(Received for publication, April 3, 1995; and in revised form, September 6, 1995)
From the
The human receptor for the potent eosinophilopoietic cytokine
interleukin-5 (IL-5) consists of two components: a 60-kDa
ligand-binding chain (IL-5
R) and a 130-kDa
chain
(IL-5
R). Three ectodomain constructs of the
chain (
RED)
bearing C-terminal epitope tags were engineered and expressed in
baculovirus-infected Sf9 cells. Each recombinant
chain was
secreted into the medium, maximum expression occurring 72 h
post-infection. The various soluble
chains were shown by affinity
cross-link labeling and competition with unlabeled IL-5 to bind
recombinant human (rh)
I-IL-5 specifically with an
ED
of 2-5 nM. The epitope tag provided a
simple purification of the receptor from conditioned medium using
immunoaffinity chromatography. The purified material had an apparent
molecular mass of 43 kDa and was heterogeneously glycosylated.
Sedimentation analysis revealed a 1:1 association of the purified
epitope-tagged soluble receptor with its ligand, resulting in the
formation of a 70-74-kDa complex. Circular dichroism analysis
revealed that the soluble
chain existed with a significantly
ordered structure consisting of 42%
-sheet and 6%
-helix.
Such analyses combined with fluorescence spectrometry suggested that
ligand-receptor complex formation in solution resulted in minimal
conformational changes, consistent with the suggestion that the
membrane-associated form of the
chain itself has minimal signal
transduction capability. Surface plasmon resonance studies of the
interaction of the purified
RED with immobilized rhIL-5 revealed a
specific, competable interaction with a dissociation constant of 9
nM. Preincubation of an IL-5-dependent cell line with the
epitope-tagged
RED also dose-dependently neutralized
rhIL-5-induced proliferation. These data demonstrate that biologically
active epitope-tagged recombinant soluble IL-5 receptors are facile to
produce in large quantities and may have therapeutic utility in the
modulation of IL-5-dependent eosinophilia in man.
Interleukin-5 (IL-5) ()is a pleiotropic cytokine with
demonstrated activatory and proliferative effects on murine B cells, T
cells, and eosinophils. In humans, IL-5 activity appears to be
restricted to eosinophil/basophil lineages and may play a role in the
regulation of eosinophilic inflammation associated with various
diseases, notably bronchial asthma(1) .
The human receptor
for IL-5 consists of two components: a 60-kDa ligand-binding
chain (IL-5
R) and a 130-kDa
chain (IL-5
R), which does
not by itself bind IL-5 but forms a high affinity signal transducing
receptor with the IL-5/
chain complex(2) . The cloning and
characterization of the IL-5 receptor
chain identified two mRNA
species that correspond to alternatively spliced soluble isoforms of
the full-length transmembrane
chain(2, 3) . One
of these alternatively spliced mRNA species is the major transcript
expressed in eosinophilic HL-60 cells (4) and in eosinophils
grown from cord blood(2) . These soluble receptors may play a
role in the immunoregulation of eosinophilia by neutralizing the
biological activity of secreted IL-5.
This report describes the
baculovirus expression of three ectodomains of the ligand-binding IL-5
receptor chain (S1-, S2- and S3-
RED). Epitope tags were
engineered onto the C termini of the various constructs allowing
purification in a mild single-step procedure. The purified proteins
were then shown to bind IL-5 and to neutralize the biological activity
of this potent pro-inflammatory hematopoietin in a cellular
proliferation assay, suggesting that such recombinant soluble receptor
forms may have utility in the therapy of eosinophilic inflammation.
The
oligonucleotides GO78 (ATGATCATCGTGGCGCATGTATTA) and GO98
(CTACTTACCCACATAAATAGGTTGGCTCCACTCACT) were used to amplify a 1002-base
pair fragment corresponding to clone lL5R.25(3) . A second pair
of oligonucleotides, GO80 (CCTCCACTGAATGTCACAGCAGAG) and GO79
(TCAAAACACAGAATCCTCCAGGGT) amplified a 542-base pair fragment spanning
nucleotides 721-1263 of the published IL-5 receptor chain
sequence (clone lh5R.12; 3). Amplified bands of the appropriate size
were electrophoretically transferred to DEAE-cellulose (NA-45
DEAE-cellulose, Schleicher & Schuell, Keene, N.H.) and then
directly cloned into EcoRV-digested, T-modified pBluescript II
SK
(Stratagene Cloning Systems, La Jolla, CA). Escherichia coli XL-1 cells were transformed, restriction
digests performed on purified plasmid preparations, and
insert-containing preparations sequenced using the dideoxy chain
termination method (Sequenase, U. S. Biochemical Corp., Cleveland, OH)
according to standard procedures(10) . The resulting plasmids
containing the 5`-1002 base pairs
chain (clone pBSK-IL5R-
3)
and the 3`-542 base pairs of the receptor (clone pBSK-IL-5R-
11)
were then used to reconstruct the full-length IL-5 receptor
chain
by restriction digestion of both pBSK-IL-5R-
3 and
pBSK-IL-5R-
11 with BamHI and SacI and ligation
into BamHI-digested pBSK to generate pBSK-IL-5R-
.
The sensor surface was prepared by covalent coupling of recombinant human IL-5 (carrier-free, R& Systems; 10 µg/ml in 20 mM NaOAc, pH 4.5) at a flow rate of 5 µl/min using the amine coupling kit containing N-hydroxysuccinimide, N-ethyl-N`-(3-diethylaminopropyl)carbodiimide hydrochloride and 1 M ethanolamine hydrochloride, pH 8.5, as described by the manufacturer (Pharmacia Biosensor AB). The extent of coupling was controlled by varying the time of sensor chip activation, IL-5 exposure, and ethanolamine quenching from 1 min/step to 7 min/step.
Sensorgrams were generated by passing RED-containing
analyte solutions across the IL-5-modified sensor surface from which
the rate of association (k
) could be derived.
Dissociation rate constants (k
) were similarly
derived by then passing D-PBS across the IL-5 sensor/
RED complex
surface according to the following reaction for a simple bimolecular
interaction.
On-line formulae not verified for accuracy
Since IL-5 was immobilized to the sensor chip, the increase in R
upon RED binding was a reflection of the amount of IL-5/
RED
complex formed and the maximum amount of IL-5/
RED complex that
could form was determined by the amount of active IL-5 present and
accessible for interaction with the analyte (R
).
On-line formulae not verified for accuracy
The association rate constant was derived from analysis of the
association phase of sensorgrams obtained by passing varying
concentrations of analyte (RED) solutions across a single IL-5
sensor surface and plotting the changing response with time
(dR/dt) against R at that time so that a
line with slope of -(k
[
RED]
+ k
) or -k
,
results. When k
is then plotted against the
concentration of
RED in each of the solutions the slope of the
resulting line is equal to the association rate constant (k
or k
).
The
dissociation rate constant was derived from the portion of the
sensorgram where the analyte solution was replaced with buffer and the
dissociation of RED from the IL-5 sensor surface monitored as a
decrease in the response units. Integration of the first order rate
equation dR/dt = k
Rt and plotting the initial phase of
the dissociation sensorgram as ln (Rt
/Rt
) versus time
resulted in a line with a slope equal to the negative of the
dissociation rate constant (-k
or
-k
). The equilibrium dissociation constant (K
) was obtained from the equation K
= k
/k
. The IL-5 sensor
surface was regenerated with a 5-µl pulse of 20 mM HCl
between exposure to successive
RED-containing analyte solutions.
Figure 1:
Engineering of epitope-labeled
RED. Panel A, a schematic representation of the
full-length human IL-5 receptor
chain (IL-5R
) polypeptide
shows the ligand-binding domain (shaded box), the
transmembrane domain (hatched box), and the intracellular
domain (open box). The SacI restriction site (
)
was used to reconstruct the full-length cDNA from two PCR-derived
clones (see ``Experimental Procedures''). The position of the
IL-5 receptor chain amino acid at the junction with the sequence
encoding the FLAG epitope is indicated for the S1, S2, and S3 forms of
the soluble receptor. The numbers refer to the amino acid
residue with reference to the N terminus. Panel B, the
nucleotide sequence and deduced amino acid sequence of the 5`-region of
the three soluble receptor isoforms is shown with that part
corresponding to the PCR-priming oligonucleotide DN265 which is boxed. Panel C, the nucleotide sequences and deduced
amino acid sequences of the 3`-regions of the three soluble receptor
isoforms are shown with those parts corresponding to the PCR-priming
oligonucleotides PB22, PB25, and PB27 (boxed) used to generate
the S1-, S2-, and S3-
RED constructs,
respectively.
Figure 2:
Time course of baculovirus expression of
RED isoforms. Conditioned media (M) and Sf9 lysates (C) obtained 0, 24, 48, and 72 h after infection were
subjected to immunoblot analysis using the FLAG M2 monoclonal antibody
(see ``Experimental
Procedures'').
Figure 3:
Affinity cross-link labeling of I-rhIL-5 to S1-S2- and S3-
RED. Panel A,
aliquots of medium harvested 72 h postinfection with each of the
baculovirus constructs were incubated with 0.5 nM
I-rhIL-5, and unlabeled rhIL-5 (0, or 30 pM to 200 nM) as described under ``Experimental
Procedures.'' An aliquot (50-fold dilution) of each was subjected
to SDS-PAGE and autoradiography to assess competition. Panel
B, the resulting autoradiograms were scanned with a laser
densitometer (Molecular Devices) and the integral of the density of the
bands corresponding to the ligand-receptor complex obtained. The values
were expressed as a percentage of the labeling in the absence of
competing IL-5, and the EC
for competition calculated by
linear interpolation.
Figure 4:
Purification of S1-RED FLAG from 72 h
postinfection conditioned medium. Panel A, conditioned medium
from Sf9 cells infected with the S1-FLAG construct prior to
purification (lane 1), after a single pass over the affinity
column (lane 2) and fractions eluted from M2 anti-FLAG
immunoaffinity column with 100 mM glycine, pH 3.0 (lanes
3-10) were subjected to SDS-PAGE immunoblot analysis. Panel B, deglycosylation of purified S1-
RED FLAG with 2
units of N-glycosidase F for 16 h at 22 °C in the absence (lane 1) and presence (lane 2) of the
enzyme.
To evaluate the
secondary structure of the RED, its far-UV circular dichroism
spectrum was obtained between 195 and 260 nm (Fig. 5). The
spectrum manifested a broad, weak negative double minimum at 210 and
200 nm. Analysis of this spectrum by the self-consistent singular value
decomposition algorithm of Sreerama and Woody (18) yielded an
estimated secondary structure content of 42%
-sheet, 20% turn, 32%
disordered, and 6%
-helix. Thus, this portion of the IL-5 receptor
appears to consist primarily of
-structure. Also shown in Fig. 5is the CD spectrum of IL-5, which displayed the
characteristic double minimum at 208 and 222 nm expected for this
-helix rich protein. When a 1:1 molar complex was formed between
IL-5 and the extracellular portion of its receptor, the resulting
spectrum was dominated by the much stronger spectrum of the interleukin (Fig. 5). When the spectrum of the complex was compared to the
numerical sum of the spectra of the individual spectra of IL-5 and the
receptor domain, little difference was seen. This suggests that no
major reorganization of secondary structure occurs in either protein
when the complex is formed.
Figure 5:
Circular dichroism analysis of
IL-5/RED interactions. The far-UV circular dichroism spectrum of
recombinant human IL-5 gave the characteristic profile of this species
(
). The recombinant IL-5 receptor extracellular
domain construct S1-
RED spectrum(- - -)
suggested that the major structural feature of this protein was
-sheet (42%), with only 6% consisting of
-helix (see
``Results''). The spectra of a 1:1 molar complex of the
receptor domain and IL-5 (-) and the theoretical sum of
the individual spectra of the receptor domain and IL-5(- - -) were
essentially superimposable, implying that the conformational changes
occurring on receptor-ligand interaction were modest and subtle. The
spectra of the free receptor domain and IL-5 were obtained at a protein
concentration of 2.3 M, while that of the complex and the
mathematical sum at 1.15 M. The spectrum of the receptor
domain is multiplied by 4 for illustration purposes. All spectra were
measured in 6 mM sodium phosphate, 0.15 M NaCl, pH
7.0, at 22 °C.
To probe the possibility that a change in stability might occur when the two proteins bind, the thermally induced unfolding of IL-5, the receptor domain and the complex was measured by monitoring the CD at 208, 215, 222, 230, and 350 nm as a function of temperature. The CD spectrum of the complex was dominated by that of IL-5, and thus only a change in stability of this component could be unambiguously detected by this method. In fact, both the complex and IL-5 itself displayed midpoints of their unfolding temperatures at 72-74 °C (not illustrated), implying that there was no major change in the thermal stability of the IL-5 component upon complex formation.
To determine if an alteration in
tertiary structure might be induced by complex formation, the
tryptophan fluorescence emission spectra of the same proteins were
examined. The IL-5 receptor ectodomain (S1-RED) displayed a
fluorescence emission maximum at approximately 338 nm, suggesting that
its nine tryptophan residues are on the average relatively buried (Fig. 6). The single tryptophan of rhIL-5 appeared somewhat more
exposed, manifesting a fluorescence peak at 345 nm. The complex
produced a maximum at an intermediate location near 342 nm. No
difference was seen when the fluorescence emission peak of the complex
was compared to the numerical sum of the peaks of the receptor domain
and IL-5 themselves, again suggesting little structural alteration upon
complex formation (Fig. 6).
Figure 6:
Spectrofluorimetric analysis of
IL-5/RED interactions. Intrinsic fluorescence emission spectra of
IL-5 (
), the recombinant IL-5 receptor extracellular
domain, (- - -), a 1:1 molar complex of the receptor
domain and IL-5 (-), and the theoretical sum of the
individual spectra of the receptor domain and IL-5(- - -) are shown.
Protein concentrations and solution conditions were the same as those
shown in Fig. 5. The intensity of the spectra of the complex and
the sum of the proteins were divided by 2 to enhance comparison.
Proteins were excited at 295 nm to produce primarily tryptophan
emission and are corrected for Raman
scattering.
Fig. 7(panel A) shows an
overlay of the sensorgrams obtained after interaction of various
concentrations of the purified S1-RED protein with the IL-5
sensor-chip surface as described under ``Experimental
Procedures.'' Panel B illustrates the specificity of the
RED interaction with IL-5 since preincubation of the receptor
solution with IL-5 reduced the interaction of the soluble receptor with
the IL-5 sensor. With this experimental format, the K
of the
RED was calculated using the association and
dissociation rate constants (2.8
10
M
s
and 2.56
10
s
, respectively) derived from
the BIAcore sensorgrams and the equation K
= k
/k
to be 9
nM at 25 °C. This value did not differ significantly from
values determined for the S2- and S3-
RED forms (data not shown).
The effects of various conditions for the elution of S1-
RED from
the affinity column on the binding interactions of the resulting
purified
RED proteins were also studied using the IL-5 surface. No
differences in the association or dissociation rate constants were
detected when the S1-
RED was eluted either with 100 mM glycine, pH 3.0, or with an excess of the FLAG peptide (data not
shown), suggesting that exposure to low pH did not significantly affect
the tertiary structure of these proteins. Accordingly, glycine elution
was utilized for all additional experiments, as no further separation
(from the FLAG peptide) was required.
Figure 7:
BIAcore analysis of non-equilibrium
analysis of IL-5/RED interactions. Panel A, the relative
mass response (vertical axis, arbitrary units) was measured
for the interaction of purified S1-
RED FLAG (90 nM to
1.35 µM) with immobilized rhIL-5 (see ``Experimental
Procedures''). At 100 s (horizontal axis), PBS control
buffer (5 ml/min) was replaced with buffer containing the recombinant
receptor to assess the association rate of the complex. After 340 s,
this was in turn replaced by the control buffer to determine the
dissociation rate. The IL-5 surface was regenerated after each exposure
to the receptor (see ``Experimental Procedures''). Repeated
regeneration had no effect on the kinetics or magnitude of
interactions. Panel B, the relative mass response (vertical axis, arbitrary units) was measured as above for the
interaction of 230 nM of the
RED in the absence (upper trace) and presence (lower trace) of an excess
(500 nM) of IL-5. In the latter experiment, the high affinity
association was abolished, demonstrating the specificity of the
response. Panel C, the slopes (vertical axis) of the
association phase curves for each
chain concentration (panel
A) were plotted against the mass response (R; horizontal axis) for 10 s after exposure to the soluble
receptor at 100 s. The slope of each individual plot represents the
negative value for k
at each concentration of the
S1-
RED FLAG construct. Panel D, the values for k
were then plotted (vertical axis)
against the receptor concentration (horizontal axis; nM), and
an association rate constant (k
or k
) of 2.8
10
M
s
derived from the
slope of the line of best fit. An equivalent analysis (see
``Experimental Procedures'') for the dissociation phase of
the analysis (after 350 s; panel A) resulted in a dissociation
rate constant (k
or k
) of 2.56
10
s
. An equilibrium dissociation constant (K
) of 9 nM was then calculated
from k
/k
)
Figure 8:
Inhibition of rhIL-5-induced BCL-1
proliferation by RED. 3
10
murine BCL-1 cells
(
) responded by proliferation (MTT conversion measured by OD at
550 nm; vertical axis) to the presence of rhIL-5 (0-500
nM; horizontal axis). Preincubation (see
``Experimental Procedures'') with the purified S1-
RED at
30 nM (
), 100 nM (
), and 300 nM (
) shifted the IL-5 response curve to the right. Values are
the mean ± S.E. of 4-6 replicates from one of three
separate experiments with three S1-
RED preparations. Inset, The IL-5 proliferative response (vertical
axis) at 5 nM (
) and 20 nM (
)
agonist was plotted against the concentration of neutralizing
S1-
RED to demonstrate dose dependence.
Eosinophilic pulmonary inflammation has been recognized as a hallmark of chronic bronchial asthma for over 100 years, and the presence of eosinophils in the lung has recently been correlated with the degenerative pathophysiology characteristic of this disease(22) . The identification of IL-5 as a potent in vitro eosinophilopoietin(1) , is consistent with the ability of IL-5 overexpression in transgenic mice to induce tissue eosinophilia (23, 24) and of IL-5 antibodies to inhibit pulmonary eosinophilia in animals (25) and suggests that this cytokine plays a critical role in the development and tissue targeting of pro-inflammatory eosinophils. Thus, disruption of the actions of interleukin-5 offers a significant opportunity for novel therapeutic intervention in asthma. Accordingly, we have used the baculovirus expression system to produce recombinant epitope-labeled form of the extracellular domain of the human IL-5 receptor to allow detailed analyses of the binding and biological interactions of this component of IL-5 function.
The cDNA encoding the full-length IL-5
receptor chain (Fig. 1A) was isolated from a
strain of eosinophilic human erythroleukemic cells, shown previously to
possess high affinity IL-5 receptors, and to be activated by
pM concentrations of rhIL-5(26) . Alternative isoforms
corresponding to soluble forms of the extracellular domain of the
chain truncated prior to the transmembrane spanning region (3) were then used to engineer three epitope-labeled forms of
the ligand-binding domain of the
chain (Fig. 1, B and C). Transfection of these constructs into Sf9 cells
using recombinant baculoviral vectors resulted, after 48 h, in the
secretion into the medium of 43-kDa molecular mass proteins bearing the
FLAG epitope (demonstrated by immunoblot; Fig. 2). In the case
of the S1- and S2-
RED constructs, the protein was predominantly
secreted, with minimal immunoreactive tagged protein being detectable
in insect cell lysates. This high level of expression and secretion of
the
RED allowed demonstration of IL-5 binding directly in
conditioned medium by affinity cross-link labeling. In these
experiments, the ED
for competition of 0.5 nM of
I-IL-5 binding was found to be 2-5 nM,
with no significant differences observed between the various forms (Fig. 3).
The relatively high level of expression and
secretion of the RED also allowed rapid and mild immunoaffinity
purification with an anti-FLAG column (Fig. 4A).
Glycine-eluted material was heterogeneously glycosylated, the extent of
which did not appear to affect ligand binding. The affinity-purified
S1-
RED was found by sedimentation analysis to form a 1:1
ligand-receptor complex with a 70-74-kDa molecular mass as
predicted, demonstrating the utility of epitope-tagging in the
production of a soluble cytokine receptor subunit.
Circular
dichroism analysis (Fig. 5) and intrinsic fluorescence emission
spectroscopy (Fig. 6) indicated that the S1-RED existed
with a defined secondary and tertiary structure, which could be altered
to a substantially disordered form by elevated temperatures. The
S1-
RED consisted primarily of
-structure with a substantial
portion of its Trp residues buried in the protein's interior.
Comparison of the spectra of the interleukin/receptor domain complex to
that of the mathematical sum of the free ligand and receptor suggested
that little structural alteration occurred upon complex formation
(although small alterations in side chains might not be detectable by
these analyses). This absence of major conformational change upon
interaction with the ligand implies that direct signal transduction is
unlikely to occur via a membrane-associated
chain alone, but
requires further association with the
-subunit to activate the
latter's large intracellular domain. The
RED proteins were
also amenable to biophysical analysis using surface plasmon resonance
detection. Analysis of the kinetic parameters derived from
non-equilibrium binding using this technique (Fig. 6) gave a
calculated dissociation constant of 9 nM at 25 °C. This
value is within 2-fold of that estimated from the affinity cross-link
labeling experiments (which were performed at 4 °C), and is
consistent with an increase in affinity with reduced temperature as
shown by microcalorimetric determinations(21) . Indeed, this
value is in excellent agreement with that reported in analogous
immobilized IL-5 experiments (5.5 nM) for the non-tagged
soluble
chain expressed in Drosophila cells(21) . Thus, the process of FLAG epitope tagging does
not appear to affect IL-5/
RED interactions, although adding
tremendous utility for the expression and purification of these, and
additionally mutated forms of the soluble domain.
The
affinity-purified RED described above showed significant
inhibitory activity against an IL-5-induced proliferative response in
the murine B-cell lymphoma BCL-1 in a serum-free medium (Fig. 8). At a 60-fold molar excess, the soluble form of the
receptor completely ablated the proliferative response to IL-5 (Fig. 8, inset). This is consistent with the suggestion
that naturally occurring soluble forms of cytokine receptors act to
modulate the biological activity of these potent hematopoietins.
Whereas the potency of this modulation in the BCL-1 bioassay was
significantly less than that observed for the avidity of the soluble
receptor for IL-5 as measured by affinity cross-link labeling, and
non-equilibrium interactions, little is known about the stability of
this recombinant protein in cellular incubates, or the degree or
duration of IL-5 receptor occupancy required for signal transduction.
In this respect, a large molar excess of the soluble receptor may be
necessary to completely inhibit the biological response to IL-5 by
vastly reducing the frequency of reversible IL-5-receptor interactions
which could otherwise trigger irreversible cellular proliferation.
In summary, we have engineered three epitope-tagged soluble forms of
the extracellular domains of the IL-5 receptor -subunit and
demonstrated their equivalent abilities to interact with rhIL-5 at
nanomolar concentrations. After simple affinity purification, one of
these isoforms was shown to inhibit the proliferative activity of
rhIL-5 on murine B-lymphoma cells, suggesting that this novel construct
itself may have therapeutic potential in the treatment of eosinophilic
inflammation. Furthermore, circular dichroism and fluorescence spectral
analysis of this recombinant protein demonstrated that the
conformational changes associated with ligand binding were
comparatively minor and subtle. Therefore, with additional structural
information, it appears possible that molecules could be designed to
disrupt the interaction of IL-5 with its receptor and thus act as IL-5
receptor antagonists.