From the I. Medizinische Klinik, Abteilung Pathophysiologie,
Johannes Gutenberg Universität Mainz, Obere Zahlbacher Str. 63, 55101 Mainz, Germany, the Institut für Biochemie,
Klinikum der RWTH Aachen, Pauwelsstr. 30, 52057 Aachen, Germany, and
§ INSERM, C.J.F. 97-08, 4 rue Larrey, CHU Angers,
49033 Angers Cedex, France
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ABSTRACT |
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Interleukin-6 (IL-6) and ciliary neurotrophic
factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type
family of neuropoietic and hematopoietic cytokines. IL-6 signals by
induction of a gp130 homodimer (e.g. IL-6), whereas CNTF
and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130
and LIF receptor (LIFR). Despite binding to the same receptor component
(gp130) and a similar protein structure, IL-6 and CNTF share only 6%
sequence identity. Using molecular modeling we defined a putative LIFR
binding epitope on CNTF that consists of three distinct regions
(C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal
CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6
was exchanged with this epitope. The resulting IL-6/CNTF chimera lost
the capacity to signal via gp130 on cells without LIFR, but acquired
the ability to signal via the gp130/LIFR heterodimer and STAT3 on
responsive cells. Besides identifying a specific LIFR binding epitope
on CNTF, our results suggest that receptor recognition sites of
cytokines are organized as modules that are exchangeable even between
cytokines with limited sequence homology.
Ciliary neurotrophic factor
(CNTF)1 is a survival and
differentiation factor for a variety of neuronal and glial cells (1). Several groups have demonstrated its ability to prevent or slow down
neuronal degeneration in animal models of neuropathic diseases (2-4).
Non-neuronal effects of CNTF include maintenance of embryonic stem
cells in an undifferentiated state (5), initiation of an acute-phase
response in liver cells (6), and a myotrophic effect on denervated
skeletal muscles of mice (7).
CNTF belongs to the IL-6 type family of hematopoietic and neurotrophic
cytokines that also encompasses interleukin 6 (IL-6), leukemia
inhibitory factor (LIF), oncostatin M, cardiotrophin-1, and interleukin
11 (IL-11). All IL-6 type cytokines use a membrane spanning 130-kDa
glycoprotein, gp130, as a signal transducing subunit (8, 9). Some IL-6
type cytokines also use the LIF receptor (LIFR) and the oncostatin M
receptor for signaling. Despite very limited sequence homology, the
IL-6 type cytokines were predicted to share a common structure
consisting of four anti-parallel The biological response to CNTF is elicited by formation of a multiunit
receptor complex (15). CNTF first binds in a 1:1 stoichiometry to a
glycosylphosphatidylinositol-anchored ligand binding Immunoprecipitation experiments in solution as well as biophysical
evidence suggested that the IL-6 and CNTF receptor complexes are
hexamers consisting of IL-6, IL-6R Mutagenesis studies of CNTF, IL-6, and LIF have identified contact
sites of these cytokines with the subunits of their respective receptor
complexes. For IL-6 and CNTF the contact site with the receptor
Considering the conserved four-helical bundle structure of most
cytokines we reasoned that receptor recognition sites of cytokines might have evolved as discontinuous modules which should principally be
exchangeable between different cytokines. A comparison of the homology
based IL-6 model and the x-ray structure of CNTF (13, 27) prompted us
to define boundaries of the potential LIFR binding epitope of CNTF
which encompasses residues of the C-terminal A-helix, the N-terminal AB
loop, the BC loop, the C-terminal CD-loop, and the N-terminal D-helix.
The transfer of this putative "LIFR binding module" from CNTF to
IL-6 resulted in a chimeric IL-6/CNTF molecule that binds to IL-6R Cells and Reagents--
Human SK-N-MC neuroblastoma, HepG2 and
Hep3B hepatoma and COS-7 cells (bought from ATCC (Manassas, VA)) were
routinely grown in RPMI or Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. BAF/3 cells transfected with
human gp130 were a kind gift from Immunex (Seattle, WA). BAF/3-(gp130)
cells were stably transfected with cDNAs coding for human IL-6R,
human CNTFR, or human LIFR as described elsewhere (17). Thus four different cell lines were generated: BAF/3-(gp130,IL-6R) cells, BAF/3-(gp130,LIFR) cells, BAF/3-(gp130,LIFR,IL-6R) cells, and BAF/3-(gp130,LIFR,CNTFR) cells. The relative expression of the receptors of the IL-6 family on the different BAF/3 cell lines as well
as the SK-N-MC cells was analyzed by fluorescence-activated cell sorter
analysis using the murine monoclonal antibodies B-S12 (anti-gp130),
B-R6 (anti-IL-6R), AN-E1 (anti-LIFR), and AN-D3 (anti-CNTFR) and is
shown in Table I. B-S12 and B-R6 have been described in detail before
(36), AN-E1 and AN-D3 are newly developed murine monoclonal anti-LIFR
and anti-CNTFR antibodies.2
Anti-STAT3 mAb was obtained from Transduction Laboratories (Lexington, KY), anti-phosphotyrosine mAb 4G10 was bought from UBI (Lake Placid, NY), and the anti-mouse peroxidase-coupled mAb was from
BIOSOURCE (Calmarillo, CA). The restriction
enzymes NcoI, HindIII, and XbaI were
obtained from AGS (Heidelberg, Germany); calf intestinal phosphatase
was bought from Boehringer Mannheim (Mannheim, Germany). The
restriction enzyme AccI, Vent DNA polymerase, and T4 DNA
ligase were purchased from New England Biolabs (Schwalbach, Germany), the T7 sequencing kit was from Pharmacia (Freiburg, Germany). [ Construction of Chimeras of Human IL-6 and CNTF--
The
discrete regions of human IL-6 and CNTF used to construct the chimeras
are given in Fig. 1. Construction of chimeras IC1, IC2, and IC3 relied
heavily on PCR-ligation-PCR (37). cDNAs of human IL-6 and CNTF
cloned into the pRSET5d bacterial expression vector via NcoI
and HindIII restriction sites served as PCR templates (27,
28). To construct chimera IC1, an IL-6 cDNA fragment encoding the
N-terminal part of the molecule to Arg40 and a C-terminal
CNTF cDNA fragment starting at the codon for Asp36 were
amplified by standard PCR. The PCR products were ligated (37) and the
ligation product subsequently amplified by PCR to produce the fragment
IL-6(Pro1-Arg40):CNTF(Glu36-Met56).
This fragment was purified from a 1% agarose gel and ligated to the
amplified C-terminal IL-6 cDNA fragment starting at the codon for
Asn60. The ligation product, IC1, was amplified by PCR and
subsequently cloned into the bacterial pRSET5d expression vector after
digestion with NcoI and HindIII. Chimeras IC2 and
IC3 were constructed analogously. The sequences of all primers used are
available on request. Chimeras IC4 to IC6 were produced from chimeras
IC1 to IC3 using NcoI, AccI, XbaI, and
HindIII restriction enzymes. IC7 was produced from IC1 and
IC6. The integrity of all constructs was verified by restriction
fragment analysis and DNA sequencing according to standard methods
(38).
Molecular Modeling--
The boundaries of the IL-6 and CNTF
regions exchanged were derived from a molecular model of IL-6 (27) and
the x-ray structure of CNTF as taken from the Brookhaven data bank
(accession code 1cnt). Recently, the x-ray as well as the NMR structure
of human IL-6 were solved (12, 39), the regions interchanged are color
coded on the ribbon models of the IL-6 NMR and CNTF x-ray structures.
Structure comparisons and all computer graphic work were performed with
the WHATIF program package running on an SGI-Indigo2 (40). For
graphical representation the program Grasp was used (41).
Preparation and Quantification of Mutant
Proteins--
Escherichia coli bacteria (strains BL21-DE3
and BL21 pLysS) were transformed with the expression vector pRSET5d
containing human IL-6, human CNTF, and chimeric cDNAs. Transformed
bacteria were grown to an A600 of approximately
0.5-0.7 and induced to produce recombinant protein by addition of 0.4 mM isopropyl-1-thio- CD Spectra--
CD spectra of all chimeras were taken on an AVIV
CD spectrometer 62DS and on a Jasco J-600 spectropolarimeter. Both
instruments were calibrated with an aqueous solution of
10-camphosulfonic acid at 25 °C. The spectral band width was 1.5 nm.
Protein samples were dissolved in water, the pH was adjusted to
3.5.
Bioassays--
Proliferation of at least two different clones of
the transfected BAF/3-(gp130) cell lines in response to human IL-6,
human CNTF, human LIF, and the chimeras IC1 to IC7 was measured in
96-well microtiter plates. The cells were exposed to test samples for 72 h and subsequently pulse-labeled with
[3H]thymidine for 4 h. Proliferation rates were
measured by harvesting the cells on glass filters and determination of
the incorporated radioactivity by scintillation counting. For each
mutant and each cell line the proliferation assay was performed at
least three times in triplicate. Haptoglobin production by HepG2 and
Hep3B cells in response to stimulation with the above cytokines and chimeras was measured by a sandwich enzyme-linked immunosorbent assay
as described recently (43).
cDNAs and COS-7 Cell Transfection--
COS-7 cells were
simultaneously transfected with cDNAs for human gp130, LIFR, IL-6R,
and STAT3 with the DEAE-dextran method as described (44). gp130 and
LIFR were subcloned into the p409 expression vector (a kind gift from
Immunex). The human IL-6R was in the pCDM8 expression vector, STAT3 in
pSVL. After transfection, COS-7 cells were cultured for 2 days in RPMI
plus 10% fetal calf serum before starvation in serum-free medium.
Tyrosine Phosphorylation Analysis--
SK-N-MC, HepG2, and
transfected COS-7 cells were starved overnight in serum-free medium
before stimulation with cytokines. After stimulation, cells were lysed
in 50 mM Tris, pH 7.5, 100 mM NaCl, 50 mM sodium fluoride, and 3 mM sodium
orthovanadate containing 1% Brij-96. Insoluble material was pelleted
and the supernatants immunoprecipitated overnight with B-S12 anti-gp130 (2 µg/ml) or AN-E1 anti-LIFR mAb (10 µg/ml). The complexes were isolated with Protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) and the supernatants subjected to a second immunoprecipitation with
anti-phosphotyrosine mAb 4G10 (5 µg/ml) after addition of 1% Nonidet
P-40. The precipitates were subjected to SDS-PAGE and transferred to an
Immobilon membrane (Millipore, Bedford, MA). The membranes were
incubated with an appropriate primary antibody before being labeled
with a secondary antibody coupled to peroxidase. Subsequently, the
membranes were developed using the Amersham ECL kit.
Structural Comparison of IL-6 and CNTF--
The four-helix bundle
fold (Fig. 1A) is the
characteristic structure of most cytokines (11). A schematic
representation of the backbone atoms of the IL-6 NMR and the CNTF x-ray
structure is shown in Fig. 1B. Both structures were
superimposed onto each other using the C
A structural analysis of CNTF suggested that amino acid residues
situated in the C-terminal A-helix
(Glu36-Gln42) and N-terminal AB loop
(Gly43-Met56) form site IIIA, while the BC loop
(His97-Asp104) with adjacent residues of the B-
(Leu91-Val96) and C-helix
(Phe105-Ile109) represents site IIIB. Together
with site IIIC which closely corresponds to Bazan's D1 motif (10) and
consists of the C-terminal CD loop
(Gly147-Leu151) and the N-terminal D-helix
(Phe152-Leu162) they constitute the putative
LIFR-binding epitope on CNTF (site III). Potential site III residues of
IL-6 were substituted by these CNTF amino acids. The precise amino acid
sequences of the resulting chimeric molecules are given in Fig.
1C. Considering the extensions of helices and loops in IL-6
and CNTF (12, 13), site IIIB of CNTF extends the B-helix of the
resulting chimeras by five amino acids, whereas the BC loop is
lengthened by two amino acids. Site IIIA and IIIC differ in two and one
amino acid residues, respectively. The transferred part of the CNTF AB
loop (Gly43-Met56) includes the CNTF mini-helix
(Lue50-Ala53) (13), whereas the putative
E-helix of IL-6 (Pro141-Gln152) (12) is not
affected by the exchange of site IIIC.
The chimeras were expressed in E. coli, refolded, and
purified to near homogeneity as shown in Fig. 1D. Correct
folding of each chimera was checked by CD spectroscopy (data not
shown). Despite small differences, the CD spectra of all molecules
exhibited band shapes typical of four-helix bundle proteins (28, 45) indicating successful refolding of the chimeric proteins. Chimera IC6
could not be expressed in any of our bacterial expression systems.
Protein concentrations were estimated from Coomassie Blue staining,
silver staining (data not shown), and amino acid analysis of the
individual proteins.
Chimeras IC5 and IC7 Induce Phosphorylation of the gp130/LIFR
Heterodimer--
To verify the successful transfer of a LIFR binding
epitope from CNTF to IL-6 we assessed the ability of the chimeras
carrying the most extended amino acid stretches from CNTF, IC5, and
IC7, to induce heterodimerization and phosphorylation gp130 and LIFR in
SK-N-MC neuroblastoma cells (Fig. 2,
A and B). SK-N-MC cells express gp130, LIFR, and
CNTFR, but no IL-6R on the cell surface (Table
I) and display particularly strong
tyrosine phosphorylation after stimulation with IL-6-type cytokines (9,
46). Phosphorylation of SK-N-MC gp130 was observed after stimulation
with CNTF and also with IL-6, IC5, and IC7 in the presence, but not the
absence of the soluble IL-6R (Fig. 2a). IC5, IC7, and CNTF also induced phosphorylation of a protein of around 190-kDa molecular mass coprecipitating with gp130, most likely the LIFR. Immunoprecipitation of SK-N-MC with the anti-LIFR mAb AN-E1 confirmed phosphorylation of
LIFR after treatment with IC5, IC7, and CNTF, but not IL-6 (Fig.
2B). The slight band apparent after stimulation with IL-6 in
the presence of the soluble IL-6R was due to background
phosphorylation, in two further experiments a similar band was seen in
the control lane (data not shown). Chimeras IC5 and IC7 and CNTF also
induced coprecipitation of phosphorylated gp130. In the presence of the soluble IL-6R IL-6, IC5 and IC7 triggered STAT3 phosphorylation as did
CNTF (Fig. 2C), thus demonstrating activation of the
signaling cascade downstream of gp130 and LIFR. Phosphorylation of
gp130, LIFR, and STAT3 after stimulation with IC7 equaled that observed after stimulation with CNTF, whereas we consistently observed weaker
phosphorylation of these proteins after application of IC5, although
10-fold higher concentrations of IC5 were used (Fig. 2). Together,
these experiments strongly suggest that upon binding to the IL-6R, IC5
and IC7 cause heterodimerization and phosphorylation of gp130 and LIFR
as well as activation of STAT3.
The phosphorylation pattern of HepG2 cells after stimulation with IL-6,
IC5, IC7, and LIF confirmed the results on SK-N-MC cells (Fig.
3, A-C). gp130 phosphorylation
was clearly weaker after treatment with IC5 and IC7 than after IL-6,
but stronger than LIF induced gp130 phosphorylation (Fig.
3A). Coprecipitation of an activated LIFR was clearly
discernible after stimulation with IC7 and LIF, whereas much longer
exposures of the x-ray film were necessary to detect the phosphorylated
LIFR after stimulation with IC5 (data not shown). Immunoprecipitation
of HepG2 cells with anti-LIFR mAb AN-E1 demonstrated LIFR
phosphorylation only after treatment with LIF, IC7, and IC5, but not
IL-6 (Fig. 3B). With the former cytokines, phosphorylated
gp130 was also coprecipitated, which in the case of IC5 became visible
only after longer exposure of the x-ray film. Although IC5 induced
weaker phosphorylation of gp130 and LIFR than LIF or IC7 this
difference was not observed with regard to the phosphorylation of STAT3
suggesting amplification of the activation signal (Fig.
3C).
Furthermore, we reconstituted the cellular response to IC7 in COS-7
cells by transfecting these cells with cDNAs coding for IL-6R,
LIFR, gp130, and STAT3. Stimulation of the transfected COS-7 cells with
LIF and IC7 resulted in appearance of the double band typical of the
phosphorylated gp130/LIFR heterodimer after immunoprecipitation, while
IL-6 only induced phosphorylation of gp130 (Fig.
4A). All three cytokines were
able to trigger phosphorylation of STAT3 (Fig. 4B).
Biological Activity of the IL-6/CNTF Chimeras--
To assess the
biological activity of the IL-6/CNTF chimeras we first measured
secretion of the acute-phase protein haptoglobin by stimulated HepG2
and Hep3B human hepatoma cells. In contrast to HepG2 cells, Hep3B cells
express gp130 and IL-6R, but not the LIFR (6). Chimeras IC2, IC5, and
IC7 achieved virtually the same activity as IL-6 on HepG2 cells (Fig.
5A) which accords well with
the almost equal phosphorylation of STAT3 induced by IL-6, IC5, and IC7
in these cells (Fig. 3C). IC2 was clearly active on Hep3B
cells, although around 100-fold higher concentrations were needed to
exert the same half-maximal activity as IL-6. In contrast, IC5 and IC7
were completely inactive on Hep3B cells (Fig. 5B). Thus
chimeras IC5 and IC7 have lost the ability to elicit cellular responses
via gp130 alone. Biological activity of IC1 and IC3 on HepG2 cells was
only observed at very high concentrations that even exceeded those
needed for unspecific stimulation by CNTF (Fig. 5A). These
two chimeras also showed some minor activity on Hep3B cells.
In a second bioassay we investigated the proliferative response of
transfected BAF/3 cells to the chimeras. Murine BAF/3 cells display a
strong proliferative response to IL-3, but do not respond to human IL-6
type cytokines, since they neither express gp130 nor any other receptor
of this family (47). However, BAF/3 cell lines transfected with human
gp130 and human IL-6R proliferate upon treatment with human IL-6 (Fig.
6A) and after additional transfection of the human LIFR also upon human LIF (Fig.
6B). BAF/3-(gp130,IL-6R) cells cannot be stimulated to
proliferate by human LIF (Fig. 6A). The response of the
above cell lines to our IL-6/CNTF chimeras is more diverse. Hardly any
proliferation was observed in response to IC3 and IC4, whereas IC2 was
almost as active as IL-6 on both cell lines. IC1 was also active on
both cell lines, but 300-1000-fold higher concentrations than those of
IL-6 were needed to achieve half-maximal activity. The most significant
findings, however, concern chimeras IC7 and IC5: on BAF/3-(gp130,IL-6R,LIFR) cells IC7 had virtually the same activity as
LIF, while roughly 100-fold higher concentrations of IC5 were needed to
achieve the same half-maximal activity as IC7. Absence of the LIFR as
in BAF/3-(gp130,IL-6R) cells (Fig. 6A) prevented a
proliferative activity of IC5 and IC7 on these cells.
Cells that only possess gp130 and LIFR, but not CNTFR
The preparation of IC7 contained trace amounts of LPS which amounted to
30-300 pg/ml at IC7 concentrations (10-50 ng/ml) where maximal
biological activity of IC7 was reached. However, it is highly unlikely
that our assays were distorted by a direct LPS effect, since SK-N-MC
cells only react to IC7 in the presence, but not the absence of the
soluble IL-6 receptor (Fig. 2). Furthermore, BAF/3-(gp130,LIFR) cells
served as the parent cell line to BAF/3-(gp130,IL-6R,LIFR) cells and in
contrast to the latter are unresponsive to IC7 (Fig. 6). These
differences cannot be explained by a direct LPS effect.
Our experiments define a specific LIFR binding epitope on CNTF
that corresponds to site III in the terminology established by the
growth hormone receptor complex paradigm (13, 29). The epitope consists
of amino acid residues located in the C-terminal A-helix, the
N-terminal AB loop (Glu36-Met56), the BC loop
with adjacent parts of B- and C-helix
(Leu91-Ile109), and the C-terminal CD loop with
the adjoining N-terminal D-helix (Gly147-Leu162). This accords well with an
analysis of the x-ray structure of human CNTF by McDonald et
al. (13) who predicted a LIFR binding epitope on CNTF consisting
of the AB, BC, and CD loops. We could successfully substitute one of
the two established gp130-binding sites on IL-6, namely site III (33),
by this LIFR binding epitope. In contrast to IL-6, the resulting
chimera IC7 induced heterodimerization of gp130 and LIFR (Figs. 2,
A and B; 3, A and B; and
4A). IC7 induced phosphorylation of gp130 and LIFR as
strongly as CNTF (Fig. 2) and only slightly weaker than LIF (Figs. 3
and 4). Biological activity on responsive cells was practically
undiminished compared with human LIF (Fig. 6B).
Consequently, the transferred LIFR binding epitope of CNTF appears to
be complete, i.e. it contains all amino acids necessary for
LIFR binding and activation. The successful reconstitution of the
complete LIFR binding epitope of CNTF on IL-6 suggests that the
receptor recognition sites of hematopoietic and neuropoietic cytokines
can be regarded as discontinuous modules which could, in principle, be
exchanged between different cytokines.
While IC7 contains the complete LIFR binding epitope our data do not
allow to decide whether it is the minimal epitope sufficient for a
fully active IL-6/CNTF chimera. The minimal epitope sufficient to
stimulate cells via gp130 and LIFR was the combination of CNTF sites
IIIA and IIIC in chimera IC5 (Figs. 5A and 6B).
At 10-fold higher concentrations than IL-6, IC7, CNTF, and LIF chimera
IC5 also induced phosphorylation of the gp130/LIFR heterodimer and the
downstream effector STAT3 (Figs. 2 and 3). Consequently, CNTF site IIIB
(BC loop, adjacent residues of B-/C-helices) does not appear to be an
essential part of the LIFR binding epitope. Nevertheless, the BC loop
region is an important part of the LIFR binding epitope, since its
presence in IC7 greatly enhances interaction of gp130 and LIFR (Figs. 2
and 3) and biological activity (Fig. 6B) compared with IC5.
In contrast, the BC loop seems to be less important for the site III
gp130 binding epitope on IL-6, since IC2 which carries the BC loop of
CNTF shows only slightly diminished activity compared with IL-6 (Figs.
5B and 6A).
Even at very high concentrations none of the IL-6/CNTF chimeras could
elicit biological effects on cells that lacked the IL-6 receptor (Fig.
6, C and D). Likewise, IC5 and IC7 were unable to
induce formation of the phosphorylated gp130/LIFR heterodimer in the
absence of the soluble IL-6R (Fig. 2). Consequently, binding to the
IL-6R is essential for biological activity of the chimeras. Furthermore, the CNTFR cannot replace the IL-6R as receptor Two residues of CNTF site IIIC, Phe152 and
Lys155, are conserved in all members of the IL-6 family
that signal via LIFR (34). Mutation of these residues abolished binding
of CNTF and LIF to LIFR (30, 34). Di Marco et al. (34)
therefore restricted a putative LIFR binding epitope of CNTF to the
C-terminal CD loop/N-terminal D-helix. Studies on the binding of
chimeric murine and human LIF to human and murine LIFR concluded that
six residues of human LIF (Asn57, Ser107,
His112, Val113, Val155, and
Lys158) located in the short BC loop, the N-terminal
C-helix, and the CD loop contribute most of the binding energy to human
LIFR (35, 48). With the exception of Asn57 equivalent
residues on CNTF are part of the LIFR binding epitope transferred to
IC7. The biological inactivity of IC4 (Fig. 5A and
6B) which lacks the CNTF site IIIC is in line with these
concepts. However, the inactivity of IC3 reveals that site IIIC alone
is unable to recruit the LIFR, whereas the combination of site IIIA and
IIIC as in IC5 is active. Thus the C-terminal A-helix and N-terminal AB
loop are an indispensable and important part of the LIFR binding
epitope of CNTF (Figs. 5A and 6B). Contributions of the same region of LIF to LIFR binding might have escaped the approach of Owczarek and Layton due to the high homology of murine and
human LIF in their AB loops (35). The importance of site IIIA for
binding of IL-6-type cytokines is further indicated by a comparison of
IC2 and IC1. Exchange of the BC loop (site IIIB) as in IC2 has an only
small effect on biological activity, whereas exchange of site IIIA in
IC1 substantially reduces, but does not abolish, bioactivity of IC1
compared with IL-6. The combined sites IIIB and IIIC (BC loop,
C-terminal CD loop/N-terminal D-helix) might therefore constitute a
minimal epitope for contact to the second gp130 molecule.
Unfortunately, our inability to express chimera IC6 leaves open whether
combined CNTF sites IIIB and IIIC constitute an epitope sufficient to
induce signaling via LIFR.
Chimera IC7 is fully active on cells that express human IL-6R
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices (A, B, C, and D) connected
by two long cross-over loops (AB, CD) and one short loop (BC) (10, 11).
This so-called "four-helix bundle" structure represents a
fundamental protein fold characteristic of most cytokines (11).
Crystallographic and NMR studies have confirmed this structure for
IL-6, CNTF, and LIF (12-14).
-unit, CNTF
receptor (CNTFR
), which is not involved in signal transduction (16).
This is followed by the recruitment of gp130 and LIFR as membrane
spanning signal transducing
-units (15), which in turn form a
disulfide-linked heterodimer that activates the JAK/STAT and the
Ras/MAP kinase pathways (8). Signaling of cardiotrophin-1, LIF, and
oncostatin M also occurs via a gp130/LIFR heterodimer. Similar to CNTF,
cardiotrophin-1 first binds to non-signaling receptor
-unit (8),
whereas LIF and oncostatin M directly bind to LIFR and gp130,
respectively (17). In contrast, binding of IL-6 to a non-signaling
-unit, gp80 (IL-6R
), induces gp130 homodimerization and
subsequent activation of Jak/Tyk kinases (8).
, and gp130 in a 2:2:2 stoichiometry (18, 19) or CNTF, CNTFR
, gp130, and LIFR in a 2:2:1:1
ratio (20). However, recent analyses of crystallographic and
mutagenesis data of CNTF suggested a tetrameric complex as the simplest
model of the CNTFR complex (13, 21). Furthermore, an arrangement of
cytokine and cytokine receptors as in the hexameric models of Paonessa
and de Serio (18, 20) has been considered to be inconsistent with the
known dimensions of IL-6 and CNTF (22). Other authors argued that
structural constraints of the above hexameric models would lead to
contradictions with the hitherto observed general conservation of the
structural arrangement of cytokine receptors (23). A new hexameric
model for the IL-6 receptor complex could alleviate these structural
objections (23). Adapted to the CNTFR receptor complex it nevertheless
implies, as does the model suggested by de Serio et al. (20)
that the same site of CNTF is able to contact either gp130 or LIFR
which rules out the existence of a genuine and specific LIFR-binding site on CNTF.
-unit could be mapped to a site that includes residues of the
C-terminal AB loop and the C-terminal D-helix (13, 21, 22, 24-28).
This site corresponds to site I of growth hormone in its receptor
complex (29). Residues of the A- and C-helices of CNTF, LIF, and IL-6
constitute a gp130-binding site which is equivalent to site II of
growth hormone in its receptor complex (21, 30-32). In IL-6, a second
gp130-binding site consists of amino acids residues of the N-terminal
AB loop, the C-terminal CD loop, and the N-terminal D-helix (27, 33).
This site is now termed site III in continuation of the growth hormone
terminology. Crystallographic and mutagenesis studies of CNTF and LIF
indicated that residues of the C-terminal B-helix, possibly the BC
loop, CD loop, and the N-terminal D-helix constitute site III in these cytokines (13, 21, 30, 34, 35). These experiments also suggested site
III as a potential LIFR binding epitope in LIF and CNTF.
and signals via a heterodimer of gp130 and LIFR. Effectively, this
"module swap" created a new cytokine with LIF-like, but IL-6R
dependent activity on cells expressing gp130, IL-6R
, and LIFR. On a
more general basis, our results indicate that cytokines are organized
as a set of modules, making specific contacts to different receptors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP, [thio-
-35S]dATP, and
[3H]thymidine were obtained from Amersham International.
Oligonucleotides were bought from Eurogentec (Seraing, Belgium),
Brij-96 and Nonidet P-40 from Sigma (Munich, Germany).
-D-galactopyranoside. After 2 h, purification of inclusion bodies and denaturation with 6 M guanidinium chloride was performed as described before
(42). Refolding of proteins was achieved by dialysis against refolding buffer (1 M guanidinium chloride, 3 mM oxidized
glutathione, 0.6 mM reduced glutathione, 12 h) and 20 mM Tris-Cl, pH 6.8 (12 h). The purity of the recombinant
proteins was ascertained by 12.5% SDS-PAGE, stained with Coomassie
Blue or silver. In addition, protein concentrations were determined by
hydrolysis of the proteins in 6 M HCl and subsequent amino
acid analysis. LPS concentrations in the purified protein preparations
were ascertained with the Limulus amobecyte lysate assay (Biowhittaker,
Walkersville, MD).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-atoms of the
helices to identify putative components of the site III receptor
binding epitope. The segments of IL-6 and CNTF which participated in
the "epitope shuffle" of site III are color coded and designated as
IIIA, IIIB, and IIIC, respectively (Fig. 1, A and
B).
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Fig. 1.
The site III gp130 and LIFR binding epitope
of IL-6 and CNTF, respectively. A, schematic drawing of the
common four-helix bundle cytokine fold. B, ribbon models of
the recently solved IL-6 NMR and CNTF x-ray structures. The different
parts of site III are color coded: yellow (site IIIA),
green (site IIIB), and blue (site IIIC).
C, bar representation of IL-6, CNTF, and chimeras
IC1 to IC7. Sequence stretches that are part of the exchanged epitopes
of IL-6 and CNTF are hatched. On CNTF the N- and C-terminal
amino acid residues of the transferred stretches are designated in
single letter code, on IL-6 the residues adjacent to the
transferred CNTF stretches are denoted. The symbols located
left of the bars are kept throughout the figures
of this article to mark the respective mutant or natural cytokine.
D, SDS-PAGE (12.5%) of the purified IL-6/CNTF chimeras
after staining with Coomassie Blue. IC6 could not be expressed in our
bacterial expression systems.
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Fig. 2.
Tyrosine phosphorylation of gp130, LIFR, and
STAT3 in SK-N-MC human neuroblastoma cells. SK-N-MC cells were
stimulated for 10 min with CNTF and with IL-6, IC5, and IC7 in the
absence or presence of the soluble IL-6R (sIL-6R). IC5 and
sIL-6R were used at a concentration of 500 ng/ml; CNTF, IL-6, and IC7
were used at 50 ng/ml. A, cells were lysed and precipitated
with anti-gp130 mAb B-S12, precipitates were blotted with anti-Tyr(P)
mAb 4G10 after SDS-PAGE. B, cells were lysed and
precipitated with anti-LIFR (gp190) mAb AN-E1, precipitates were
blotted with anti-Tyr(P) mAb 4G10 after SDS-PAGE. C, after
precipitation with anti-gp130 mAb B-S12 supernatants were subjected to
a second immunoprecipitation with anti-Tyr(P) mAb 4G10. After SDS-PAGE
of the precipitates, membranes were blotted with anti-STAT3. Virtually
identical results were obtained in two further experiments.
Relative expression of receptors of the IL-6 family present in the
BAF/3 cell lines and SK-N-MC cells used in this study
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Fig. 3.
Tyrosine phosphorylation of gp130, LIFR and
STAT3 in HepG2 human hepatoma cells. HepG2 cells were stimulated
for 10 min with IL-6, IC5, IC7, and LIF. Cytokine concentrations were
50 ng/ml, except for IC5 which was used at 500 ng/ml. A,
cells were lysed and precipitated with anti-gp130 mAb B-S12,
precipitates were blotted with anti-Tyr(P) mAb 4G10 after SDS-PAGE.
B, cells were lysed and precipitated with anti-LIFR (gp190)
mAb AN-E1, precipitates were blotted with anti-Tyr(P) mAb 4G10 after
SDS-PAGE. C, after precipitation with anti-gp130 mAb B-S12
supernatants were subjected to a second immunoprecipitation with
anti-Tyr(P) mAb 4G10. After SDS-PAGE of the precipitates, membranes
were blotted with anti-STAT3.
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Fig. 4.
Reconstitution of the IL-6R/gp130/LIFR/STAT3
signaling system in COS-7 cells. Phosphorylation of gp130, LIFR,
and STAT3 was measured in COS-7 cells after transfection with human
cDNAs for IL-6R, gp130, LIFR, and STAT3 and stimulation with IL-6,
IC7, and LIF at 50 ng/ml. A, cells were lysed and
precipitated with anti-gp130 mAb B-S12, precipitates were blotted with
anti-Tyr(P) mAb 4G10 after SDS-PAGE. B, the supernatant of
A was subjected to a second precipitation with anti-Tyr(P)
mAb 4G10 and blotted with anti-STAT3 after SDS-PAGE.
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Fig. 5.
Secretion of the acute-phase protein
haptoglobin induced by IL-6/CNTF chimeras. A,
haptoglobin secretion induced by IL-6, CNTF, and the chimeras IC1 to 5 and IC7 on human HepG2 cells. B, haptoglobin secretion
induced by IL-6, LIF, and the chimeras IC1 to 5 and IC7 on Hep3B cells.
LIF is symbolized by open squares, the other symbols are as
described in the legend to Fig. 1C.
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Fig. 6.
Proliferative response of transfected BAF/3
cells to the IL-6/CNTF chimeras. A, effect of IL-6,
LIF, and the chimeras IC1 to 5 and IC7 on BAF/3 cells transfected with
gp130 and IL-6R . LIF is symbolized by open squares, the
other symbols are as described in the legend to Fig. 1C.
B, effect of IL-6, LIF, and the chimeras IC1 to 5 and IC7 on
BAF/3 cells transfected with gp130, IL-6R
, and LIFR. C,
effect of IL-6, LIF, and the chimeras IC1 to 5 and IC7 on BAF/3 cells
transfected with gp130 and LIFR. D, effect of IL-6, CNTF and
the chimeras IC1 to 5 and IC7 on BAF/3 cells transfected with gp130,
LIFR, and CNTFR.
, are usually
inert to stimulation by CNTF. However, at high concentrations CNTF can
stimulate these cells without requirement of CNTFR
(6, 16, 17). To
test whether the transferred amino acid residues of CNTF could confer
this ability to chimeras IC1 to IC7, the chimeras were tested on BAF/3
cells transfected either with gp130 and LIFR alone (Fig. 6C)
or on cells transfected with gp130, LIFR, and CNTFR
(Fig.
6D). None of the chimeras showed any activity on either of
the two cell lines, while LIF and CNTF were fully active. Thus, the
chimeras do not possess the ability to elicit LIF-like responses on
cells without the IL-6R (Fig. 6C), nor can the CNTFR
substitute for the IL-6R (Fig. 6D).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-unit for the chimeras (Figs. 2 and 6D). Contrary to a previous
prediction based on structural considerations (21) CNTF amino acids
36-56, which are roughly equivalent to the transferred CNTF site IIIA, may thus not be involved in binding to the CNTFR.
,
gp130, and LIFR (Figs. 5A and 6B), whereas it is
virtually inactive on cells that only possess IL-6R
and gp130 (Figs.
5B and 6A). This observation indicates that the
epitope transferred from CNTF to IL-6 cannot bind gp130 which otherwise
would enable chimera IC7 to generate a gp130 homodimer and hence to
signal. Therefore, site III of CNTF as defined by our experiments
constitutes a specific binding site for LIFR. This finding confutes the
existence of a versatile gp130/LIFR binding epitope at site III on CNTF (49) which is a prerequisite of a hexameric CNTFR complex (Fig. 7, A and B).
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Fig. 7.
Different models of the CNTFR complex.
A, De Serio's model of the CNTFR complex. The different
receptor binding epitopes in CNTF are denoted as in Paonessa's model
of the IL-6R complex. B, Simpson's model of the IL-6R
complex adapted to CNTF. Note that there is no information available on
domains of the LIF receptor involved in binding to cytokines.
C, tetrameric model of the CNTFR complex. The LIFR
"shields" the top of CNTF site III. The only possible dimerization
site left is on the rear side of CNTF, consisting of helices B and
C.
Immunoprecipitation experiments (18, 20) and gel size exclusion chromatography coupled to sedimentation equilibrium studies (19) indicated that in solution the high affinity receptor complexes of IL-6 and CNTF are hexameric (Fig. 7, A and B). Other authors, however, have argued (22) that the known dimensions of IL-6 and CNTF are hardly compatible with the arrangement of cytokine and cytokine receptors as suggested by the models of Paonessa and de Serio (18, 20). Based on structural considerations Simpson et al. (23) developed a new hexameric model of the IL-6R complex which also accounts for the observation that antibodies against the gp130 Ig-domain neutralize IL-6 bioactivity. In Fig. 7B, we have adapted the Simpson model to the CNTFR complex. Both models of the hexameric CNTFR complex imply that site II and site III are nonspecific binding sites for gp130 and LIFR. In contrast, our data indicate that CNTF site III is a specific LIFR-binding site. It should be noted that the co-immunoprecipitation experiments (18) leading to the concept of a hexameric CNTFR complex were performed with soluble recombinant receptor proteins at cytokine concentrations of around 200 µM. It has been reported that recombinant CNTF forms an antiparallel dimer at concentrations higher than 40 µM with the contact site mainly consisting of amino acid residues from the B- and C-helix (13). In our view the concept of a hexameric CNTFR complex in solution might thus not necessarily reflect the composition of the membrane bound receptor complex.
An alternative tetrameric model of the CNTF receptor complex is
presented in Fig. 7C. Site I is occupied by CNTFR and
encompasses the C-terminal D-helix, which is in keeping with the
affinity enhancing effect of the D-helix substitutions S166D, Q167A,
and V170R (26). Analogously to IL-6 (27) the C-terminal AB loop of CNTF
is involved in this epitope. gp130 binds to site I while LIFR locates
to site III and shields the top of the molecule. This architecture is
very reminiscent of the IL-2 receptor complex: the - and
-subunits of the IL-2 receptor bind to sites I and II, respectively,
whereas site III is occupied by the
-subunit (50). The tetrameric
model of CNTFR complex only requires the minimal number of complex
components, i.e. CNTF, CNTFR
, gp130, and LIFR in 1:1:1:1
stoichiometry. Such a minimal composition of the receptor complex has
recently also been demonstrated for the LIFR complex which is a trimer
consisting of LIF and one molecule each of gp130 and LIFR (51).
By transferring the specific LIFR binding epitope from CNTF to IL-6, we created an artificial cytokine with novel biological characteristics, i.e. a cytokine that signals like LIF, but needs the IL-6R for binding. The existence of such an exchangeable LIFR binding module could not be expected with regard to the primary sequence of CNTF and IL-6. It could only be predicted from structural analyses of CNTF and IL-6, since their low sequence identity of only 6% (10) is that of unrelated proteins. A modular structure of receptor recognition site seems to also exist in the neurotrophins, a family of neural growth factors which are structurally unrelated to four-helical bundle cytokines. By combining active domains of the neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 into an neurotrophin 3 backbone a synthetic "pan-neurotrophin" was constructed that activates all known neurotrophin receptors (52). However, in contrast to four-helical bundle cytokines the neurotrophins have a high sequence identity of around 50%.
The organizational principle underlying the molecular structure of
cytokines and growth factors may therefore be regarded as a set of
modules that are discontinuous with regard to the primary amino acid
sequence. Functional diversity among cytokines could therefore have
arisen from recombination of modules encoded by naturally occurring
homologous genes. Both the IL-6 and CNTF gene possess an intron-exon
junction near the end of the A-helix, close to the start of site IIIA.
However, the CNTF gene possesses only two exons, in contrast to the
IL-6 gene which consists of four coding exons (10). Thus, the concept
of a modular cytokine structure does not simply reflect the
organization of the cytokine gene. Nevertheless, our interpretation is
strikingly similar to results obtained recently by directed evolution
(DNA shuffling) of cephalosporinase genes from four different bacterial
species (53). With only a few point mutations, segmental recombination of the four cephalosporinase genes evolved into a powerful
moxalactamase resistance. In principle, modules responsible for
receptor recognition should also be identifiable on other cytokines,
e.g. IL-2. Exchanging such ligand modules should allow the
engineering of new designer cytokines or growth factors almost at will
to meet prespecified receptor requirements on target cells and thus
exert novel biological activities. Such designer cytokines could be of
therapeutical value (44, 54, 55), but will also be useful tools to
analyze the formation of cytokine receptor complexes.
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ACKNOWLEDGEMENTS |
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We thank Prof. Peter Galle and Dr. Heidi Schooltink for critically reading the manuscript. Dr. Birgit Oppmann kindly provided BAF/3-(gp130,LIFR) cells and recombinant LIF, recombinant IL-6, and CNTF were expressed by Martina Fischer and Dr. Barbara Krebs. Recombinant soluble IL-6R was a kind gift from Dr. Mark Stahl (Genetics Institute, Cambridge, MA). Mariam Klouche and Monika Hemmes are thanked for their help with LPS quantifications.
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FOOTNOTES |
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* 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: I. Medizinische Klinik, Abteilung Pathophysiologie, Johannes Gutenberg Universität Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany. Tel.: 06131-173363; Fax: 06131-173364; E-mail: rosejohn{at}mail.uni-mainz.de.
Supported by a fellowship from the "Association
Française contre les myopathies".
2 H. Gascan, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: CNTF, ciliary neurotrophic factor; IL-6, interleukin-6; IL-6R, IL-6 receptor; CNTFR, CNTF receptor; LIF, leukemia inhibitory factor; LIFR, LIF receptor; LPS, lipopoysaccharide; mAb, monoclonal antibody; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
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