(Received for publication, July 19, 1994; and in revised form, September 28, 1994)
From the
Agents that antagonize the functions of interferon
(IFN
) are potential pharmaceuticals against several immunological
and inflammatory disorders. IFN
receptor-immunoglobulin G fusion
proteins (IFN
R-IgG) function as antagonists of endogenous IFN
and have longer half-lives in vivo in comparison with soluble
IFN
receptors (sIFN
R), consisting of the extracellular region
of the native sequence. A fusion protein comprising the extracellular
domain of the human IFN
receptor and the hinge, CH
and
CH
domains of the human IgG3 constant region, was expressed
in Chinese hamster ovary cells. The IFN
R-IgG3 fusion protein was
secreted into the culture medium as a 175-kDa glycoprotein and was
purified over Protein G-Sepharose, DEAE-Sepharose, and size exclusion
chromatography. IFN
R-IgG3 bound IFN
in solid phase assays and
ligand blots, competed for the binding of radiolabeled IFN
to the
cell surface receptor of Raji cells, and inhibited the
IFN
-mediated antiviral activity with an efficiency at least one
order of magnitude higher than that of the soluble receptor produced in
the same expression system. Two IFN
R-IgG3 fusion proteins bound
two IFN
dimers forming a complex of approximately 380 kDa. In
immunodiffusion assays, the IFN
R-IgG3 fusion protein did not
precipitate IFN
. Dissociation of bound IFN
from
IFN
R-IgG3 was 2-fold slower than from the sIFN
R produced in
insect cells.
Interferon (IFN
) (
)is a cytokine, produced
by activated T lymphocytes and natural killer cells, that exerts
complex functions in the control and modulation of nearly all phases of
immunological and inflammatory
responses(1, 2, 3) . The active protein is a
homodimer with a predominant
-helical structure, the two subunits
showing an anti-parallel organization(4) . IFN
exerts its
functions by interacting with a ubiquitous, specific cell
receptor(5, 6, 7) , a 90-kDa
glycoprotein(8, 9, 10) . In addition to
IFN
and IFN
R, accessory proteins are required for signal
transduction (11, 12, 13, 14, 15) .
Recently the cloning of such an accessory factor or IFN
R
-chain was reported(16, 17) . It is not clear at
present whether the accessory protein participates in ligand binding.
The IFN
R does not possess intrinsic tyrosine kinase activity,
suggesting receptor association with specific kinases following ligand
binding(18, 19, 20) . The signal transduction
pathway of IFN
involves phosphorylation of p91, a subunit of the
IFN-stimulated gene factor-3(21, 22) .
In certain
immunological disorders, IFN acts as a disease-promoting agent,
and substances that antagonize its functions are potential
pharmaceuticals(23, 24) . In order to obtain IFN
antagonists, we engineered soluble forms of the IFN
receptor
(sIFN
R), comprising the extracellular domain of the native protein
and retaining full capacity for binding
IFN
(25, 26) . When administered to animals, the
sIFN
Rs inhibited the activity of IFN
, but they showed
relatively short half-lives of 1-3 h in the blood and 6 h in
lymphoid organs(28, 29) . Fusion proteins comprising
the extracellular domain of the mouse IFN
receptor and sequences
of the mouse constant IgG region were active in vivo and
showed an increased blood persistency (30, 31) .
Because administered human sIFN
R is expected to have a short
half-life, we expressed in Chinese hamster ovary (CHO) cells a fusion
protein comprising the extracellular domain of the human IFN
R and
parts of the human IgG constant chain. Here we report on the
purification and characterization of this human fusion protein and the
stoichiometry of its interaction with IFN
.
Figure 1:
SDS-PAGE analysis of the human
IFNR-IgG3 fusion protein after each purification stage (A) and after a second size exclusion chromatography step (B). The fusion protein was purified as described under
``Experimental Procedures.'' Electrophoresis was in the
presence or absence of 10%
-mercaptoethanol (as indicated) on 7.5%
SDS gels stained with Coomassie Blue. A, lanes 1 and 6, bovine IgG (reference, 2 µg). In lane 6 only
the heavy chain of bovine IgG is seen. In lanes 2-5 and 7-10, 10 µg of total protein were loaded. Lanes
2 and 7, supernatant of culture medium. The strong band
represents BSA, which shows a shift in mobility under reducing
conditions (lane 7). Lanes 3 and 8, eluate
from Protein G-Sepharose. Lanes 4 and 9, eluate from
DEAE-Sepharose. Lanes 5 and 10, eluate from
Superose-12 H/R 10/30 column. M, high molecular mass markers. B, the eluate from the size exclusion step was concentrated by
ultrafiltration and loaded a second time on the sizing column. Lanes 1 and 4, bovine IgG (reference). Lanes
2-3 and 5-6, eluate from the second
Superose-12 step (lanes 2 and 5, 10 µg; lanes
3 and 6, 20 µg). M, high and low molecular
mass markers.
Chromatography on DEAE-Sepharose,
following the Protein G-Sepharose step, removed most of the bovine IgG,
eluted at pH 4.5. The fusion protein was eluted at pH 3.5 and 3.0, and
it still included small amounts of bovine IgG (Fig. 1A, lanes 4 and 9). Size exclusion chromatography, which
followed the ion exchanger step, delivered two protein peaks, a and b, corresponding to proteins of apparent molecular
masses 600 and 160 kDa, respectively (Fig. 2A). The
protein bands of the fractions of peak a barely entered the
native gel (Fig. 2B, lanes 2-4). When
these fractions were analyzed on SDS-PAGE, the proteins co-migrated
with the fusion protein of peak b (Fig. 2C, lanes 7-9), indicating that peak a included
oligomeric forms of IFNR-IgG3 (Fig. 2C, lanes
2-4). The oligomeric forms were noncovalently linked
(nonreducing SDS-PAGE is not shown). The SDS-PAGE analysis additionally
revealed the presence of bovine IgG in the fractions of peak a (Fig. 2C, lanes 2-4; the 55-kDa
band). Peak b comprised highly purified fusion protein (Fig. 2B, lanes 8-10, and Fig. 2C, lanes 7-9), still contaminated,
however, with approximately 2% bovine IgG (Fig. 1A, lanes 5 and 10). A second size exclusion
chromatography cycle of the fusion protein of peak b yielded a
more than 99% pure fusion protein preparation with less than 1% bovine
IgG (Fig. 1B, lanes 2-3 and 5-6). The overall recovery was approximately 60%, as
judged by ligand blots. The described method did not completely remove
bovine IgG. Bovine IgG was completely removed when Protein G-pretreated
FBS was used for cell culture and the fusion protein was purified
following the described purification scheme over Protein G-Sepharose,
DEAE-Sepharose, and sizing steps (data not shown).
Figure 2: Protein elution profile (A), native (B), and SDS-PAGE analysis (C) of the proteins eluted from the sizing column. A, the proteins were loaded on a Superose-12 H/R 10/30 column developed with PBS. B, analysis of selected fractions on a 5% native gel stained with Coomassie Blue. Lane 1, starting material loaded; lanes 2-10, fractions eluted from the column. Fractions 20-22 (lanes 2-4) contained oligomeric forms of the fusion protein. *, fusion protein, homodimer. C, the proteins were analyzed under reducing conditions on a 7.5% SDS gel stained with Coomassie Blue. Lane 1, proteins loaded. The 55-kDa band represents bovine IgG (heavy chain). Lanes 2-9, fractions eluted from the sizing column. M, molecular mass markers.**, fusion protein, single chain.
Figure 3:
Competition of binding of radiolabeled
IFN to Raji cells (A) and inhibition of IFN
-mediated
antiviral activity (B). The assays were performed as described
in (27) . A, IFN
R-IgG inhibited the binding of 2
ng of
I-IFN
to the receptor of 10
Raji
cells with an IC
of 0.17 nM (
) and the
sIFN
R from CHO cells inhibited with an IC
of 2.30
nM (
).
, inhibition by unlabeled IFN
;
,
inhibition by anti-IFN
antibody
69. B, the
inhibition of antiviral activity was studied on human WISH fibrostast
cells infected with encephalomyocarditis virus. IFN
R-IgG3
inhibited with an IC
of 1.20 nM (
), the
CHO cell-derived sIFN
R with an IC
of 23 nM (
), and the antibody
69 with an IC
of 0.05
nM (
). AVA, antiviral
activity.
We
investigated the exchange rate of IFN bound by IFN
R-IgG3 and
for comparative reasons by sIFN
R and anti-IFN
antibody
69. The kinetics of dissociation were studied by mixing
radiolabeled IFN
with either of the three proteins, adding the
excess of unlabeled IFN
, and measuring the time-dependent release
of the iodinated ligand. IFN
complexed with IFN
R-IgG3 was
released approximately 2-fold more slowly in comparison with that
complexed with
69 or the sIFN
R (Fig. 4). Table 1summarizes the performance of the three proteins in ligand
binding. The increased retention time may be essential if
IFN
R-IgG3 will be used as antagonist of endogenous IFN
.
Similarly, the better performing in vivo tumor necrosis factor
receptor p55 fusion protein had a significantly slower exchange rate
for bound tumor necrosis factor
in comparison with the p75 fusion
protein(39) .
Figure 4:
Dissociation of IFN bound to the
IFN
R-IgG3 fusion protein. The kinetics of dissociation of
complexed
I-IFN
(3
10
cpm) were
performed as stated under ``Experimental Procedures.''
Dissociation of
I-IFN
bound to IFN
R-IgG3
(
), to sIFN
R from insect cells (
), and to monoclonal
anti-IFN
antibody
69 (
). The bars represent
standard error of the mean of four
experiments.
After reduction, IFNR-IgG3 did not show
IFN
binding activity on ligand blots. Controlled reduction of the
175-kDa fusion protein resulted in generation of a 90-kDa single-chain
species with IFN
binding capacity suggesting that the four
disulfides of the extracellular domain of the IFN
R, all of which
are essential for ligand binding(44, 45) , are more
stable than the disulfides connecting the two IgG3 heavy chains.
Dialysis of the completely reduced fusion protein resulted in partial
reconstitution of the biological activity of the 90-kDa single chain
species only, but not of the 175-kDa IFN
R-IgG3. Thus, the
disulfide bonds of the IFN
R domain of the fusion protein were
reconstituted, whereas the disulfides between the two IgG3 constant
domains were not formed (data not shown).
Figure 5:
Size exclusion chromatography (A)
and native gel analysis (B and C) of IFNR-IgG3
fusion protein-IFN
dimer complexes. A, IFN
R-IgG3
(440 µg) and IFN
(220 µg) were mixed and chromatographed
on a Superose-12 column developed with PBS at 0.4 ml/min. The positions
of elution of the peaks of standard proteins (670, 158, and 44 kDa) are
indicated. B, 2 µg of IFN
R-IgG (f) were
mixed with increasing amounts of IFN
10 (lanes
2-5) or IFN
0 (lanes 6-8) at the
indicated ratios, and the complexes were analyzed on a 5% native gel
stained with Coomassie Blue. One major complex (c) was formed.
When IFN
was added at ratios 1:3 and 1:4, additional broad bands
were detected (*) (lanes 4-5 and 7-8). C, IFN
10 (1.3 µg) was mixed with increasing
amounts of IFN
R-IgG3 (f) as indicated. Analysis was as
stated under B. One complex (c) was formed. In excess
of IFN
, additional weak bands (*) were visible (lanes
2-4). IFN
R-IgG3 added in excess remained uncomplexed (f, lanes 6-8).
We further studied whether the
fusion protein and IFN dimer form complexes larger than the
380-400-kDa complex detected by gel filtration and analytical
ultracentrifugation. IFN
R-IgG3 and IFN
were mixed at
different ratios, and the complexes were analyzed by nondenaturing gels (Fig. 5, B and C). Fusion protein and IFN
dimer formed in all cases one major complex (c) for which
amino acid analysis revealed a ratio of 1:1 (Fig. 5B, lanes 2-8 and Fig. 5C, lanes
2-8). In excess of IFN
, additional bands migrating
between the fusion protein f and the complex c were
visible, suggesting the formation of complexes in which two IFN
dimers were bound by one bivalent IFN
R-IgG3 fusion protein (Fig. 5B, lanes 4-5 and 8, and Fig. 5C, lane 4; these bands are broad and not
clearly seen). Similar complexes were formed by one IFN
dimer
bound by one sIFN
R molecule when IFN
was added in excess to
the sIFN
R, although when the sIFN
R was available in adequate
amounts, two soluble receptor molecules always bound one IFN
dimer(41) .
When IFNR-IgG3 was added in excess, again
one complex c was formed. The excess of IFN
R-IgG3
remained uncomplexed (Fig. 5C, lanes
6-8). Thus, no complexes larger than complex c were
detected. Formation of such complexes would suggest an
agglutination-like situation in which the fusion protein and IFN
could form precipitate. In immunodiffusion assays on agarose,
IFN
R-IgG3 did not precipitate IFN
, behaving like a
nonprecipitating monoclonal antibody. IFN
was not precipitated by
the anti-IFN
monoclonal antibody
69 either (data not shown).
In previous studies, using sIFNRs produced in eukaryotic
expression systems, we found that one IFN
dimer is bound by two
receptor molecules(35, 41) . The one human IFN
dimer-two receptor relation was confirmed later by other
groups(47) . Applying chemical cross-linking and using high
concentrations of two cross-linkers simultaneously, they showed the
generation of a 240-kDa product, likely consisting of one IFN
dimer bound by two native receptor molecules, thus confirming
dimerization of the native receptor in the presence of ligand.
Based
on predicted homology between IFNR and members of the
hematopoietic receptor family (48) and in analogy to the
crystal structure of the human growth hormone receptor(49) , we
proposed a model for the IFN
ligand-receptor
interaction(45) . According to that model, the extracellular
part of the IFN
receptor consists of two Ig-like domains linked in
a V-shaped structure. The two domains are connected with one essential
disulfide (Cys
-Cys
). The region at the
convergence of the two Ig-like domains most likely includes the ligand
binding domain of the receptor. That this region is essential for
ligand binding was confirmed by studies with mutant IFN
receptors
carrying domains of both the human and mouse species(50) .
In this study, applying four approaches, we found that two
IFNR-IgG3 molecules, carrying two ligand binding sites each, bind
two IFN
dimers. Such a complex could be formed if each IFN
dimer interacts with one of the IFN
binding regions of one fusion
protein and one of the binding regions of a second fusion protein (Fig. 6A). The model of the IFN
R-IgG3 fusion
protein-IFN
dimer binding (Fig. 6A) shows that the
stoichiometry of interaction is responsible for the higher ligand
binding affinity of the fusion protein and for the better performance
in competition of binding and inhibition of antiviral activity, in
comparison with soluble receptors, in which case two receptor molecules
bind only one ligand dimer (Fig. 6B).
Figure 6:
Schematic representation of the
interaction between IFNR-IgG3 fusion protein and IFN
(A) and sIFN
R and IFN
(B). A, two
IFN
R-IgG3 fusion protein molecules bind two IFN
dimers,
forming a complex of approximately 380 kDa. For such a complex to be
formed, each IFN
dimer should be bound by one of the ligand
binding domains of either of the two bivalent fusion protein molecules. B, two sIFN
R molecules bind one IFN
dimer ( (35) and (41) ). A and B, the two
Ig-like domains of the IFN
R part of each fusion protein (A) or soluble receptor (B) are shown in black. The IgG3 part is shown in gray (A).
The connecting line between the IgG3 domains represents
disulfides. The two subunits of the IFN
dimer are shown in white (A and B).
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