a CM5 sensor
chip were activated with 50 µl of a mixture of 0.2 M
1-ethyl-3-(3 dimethylaminopropyl)carbodiimine 0.05 M N-hydroxy-sulfosuccinimide, after which 50 µl of
streptavidin (0.2 mg/ml in 10 mM acetate buffer, pH 4.2)
was injected. Unreacted groups were blocked with a 50-µl injection of
1 M ethanolamine, pH 8.5. Approximately 8000 resonance
units (RU) of streptavidin were fixed on the surface by this procedure.
Biotinylated sIFN
R (10 µl/ml in HBS) was then injected for 1 min
on one of the two streptavidin surfaces (the other one being a negative
control). Both flow cells were then conditioned with 10 2-min pulses of 10 mM HCl. This resulted in the attachment of 800 RU of
IFN
R. The conversion of RU to surface concentration of proteins was performed using a conversion factor of 1000 RU = 1 ng/mm2.
Binding Assay--
Test samples were diluted in HBS maintained
at 25 °C and injected over the IFN
R surface at a flow rate of 50 µl/min. This high flow rate was necessary to reduce mass transport
effect due to the high association rate of the proteins being studied.
Using the Kinject command, usually 200 µl of IFN
or IFN
-derived
molecules were injected across the IFN
R surface, after which the
formed complexes were washed at 50 µl/min with HBS to study the
dissociation phase. The IFN
R surface was regenerated with a 2-min
pulse of 10 mM HCl. For kinetic analysis, a complete set of
sensorgrams were recorded with eight different IFN
concentrations in
the range 0-0.5 µg/ml.
Kinetic Analysis--
Sets of sensorgrams were analyzed with the
Biaevaluation 2.1 software, provided with the machine, using both
linear transformation of the primary data and nonlinear fitting of the
sensorgrams. Briefly, the equation for the measured binding rate
(dR/dt) as a function of the binding response
(R) is dR/dt = konCRmax
(konC + koff)Rtn, where
kon and koff are the
association and dissociation rate constant, C is the
concentration of the analyte (IFN
), Rmax is
the binding capacity of the immobilized ligand (IFN
R), and
Rtn is the amount of analyte bound to ligand at time
tn. Kinetic constants (kon
and koff) can be obtained by linear
transformation of a set of sensorgrams using a plot of
ln(dR/dt) versus time for each analyte
concentration. These plots give a line of which the slope is
ks. A secondary plot of these slopes
(ks) versus C is then used to determine kon and koff from the
linear relationship ks = konC + koff.
Association and dissociation rate constants can also be extracted from
a single sensorgram (i.e. a single analyte injection). In
that case, the integrated forms of the rate equations
(r = Ro
exp(
koff(t
td)) for dissociation and r = Req (1
exp(
(konC + koff)(t
td))) for association, where Req is the steady-state binding response and
ta and td are the start times for
association and dissociation) were fitted to the experimental data by
nonlinear regression (23). In some cases to derive kinetic constants,
numerical integration, which allows fitting of data to complex
interaction models, was performed (Biaevaluation 3.0 software). Data
were analyzed by global fitting of both association and dissociation
phases for several concentrations simultaneously. Affinity
(dissociation equilibrium constants, Kd) were
calculated from the ratio of dissociation and association rate
constants (Kd = koff/kon).
 |
RESULTS |
Binding of IFN
to Sensor Chip Immobilized IFN
R--
Using
the Biacore technology, we set up a binding assay to analyze the
IFN
/IFN
R interaction, in particular the kinetic aspects. The
Biacore instrument uses surface plasmon resonance to measure changes in
refractive index when a soluble analyte (here IFN
) binds to an
immobilized ligand (IFN
R). Biotinylated sIFN
R was immobilized on
a streptavidin-activated sensor chip, at a density of 0.8 ng/mm2 (800 RU). Biotin groups had been attached to the
glycan part of the receptor (a part not required for ligand binding
(24)) to ensure that 100% of the molecules remain active upon
immobilization. When IFN
was injected over the IFN
R surface, a
typical sensorgram was obtained (Fig.
1a), with an association phase
(A), equilibrium (E), and, when IFN
was
replaced by running buffer alone, a dissociation phase (D).
Preincubation of the cytokine with increasing concentrations of its
soluble receptor completely inhibited the interaction with the
immobilized IFN
R, demonstrating the specificity of the binding (Fig.
1a). In addition, upon injection of IFN
over a control surface (containing streptavidin only) no binding was observed (Fig.
1b).

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Fig. 1.
IFN binding to immobilized IFN R.
a, IFN (0.5 µg/ml), either alone or in combination
with 2.5, 5, 10, or 20 µg/ml soluble IFN R, was injected for 4 min
(from 130 to 370 s) to an IFN R activated sensor chip, after
which the samples were replaced by running buffer. The binding response
in RU was recorded as a function of time and showed the association
phase (A), the equilibrium (E), and the
dissociation phase (D). When coincubated with soluble
IFN R, the binding of IFN was strongly reduced. b,
IFN (eight concentrations from 0 to 0.5 µg/ml) was injected on a
sreptavidin-activated sensor chip, which showed that the binding in
a was specific to the immobilized IFN R. c,
IFN (0.5 µg/ml) was injected on the IFN R-activated sensor chip
until the equilibrium was reached. After a quick wash with running
buffer, soluble IFN R (1, 2, 10, 20 µg/ml, from top to
bottom) was injected on the bound IFN . The soluble
IFN R could not bind to a preformed IFN ·IFN R complex,
demonstrating that at the surface of the sensor chip the cytokine was
already bound by two IFN R molecules.
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Maximum binding was approximately 400 RU. Since we had 800 RU of
immobilized IFN
R, this suggests that each IFN
dimer (34 kDa) has
been bound by two IFN
R molecules (32 kDa each). Dimerization of
IFN
R by its ligand also occurs both in solution and at the cell
surface (9). To further confirmed this stoichiometry at the sensor chip
surface, we also injected sIFN
R on a preformed IFN
·IFN
R
complex. As shown on Fig. 1c, the complex was unable to bind
additional IFN
R, demonstrating that it already contained two IFN
R
molecules per IFN
dimer. Nonlinear least squares regression analysis
of the sensorgram (Fig. 1a, upper curve), using a
simple A + B = AB model, gave a
koff = 1.2 × 10
3·s
1 and a kon = 4.5 × 106
M
1·s
1 and therefore an
equilibrium dissociation constant Kd = 0.26 nM. This is higher than the affinity of IFN
for its
soluble receptor (1.5 nM) reported with other methods (9).
However, it should be noted that we observed deviation of the data from the simple model we used. This may be caused by factors that derive from the biosensor method itself (25), such as rate-limiting mass
transport effect or rebinding of the ligand during the dissociation phase (see below).
Binding of IFN
to Heparin and to IFN
R Are Mutually
Exclusive--
One of our objectives was to determine whether IFN
bound to heparin was still able to interact with its receptor. For that purpose, IFN
was preincubated with increasing concentrations of
different heparin molecules and then injected over the IFN
R surface
(Fig. 2). The first set of sensorgrams
show that a 12-kDa heparin fragment bound to IFN
strongly decreased
the on rate of the reaction (Fig. 2a). As a consequence, the
amount of IFN
bound to its receptor was strongly reduced. This
heparin fragment (12 kDa, approximately 20 disaccharides in length)
represented the minimum length that binds efficiently to IFN
and
functions by bridging two IFN
monomers (19). Smaller heparin
fragments (4.5 kDa, approximately 8 disaccharides in length) displayed
a strongly reduced ability to inhibit the IFN
·IFN
R complex
formation (Fig. 2b), while a hexasaccharide (1.8 kDa) was
completely inactive, even at a 50-fold molar excess (Fig.
2c).

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Fig. 2.
IFN bound to heparin displayed a reduced
binding to IFN R. IFN (0.5 µg/ml) was preincubated with
0.5-, 5-, 10-, or 50-fold molar excess (from top to
bottom on each set of sensorgram) of different heparin
molecules. Bound to the 12.5-kDa heparin molecule (a),
IFN displayed a strongly reduced binding to IFN R. Heparin
fragments of 4.5 (b) or 1.8 kDa (c) had a reduced
ability to inhibit the IFN binding to IFN R.
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The C1, but Not the C2, Domain of IFN
Is Involved in Receptor
Recognition--
To investigate which of the two basic clusters, C1
and C2, involved in heparin binding could be implicated in receptor
interaction, IFN
preincubated with two different monoclonal
antibodies (mAb), was injected over the IFN
R surface. These two mAbs
(293-4-45 and 13-16-2) defined two overlapping sequences of the
carboxyl-terminal part of IFN
(amino acids 125-134, which encompass
the first basic domain C1 of the carboxyl-terminal part of the cytokine
for mAb 293-4-45, and amino acids 132-138, which overlap the second
basic domain C2 for mAb 13-16-2) and are described elsewhere (26). Coincubation of IFN
with mAb 293-4-45 dramatically reduced the binding of the cytokine to its receptor (Fig.
3a). Coincubation of IFN
with mAb 13-16-2 also reduced the binding but to a smaller extent (Fig.
3b). Both mAbs were also used to probe a preformed IFN
·IFN
R complex. For that purpose, a saturating dose of IFN
was injected over the IFN
R surface until equilibrium was reached. The complex was then quickly washed with running buffer, after which
mAbs were injected (Fig. 3c). These data showed that mAb 13-16-2 could bind to the IFN
·IFN
R complex, whereas mAb
293-4-45 only displayed a weak interaction. Together these results
suggest that the 125-134 sequence of IFN
becomes buried in the
cytokine-receptor complex but that the 132-138 sequence remains freely
accessible.

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Fig. 3.
The C1, but not the C2, domain of IFN is
involved in receptor binding. IFN (0.5 µg/ml) was
preincubated with 25, 50, 100, or 200 µg/ml mAb 293-4-45 (a) or mAb 13-16-2 (b) and then injected over the
IFN R surface. Both mAbs reduced the binding of IFN to its
receptor, the 293-4-45 (which defines the C1 domain) being much more
active than the 13-16-2 (which defines the C2 domain). These two mAbs
(both at 100 µg/ml) were also injected on a preformed
IFN ·IFN R complex (c). mAb 13-16-2 could bind to the
IFN ·IFN R complex, whereas mAb 293-4-45 only displayed a weak
interaction.
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Characterization of Enzymatically Processed IFN
--
To
directly analyze the role of the heparin/heparan sulfate binding
sequence in receptor interaction, we prepared various forms of IFN
lacking defined domains of the carboxyl terminus, making use of the
great enzymatic susceptiblility of this part of the cytokine (13).
Cleavage of IFN
was performed with activated factor X,
carboxypeptidase Y, or endoproteinase Arg-C (Fig.
4). The Mr of
these truncated forms of IFN
, as measured by electrospray ionization
mass spectrometry analysis (Table I),
were 16,254, 15,705, and 15,208 for activated factor X-,
carboxypeptidase Y-, and endoproteinase Arg-C-treated IFN
. This
corresponded to molecules from which 6, 10, or 14 carboxyl-terminal
amino acids were removed, and this was consistent with the known
substrate specificity of the enzymes used. These molecules therefore
ended at Arg137, Gln133, and Arg129
and will be referred to as IFN
137,
IFN
133, and IFN
129 (see Fig. 4). As a
control, IFN
143 was also analyzed, and the
Mr obtained (16,908) was exactly the
Mr expected for the full-length molecule. In
addition, deletion of six (IFN
137) or 10 (IFN
133) amino acids resulted in the expected increased
antiviral activity (13), while deletion of 14 (IFN
129)
or 19 (IFN
124) amino acids resulted in the expected
decreased activity for such truncated molecules (data not shown).

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Fig. 4.
Enzymatic cleavage of IFN . IFN was
treated with activated factor X, carboxypeptidase Y, or endoproteinase
Arg-C. Reactions were stopped with 1 mM pefablock, and the
samples were analyzed by SDS-polyacrylamide gel electrophoresis.
Lane A, molecular weight markers (97,400, 66,200, 45,000, 31,000, 21,500, 17,000, and 14,400). Lane B,
IFN 143. Lane C, activated factor X-treated
IFN . Lane D, carboxypeptidase Y-treated IFN .
Lane E, endoproteinase Arg-C-treated IFN . Lane
F, IFN 124. Lane G, a mixture of
lanes B-F. The carboxyl-terminal sequence of IFN is also
shown. The two clusters of basic amino acids C1 and C2 are in
boldface type, and the cleavage sites, as determined by
electrospray ionization mass spectrometry (see Table I), are indicated
by arrows.
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Table I
Cleavage points of the IFN truncated forms
Full-length IFN (IFN 143), activated factor X (aFX)-,
carboxypeptidase Y (CY)-, or endoproteinase Arg-C (ArgC)-treated IFN
was analyzed by electrospray ionization mass spectrometry. Cleavage
points (last amino acid) were determined from the measured relative
molecular mass (Mr).
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Kinetic Analysis of the Interaction between IFN
and IFN
R:
Role of the Carboxyl-terminal Sequence--
IFN
143
(full-length IFN
), and the carboxyl termini-deleted IFN
were
injected over the biacore IFN
R surface, each with a range of
concentrations to produce sets of sensorgrams from which association
and dissociation phases can be analyzed. The dissociation phase started
at 370 s, when the injection of IFN
was changed to a perfusion
buffer. To this part of the sensorgrams was applied a simple
AB = A + B dissociation model. A
good fit was found for IFN
124 only (Fig.
5e), which gave an off rate in the range 4.7-5.2 × 10
3·s
1. For
the others, the higher the observed on rate (see below) the worse was
the fit. Presumably, the fast on rate caused a rapid rebinding of the
dissociated molecules, a phenomenon that occurs as increasing numbers
of free immobilized ligand (IFN
R) are regenerated at the surface of
the sensor chip during the dissociation phase. Since free IFN
R, and
therefore the rebinding effect, increased with time during the
dissociation, only the first 60 s were used for the analysis. In
addition, to prevent rebinding of dissociated molecules, soluble
IFN
R was included in the perfusion buffer during dissociation
(e.g. see Fig. 1c). Including IFN
R at 10-20 µg/ml in the perfusion buffer, a simple dissociation model can now be
assumed, characterized by an off rate of 5-5.6 × 10
3·s
1. Interestingly, this
koff was similar to the
koff of IFN
124 (for which no
rebinding occurred). Therefore, the addition of soluble IFN
R in the
perfusion buffer was effective in preventing rebinding and necessary
for accurate kinetic analysis. Furthermore, these data showed that
cleavages of the carboxyl terminus of IFN
did not modify the off
rate of the reaction and, thus, that the carboxyl-terminal sequence
of the cytokine was not involved in the stability of the
complex.

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Fig. 5.
Overlay of sensorgrams showing binding of
IFN to immobilized IFN R. Role of the IFN
carboxyl-terminal sequence. IFN 143 (a),
IFN 137 (b), IFN 133
(c), IFN 129 (d), and
IFN 124 (e) were injected over an
IFN R-activated surface at a flow rate of 50 µl/ml during 4 min
(from 130 to 370 s), after which running buffer was injected, and
the response in RU was recorded as a function of time. Each set of
sensorgrams was obtained with IFN molecules at (from top
to bottom) 0.5, 0.4, 0.3, 0.25, 0.2, 0.15, 0.12, and 0 µg/ml. Association phases where the binding was not limited by mass
transport were used for the kinetic analysis either by linear
transformation of the primary data and by nonlinear fitting of the
sensorgrams (see Table II) with the Biaevaluation 2.1 software.
Global fitting of the binding curves, using numerical integration
(Biaevaluation 3.0 software) was also performed (see Table II).
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During ligand injection (130-370 s), three phases can be
distinguished. The first phase was diffusion-controlled (the reaction was limited by mass transport of the analyte), and, in our case, this
was observed (see below) for the molecules that displayed a high on
rate (IFN
143, IFN
137, and
IFN
133) but not for the others (IFN
129
and IFN
124). The second phase was determined by the
kinetics of the IFN
/IFN
R interaction, and the third one was
equilibrium. It was immediately apparent (see Fig. 5) that the binding
rate of the reaction increased for IFN
137 and
IFN
133, compared with full-length molecule (Fig. 5,
a-c) but decreased for IFN
129 and
IFN
124 (Fig. 5, d-e). Kinetic analysis of
the binding curves was first performed with the Biaevaluation 2.1 software. As indicated above, in some cases (IFN
143,
IFN
137, and IFN
133), the early phase of
the association was dominated by mass transport. However, as the
reaction proceeds, the free binding sites decrease and the binding rate
progressively depends on the intrinsic rate constant. Thus, as a first
approach, when analyzing the experiments, the mass transport-limited
parts of the sensorgrams were excluded from the data used for
evaluation, as already reported in several other cytokine-receptor
binding studies (27, 28). In this way, the kinetic part of each
sensorgram could be confidently fitted on the basis of an A + B = AB model, and deviations of the data
points from the fitted curve were quantitatively in the range expected
from the background noise. As detailed under "Materials and
Methods," analysis was performed both by nonlinear fitting and by
linear transformation of the data. Both methods gave similar kinetic
constants, and the results are summarized in Table
II.
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Table II
Kinetic parameters of the IFN ·IFN R binding reaction
Sensorgrams of Fig. 5 were analyzed as described under "Materials and
Methods." Dissociation rate constants (koff) were
measured in the absence of rebinding effect. Association rate constants
(kon) were obtained by linear transformation of the
primary data (a) or by nonlinear fitting of the sensorgrams,
using the Biaevaluation 2.1 software (b). For
IFN 143, IFN 137, and IFN 133, the early
phase of the association was dominated by mass transport, and the mass
transport-limited parts of the sensorgrams were excluded from the
analysis. Data were also analyzed by global fitting (c),
using the Biaevaluation 3.0 software, to take into account mass
transport effect (see "Results"). The on rate obtained by this
method was used for the calculation of Kd
(Kd = koff/kon).
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However, mass transport limitation has been identified as a potential
problem, leading to underestimation of the association rate constant
(29). Thus, to better assess the observed changes in on rates, caused
by cleavages of the IFN
carboxyl-terminal sequence, we also analyzed
our data by numerical integration, using the Biaevaluation 3.0 software. Here, in contrast to the previous analysis, the entire time
courses of the reactions were fitted to binding models, including mass
transport-limited binding reaction. Global fitting of binding curves,
recorded with IFN
129 and IFN
124, gave
respective association rate constants of 2.48 and 1.9 × 106 M
1·s
1. It is
noteworthy that these values are in close agreement with those
determined either by linear transformation of the data or by nonlinear
fitting (Table II). In contrast, global fitting of the binding curves
obtained with IFN
143, IFN
137, and
IFN
133 was only possible when mass transport limitation
was introduced in the model. Data are reported in Table II and showed
that these three molecules bound IFN
R with respective on rate
constants of 0.73, 1.3, and 1.57 × 107
M
1·s
1. These values are, on
average, twice as much as those determined with the first approach and
showed that kon values were underestimated when
only parts of the sensorgrams were used for the fitting procedure.
Together these analyses showed that progressive cleavages of the IFN
carboxyl terminus progressively increased the on rate of the binding to
IFN
R until the C1 domain was affected. The maximum on rate was
observed with IFN
133 (kon = 1.57 × 107
M
1·s
1). Such a high on rate
has been observed already for the interleukin-4 binding to its receptor
(30). Once the C1 domain was cleaved (IFN
129) or removed
(IFN
124), the kon decreased.
Thus, the integrity of the carboxyl-terminal sequence of the cytokine
is important for the association phase of the IFN
·IFN
R binding
reaction.
 |
DISCUSSION |
A number of growth factors and cytokines bind to heparin or
heparin-related molecules (31, 32). This include the fibroblast growth
factor family (33), vascular endothelial growth factor (34), hepatocyte
growth factor (35), heparin binding-epithelial growth factor (36), and
cytokines such as interleukin-1, -2, -3, -4, -6, -7, and -8 (37-41),
chemokines (42), and IFN
(18). Considerable data have been reported
during the past few years regarding the physiological significance of
the binding of heparin to growth factors, the prototype of which is
FGF-2. Importantly, FGF does not bind productively to its high affinity
receptor, unless it is itself bound to heparin or heparan sulfate. Such information has not been reported yet for any heparin-binding cytokine.
In this study, we set up a binding assay, suitable for kinetic
analysis, in which IFN
binds to IFN
R, with high affinity and
specificity, and with a stoichiometry identical to what occurs at the
cell surface (one IFN
for two IFN
R, Fig. 1). Using this assay, we
first showed that, in contrast to growth factors, IFN
bound to
heparin displayed a strongly reduced binding to its receptor, which was
caused by a reduction of the on rate of the reaction. As a consequence,
the mechanism by which heparin may affect IFN
activity should be
completely different from the mechanism by which heparin modulates the
activity of other growth factors (see below). Noteworthy is the fact
that only extended heparin fragments (at least 20 disaccharides) could
inhibit the IFN
/IFN
R binding (Fig. 2). This length was found to
be necessary to interact simultaneously with the two carboxyl termini
of an IFN
homodimer, and the present finding further supports the
molecular organization of the heparan sulfate binding site we
previously described (19).
The observation that IFN
binding to heparin and to IFN
R were
mutually exclusive could indicate that the cytokine carboxyl-terminal domain was also engaged in IFN
R recognition. The involvement of the
IFN
C1 sequence (one of the two basic clusters that interacts with
heparin) in IFN
R binding has been reported yet, but its possible
mechanistic role has not been established (10, 43). In particular, the
kinetic analysis performed here first indicated that IFN
molecules
from which C1 and C2 were removed (IFN
124) dissociated
from its receptor at the same off rate (5 × 10
3·s
1) as the parent molecules
(IFN
143). Thus, at equilibrium, the carboxyl-terminal
sequence of the cytokine, and especially the C1 domain, was not
involved in the stability of the IFN
·IFN
R complex. This was
also supported by the fact that mAb 293-4-45, the epitope of which is
C1, did not dissociate a preformed complex (Fig. 3c). In
contrast, progressive cleavages of the carboxyl-terminal domain of
IFN
modulated the on rate of the reaction (Fig. 5). Increased on
rate was observed for any cleavage downstream of the C1 domain
(IFN
137 and IFN
133), whereas cleavages
within or upstream of the C1 domain decreased the on rate of the
reaction (IFN
129 and IFN
124). We believed
that the functional importance of the C1 sequence, during the
association phase only, that we report here, explains the existing and
controversial data on the role of the IFN
carboxyl terminus in
bioactivity. The mechanism by which removal of the C2 domain further
increased the on rate of the IFN
·IFN
R binding reaction is not
known, but it is possible that C1 and C2 compete with each other to
bind to some sequence on IFN
R (see below).
In general, random encounters between two proteins occurs in an
orientation where the specific contact residues are not aligned, and
the two molecules diffuse away from each other after colliding nonproductively. Thus, many collisions are needed before specific association occurs (44). As stated above, the carboxyl terminus of
IFN
is highly flexible and can adopt multiple conformations. Of
interest in this regard was that one of these conformations places the
IFN
basic C1 domain in front of a cluster of acidic amino acids in
the IFN
R while the two molecules are bound to each other (11).
Attracting forces between these two charged domains could place the two
interacting molecules into a proper orientation while they are still
some distance apart. In such a way, the specific binding sites may
collide with each other more frequently, and this would result in the
increased on rate observed when C1 was present in the molecule
(IFN
143, IFN
137, and
IFN
133). Another mechanism could be proposed whereby
interacting IFN
and IFN
R are held together by relatively
nonspecific forces, (between C1 and the group of acidic amino acids
found in IFN
R) long enough to increase their chance of finding a
mutually reactive configuration. A mechanism of this sort can be viewed
as representing reorientation within an encounter complex (44) and may
account for the importance of C1 during the association phase only. It is noteworthy that the association rate can be increased for a variety
of molecules, simply by attaching weakly interacting, relatively
unstructured polymeric domains to the macromolecules involved.
Furthermore, charged residues are particularly suitable for such
mechanisms, because electric fields that surround such groups spread
out in every direction, making collision geometry less important (44).
Together, our data and the proposed mechanisms described above suggest
that C1 interaction with IFN
R is the first event of the
IFN
·IFN
R complex formation. The dissociation rate was identical
for all of the IFN
forms studied here, irrespective of the presence
of C1 and C2 domain (koff = 5 × 10
3·s
1). However, for those forms that
contained the C1 region, it was necessary to include soluble IFN
R in
the running buffer during the dissociation phase to prevent rebinding
of dissociated molecules. The observation that the C1 sequence caused
immediate rebinding of newly dissociated molecules further supports the
idea that interaction between C1 and IFN
R represents the first
contact between the two molecules.
For analytical purposes, rebinding has been eliminated in our study,
but this may have a physiological importance. IFN
is active at
extremely low concentration, well below the Kd (for
example, a few pM only are required to have an antiviral effect in cell culture). Therefore, free IFN
R are in large excess, and rebinding of dissociated molecules is likely to occur at the cell
surface. As a result, the apparent affinity of IFN
for its receptor
will be increased (see result from Fig. 1a). High affinity, and in particular a high rate of association, is necessary for achieving a high rate of product formation but also for preventing side
reactions such as inactivation.
High affinity binding of IFN
to heparin also represents a mechanism
by which IFN
is protected from inactivation (20). This binding also
supports the plasma clearance of the cytokine and localizes it in
restricted areas within tissue (45). In particular, it has been found
that in vivo heparin protected the C1, but not the C2,
domain of IFN
from degradation, a process that thus increases the
cytokine activity by as much as 600% (46). It is noteworthy that these
effects of heparin or heparan sulfate on IFN
activities did not
depend on IFN
R, and this is consistent with the fact that binding to
heparin and IFN
R are two independent events. However, since IFN
cannot bind heparin and its receptor simultaneously, it remains unclear
how the heparin-bound cytokine can be released to interact with its
receptor, and this point will be the subject of future studies.
Finally, the results of the present work together with our previous
reports clearly indicate that IFN
and growth factors (such as FGF)
belong to distinct groups regarding their regulation by heparan
sulfate/heparin-like molecules.
 |
ACKNOWLEDGEMENT |
We are grateful to Dr. Laurence Ozmen for the
generous gift of soluble IFN
receptor and to Roussel-UCLAF for the
kind gift of recombinant IFN
and monoclonal antibodies used in this
study. We also acknowledge Dr. J. E. Turnbull for critical reading
of the manuscript and the support of Dr. G. Morel. This work is
dedicated to all my past colleagues at the Pasteur Institute in
Lyon.
 |
FOOTNOTES |
*
This work was supported by CNRS, l'Association pour la
Recherche sur le Cancer, and la région Rhône-Alpes.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: CNRS UMR 5578, 43 Bd du 11 novembre 1918, 69622 Villeurbanne, France.
**
To whom correspondence should be addressed: Institut de Biologie
Structurale, Laboratoire de Biophysique Moléculaire, 41 Avenue
des Martyrs, 38027 Grenoble Cedex 01, France. Tel.: 33 476 88 95 69;
Fax: 33 476 88 54 94; E-mail: lortat{at}ibs.ibs.fr.
1
The abbreviations used are: IFN
,
interferon-
; IFN
R, interferon-
receptor; IFN
R
, IFN
receptor
-chain; FGF, fibroblast growth factor; mAb, monoclonal
antibody; RU, resonance units; sIFN
R, soluble IFN
receptor.
 |
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