From the Joint Protein Structure Laboratory, Ludwig Institute for
Cancer Research (Melbourne Tumour Biology Branch) and The Walter and
Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia and The Ludwig Institute for Cancer Research
(Melbourne Tumour Biology Branch) and The Cooperative Research Centre
for Cellular Growth Factors, Parkville, Victoria 3050, Australia
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ABSTRACT |
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The high affinity interleukin-6 (IL-6) receptor
is a hexameric complex consisting of two molecules each of IL-6, IL-6
receptor (IL-6R), and the high affinity converter and signaling
molecule, gp130. The extracellular "soluble" part of the IL-6R
(sIL-6R) consists of three domains: an amino-terminal Ig-like domain
and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have
determined the disulfide structure of the human sIL-6R by peptide
mapping in the absence and presence of reducing agent. Mass
spectrometric analysis of these peptides revealed four disulfide bonds
and two free cysteines. The disulfides
Cys102-Cys113 and
Cys146-Cys157 are consistent with known
cytokine-binding domain motifs, and Cys28-Cys77
with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like
and NH2-terminal FN III domains causing them to fold back
onto each other, has not previously been observed among cytokine
receptors. The two free cysteines (Cys192 and
Cys258) were detected as cysteinyl-cysteines, although a
small proportion of Cys258 was reactive with the alkylating
agent 4-vinylpyridine. Of the four potential
N-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and
Asn202, but not on Asn226.
Interleukin-6 (IL-6)1 is
a multifunctional cytokine that plays a central role in host defense
due to its wide range of immune and hematopoietic activities, as well
as its potent ability to induce the acute phase response (1-3). Since
overexpression of IL-6 has been implicated in the pathology of a number
of diseases (for reviews, see Refs. 4 and 5), it is anticipated that selective antagonists of IL-6 action may offer therapeutic benefits in
the treatment of IL-6-related diseases.
The biological activities of IL-6 are mediated by the IL-6 receptor
system which comprises two receptor proteins: the specific ligand-binding The cDNA of the human IL-6R encodes a protein of 468 amino acids
(7), including a signal peptide of 19 amino acids, an extracellular
region of 339 amino acids, a transmembrane domain of 28 amino acids,
and a short cytoplasmic domain of 82 amino acids. This sequence shows
54 and 57% overall amino acid identity with the cDNA sequences for
mouse (10) and rat (11) IL-6R, respectively. The mature 80-kDa IL-6R is
a glycosylated form of the predicted 50-kDa precursor (12) and contains
six potential N-linked glycosylation sites. The
extracellular region has a modular structure, consisting of three
domains of approximately 100 amino acids. The amino acid sequence of
the NH2-terminal domain is characteristic of the
immunoglobulin superfamily (Ig-like) (13, 14). Members of this family
share a common The cytoplasmic and transmembrane domains of the IL-6R are not required
for IL-6 signaling (17) and biologically active soluble forms of IL-6R
(sIL-6R) are naturally found in low concentrations in human urine (18)
and serum (19, 20) of healthy individuals. In contrast to many other
soluble cytokine receptors that act as inhibitors by competing for
ligand binding with cellular receptors (e.g. tumor necrosis
factor, IL-1, -2, -4, interferon- To elucidate the tertiary structure of the IL-6R extracellular region,
we have purified human sIL-6R using a Chinese hamster ovary (CHO) cell
expression system (29, 30). This form of the sIL-6R contains four
potential N-linked glycosylation sites and 10 cysteine
residues. Three cysteines are located in the Ig-like domain, six in the
NH2-terminal FN III domain, and one in the COOH-terminal FN
III domain. Previously, we have shown that the ligand affinity purified
sIL-6R bound IL-6 and gp130 with a 2:2:2 stoichiometry of IL-6, sIL-6R,
and sgp130 (8) and was bioactive as determined by the ability of the
IL-6·sIL-6R complex to prevent the differentiation of embryonic stem
cells (31). Here, reversed-phase HPLC peptide mapping under nonreducing
and reducing conditions, in combination with mass spectrometric and
NH2-terminal sequence analysis, was used to determine the
disulfide structure and carbohydrate attachment sites of sIL-6R. On the
basis of these results, we have created a model that depicts the
topology of the extracellular region of the IL-6R and predicts its
interactions with IL-6 and gp130.
Materials--
Trypsin (sequencing grade) and neuraminidase (EC
3.2.1.18) were obtained from Boehringer-Mannheim. An endoglycosylase
preparation obtained from Flavobacterium meningosepticum
(32) containing three Purification of the Extracellular or "Soluble" Domain of the
Human IL-6 Receptor (sIL-6R)--
sIL-6R was purified from the
conditioned medium of CHO cells transfected with an expression vector
(pECEdhfr344) which encodes the extracellular binding domain of the
IL-6R (truncated at residue 345) (29). The sIL-6R was concentrated from
CHO cell conditioned media using a Sartocon Miniapparatus (Sartorius,
Goettingen, Germany) equipped with a 30,000 molecular weight cut-off
membrane and purified by ligand affinity chromatography using an
IL-6-Sepharose column (30).
SDS-PAGE and Isoelectrofocusing--
SDS-PAGE analysis of sIL-6R
samples was performed using pre-cast 4-20% polyacrylamide/SDS gels
(Novex) according to the method of Laemmli (35). Isoelectrofocusing
(IEF-PAGE) was performed on pre-cast linear pH gradient (pH 3-10), 5%
polyacrylamide gels (Novex, San Diego, CA). Protein bands were
visualized by staining with Coomassie Brilliant Blue.
Deglycosylation of sIL-6R--
Recombinant sIL-6R (100 µg, 2 mg/ml) in 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM EDTA was treated with 2% (v/v) neuraminidase (37 °C,
overnight) and/or endoglycosylase mixture (37 °C, 3 h).
Labeling of Free Cysteine Residues--
Prior to disulfide
determination, free cysteine residues in the sIL-6R (200 µg) were
treated with a 5-fold molar excess of 4-vinylpyridine in 50 mM Tris-HCl buffer, pH 8.4, for 1 h at 25 °C in the
dark. The modified protein was purified by gel permeation chromatography on a 100 × 10-mm inner diameter fast desalting column (G-25 Sephadex, Pharmacia).
Peptide Mapping of the Tryptic Digest of
sIL-6R--
Deglycosylated sIL-6R (200 µg, 4 nmol) was digested with
trypsin (1:20 w/w, 0.05 M NaH2PO4,
pH 6.0) overnight at 37 °C. The tryptic peptide mixture was
fractionated by reversed-phase HPLC using a Hewlett-Packard Liquid
Chromatograph (model HP 1090A) and a Vydac C18 column (250 × 4.6-mm inner diameter). The column was developed at 0.5 ml/min using a
60-min linear gradient of 0-100% B, where solvent A was aqueous 0.1%
(v/v) trifluoroacetic acid and solvent B was 60% acetonitrile in
aqueous 0.09% trifluoroacetic acid (45 °C). The column eluent was
split (~1:160), post-detector, using a stainless steel Tee-union
(Upchurch catalog number U428, Upchurch, Oak Harbor, WA) directing
0.6% of the total flow at 3 µl/min to the mass spectrometer, while
the remainder (99.4%) was collected into polypropylene microcentrifuge
tubes (Eppendorf) for further analysis. To identify
disulfide-containing fractions, 25% of the digest (1 nmol) was reduced
with an equal volume of 10 mM TCEP in 0.2 M
sodium citrate buffer, pH 6.0 (65 °C, 10 min) (36), and then
re-chromatographed under identical conditions.
Ion-trap Mass Spectrometry--
On-line MS analysis of peptide
fractions was performed on a Finnigan-MAT LCQ quadrupole ion trap mass
spectrometer equipped with an ESI source (San Jose, CA). "Triple
play" experiments, consisting of MS/zoom scan/and MS/MS, were
performed as described elsewhere (37). Source CID/single ion monitoring
(sCID/SIM) was employed to identify S-pyridylethyl
cysteine-containing peptides (38). For sCID/SIM, the relative collision
energy in the source region was set at 70% (arbitrary value) and the
mass range was scanned from m/z 104.5-107.5 to detect
S-pyridylethyl fragment ions (m/z 106). Peptides
were identified using the Finnigan PEPMAPTM program, and from their
CID product ion spectra using the MS-Tag and MS-Product algorithms
(Prospector pacific rim mirror site, http://jpsl.ludwig.edu.au).
NH2-terminal Sequence Analysis--
Automated Edman
degradation of proteins and peptides was performed using a HP-G1005A
biphasic NH2-terminal protein sequencer (Hewlett-Packard,
version 3.0 chemistry) (38).
Homology Modeling--
The Ig-like domain of the IL-6R was
modeled using the structure of the mouse monoclonal antibody FAB D44.1
VL domain (39) which showed 23% sequence identity, the
highest for all known Ig three-dimensional structures. The coordinates
for the template were taken from the Protein Data Bank (40), entry 1MLB
(chain A, residues 1-109). Template structures for the CBD of IL-6R
were the CBD of gp130 (41), Protein Data Bank entry 1BQU; GHR, chain B
(first binding receptor) of Protein Data Bank entry 3HHR (42); EPOR,
Protein Data Bank entry 1EBP (43); and PRLR, Protein Data Bank entry
1BP3 (44). The sequence alignment was prepared in two parts. The
Ig-like domain of IL-6R was manually aligned with the template FAB
D44.1 VL structure. For the CBD, a structure-based multiple
sequence alignment was performed manually. The
The MODELLER program (45) was used to generate separate models of the
Ig-like domain and CBD. The quality of the models was assessed as
described previously (46); in particular using the ProsaII program
(47). MODELLER was also used to determine the relative orientations of
the Ig-like domain and CBD. A disulfide restraint between
Cys6 and Cys174 of the Ig-like domain and FN
III domain, respectively, was introduced in accordance with our
experimental results. Fifty models were generated with a range of
orientations between the Ig-like domain and the CBD. The final two
models were chosen on the basis of the quality checks described above
and agreement with experimental data. The model of the sIL-6R,
complexed with the crystal structures of IL-6 (48) (Protein Data Bank
entry 1ALU) and the gp130 CBD (41), was constructed by superimposing
these moieties over the human GH receptor complex (42).
Initial Characterization of Recombinant sIL-6
Receptor--
NH2-terminal sequence analysis of the first
20 residues of the purified sIL-6R was in agreement with the published
sequence (7) (data not shown). The purified sIL-6R yielded a single broad band on SDS-PAGE with an apparent molecular mass of ~52,000 (Fig. 1A, lane 1),
a value significantly higher than 36,368 Da calculated from the amino
acid composition (7). Upon treatment with either neuraminidase or a
combination of neuraminidase and an endoglycosidase mixture, the
Mr of the sIL-6R was reduced to ~50,000 and
~40,000, respectively (Fig. 1A, lanes 2 and
3). These data suggest that the increased
Mr of sIL-6R (~12,000) is due to glycosylation
of the CHO cell-derived protein. Pronounced charge heterogeneity of the
mature sIL-6R was observed upon IEF (Fig. 1B, lane
1). Treatment with neuraminidase (Fig. 1B, lane
2) and neuraminidase plus an endoglycosidase mixture (Fig.
1B, lane 3) reduced this complexity to one or two
major bands, respectively, indicating that the heterogeneity of the
sIL-6R preparation is primarily due to differential N-linked
glycosylation.
Labeling of Free Cysteine Residues--
Free cysteine residues in
sIL-6R (~200 µg) were modified with 4-vinylpridine at pH 8.5. Following enzymatic deglycosylation, the treated sIL-6R was digested
with trypsin at pH 6.0, and subjected to RP-HPLC/ESI-IT-MS analysis as
described under "Experimental Procedures." The total ion current
profiles of nonreduced and reduced tryptic digest of sIL-6R are shown
in Fig. 2 (panels A and
B, respectively). sCID/SIM of the nonreduced digest for
S-pyridylethyl ions (m/z 106) revealed a single
peak at retention time 31.42 min (Fig. 2C). The mass
spectrum of the peak at retention time 31.42 min (Fig. 2D)
revealed the presence of two peptides: peptides T1a and T1b with
calculated masses of 2007.3 Da and 1992.5 Da, respectively. Automated
CID MS/MS of the doubly charged ion (m/z 1003.8) of peptide
T1a (Fig. 2E) identified this peptide as residues 253-268
of the sIL-6R (DLQHHCVIHDAWSGLR) containing an additional mass of 118.9 Da located at Cys258. Upon reduction with TCEP, the mass of
peptide T1a decreased by 119.3 Da and was located at 32.20 min (peptide
T9, Fig. 2B; see Table I),
consistent with cysteinylation of Cys258.
Sequence data of peptide T1b was not available due to the low abundance
of this ion (~20% compared with peptide T1a). However, the 14.8-Da
difference observed between peptides T1a and T1b is consistent with the
difference in mass between cysteinylated (+119 Da) and
S-pyridylethylated (+105 Da) Cys258 of peptide
Asp253-Arg268. These data suggest that the peak
observed in the sCID/SIM profile (Fig. 2C) emanates from an
S-pyridylethylated form (Cys258) of peptide T1b.
Peptide T1b was also observed (in low abundance) at approximately the
same retention time in the TCEP-reduced total ion current profile
(Fig. 2B). Taken together, these data indicate that the
majority of sIL-6R purified from CHO cell conditioned medium contain a
modified Cys258 (cysteinylated), and only a small portion
(~20%) remains as the unmodified Cys258 (free sulfhydryl).
Determination of Disulfide Linkages of sIL-6R by Tryptic Peptide
Mapping--
A portion (25%) of the tryptic digest of
deglycosylated/S-pyridylethylated sIL-6R was reduced
with 10 mM TCEP at pH 6.0 and subjected to on-line
RP-HPLC/ESI-IT-MS analysis (Fig. 2B) using the same
chromatographic conditions described for the nonreduced digest.
Inspection of the nonreduced and reduced tryptic peptide maps of sIL-6R
(Fig. 2, panels A and B) revealed that upon
reduction of the digest, the retention times of five peptide fractions
(T1-T5) in the nonreduced tryptic map (Fig. 2A) changed with
the concomitant appearance of nine peptide fractions (T6-T14) in the
reduced tryptic map (Fig. 2B). A summary of peptide masses
found in fractions T1-T14 is shown in Table I.
As mentioned above, MS analysis of peptide fraction T1 revealed tryptic
peptide Asp253-Arg268 (Fig. 2, A,
D, and E) containing a cysteinyl-cysteine at
position 258 in the sequence (T1a), as well as the small proportion of the peptide that had been reacted with 4-vinylpyridine (T1b). Similarly, MS/MS analysis of nonreduced T2 (data not shown) identified this peptide as residues Thr186-Arg210 of the
sIL-6R with a cysteinylated cysteine residue at position 192. The
reduced form of this peptide (T11 in Table I) was also confirmed by
MS/MS analysis (data not shown). Modification of Cys192
with 4-vinylpyridine was not observed.
Inspection of the nonreduced and reduced total ion current profiles for
the tryptic digest of sIL-6R (Fig. 2) indicated that peptide fraction
T3 (6544.0 Da, Fig. 2A, Table I) was comprised of three
peptides (T6, Arg5-Arg13; T8,
Lys133-Lys154; and T14,
Phe155-Lys182) linked by disulfide bridges
involving Cys6, Cys146,
Cys157, and Cys174 (Fig.
2B and Table I). MS/MS analysis of peptides T6, T8, and T14
confirmed their identity (data not shown). Analysis of the CID spectrum
of the +5 ion (m/z 1309.6) of T3 (Fig.
3A) revealed two major doubly
charged fragment ions (m/z 1683.6 and 1589.5) with
calculated masses of 3365.2 and 3177.6 Da, respectively. This spectrum
is consistent with preferential cleavage at Pro162,
producing two disulfide-linked peptides: peptide T6
(Arg5-Arg13) linked to residues
Pro162-Lys182 from peptide T14 via
Cys6-Cys174 (calculated mass, 3177.6 Da), and
peptide T8 (Phe134-Lys154) linked to residues
Phe155-Val161 from peptide T14 via
Cys146-157 (calculated mass, 3365.2 Da).
Peptide fraction T4 (calculated mass, 4893.6 Da; Fig. 2A and
Table I) was tentatively identified by a comparison of peptide masses
(see Table I) as being comprised of peptides T7
(Ser66-Arg79) and T13
(Gly14-Arg44) linked by
Cys28-Cys77. MS/MS analysis of peptides T7 and
T13 (from Fig. 2B) confirmed their identity (data not
shown). Assignment of the cysteine connectivity between T7 and T13 was
provided by MS/MS analysis of the +4 charged ion (m/z
1224.3) of T4 (Fig. 3B) which revealed a major triply charged fragment ion of m/z 1441.5, corresponding to
preferential cleavage at Pro20. This yielded the peptide
Pro20-Arg44 from T13 and peptide T7
(Ser66-Arg79) linked by a disulfide bond
through Cys28-Cys77.
The mass of peptide fraction T5 (4259.9 Da, Table I) was consistent
with the summation of the masses of peptides T10 (1571.8 Da) and T12
(2689.8 Da), where the observed 1.7 Da decrease in the mass of T5
corresponded to the formation of a disulfide link between peptides T10
and T12. The identities of peptides T10
(Lys105-Arg118) and T12
(Ala80-Arg104) in Fig. 2B were
confirmed by MS/MS analysis (data not shown). Analysis of the CID
fragment ion data (m/z 1436.4 and 958.3) from the +4 charged
ion of T5 (m/z 1065.9) was consistent with fragmentation at
Pro94 resulting in residues
Pro94-Arg104 from peptide T12 and tryptic
peptide T10 (Lys105-Arg118) linked through
Cys102-Cys113 (Fig. 3C). Automated
Edman degradation of tryptic peptide fractions T1-T5 (Fig.
2A) confirmed the NH2-terminal sequence of the
peptide components in these fractions (Table I).
N-Linked Glycosylation Sites in sIL-6R--
The
N-linked glycosylation sites in sIL-6R were determined by
the non-appearance of asparagine residues during automated Edman degradation of tryptic and Asp-N endopeptidase peptides containing Asn-Xaa-(Ser/Thr) motifs (data not shown). Of the four potential N-glycosylation sites in the extracellular domain of the
IL-6R, NAT (residues 36-38), NIT (residues 202-204), and NYS
(residues 74-76) were found to be glycosylated, whereas one potential
N-glycosylation site, NSS (residues 226-228), was not
modified. Further confirmation of N-linked glycosylated
asparagine residues was provided by Asn/Asp conversion following
endoglycosidase F treatment (49); see Asn/Asp conversion at position 74 in peptide T7 (Fig. 4).
Homology Modeling--
Two separate models of the sIL-6R were
created that depict different orientations between the Ig-like domain
and the CBD (Fig. 5). In model A (Fig.
5A), the linker region between these domains follows the
gp130 template and orients the Ig-like domain in the plane of the CBD.
In model B (Fig. 5B), the linker region follows the PRLR
template and the Ig-like domain is orientated across the plane of the
CBD. The GHR and EPOR structures were not chosen as templates for the
linker region since the GHR has no linker region and the orientation of
the EPOR linker region would not allow the formation of the
Cys6-Cys174 connectivity.
The template used for the Ig-like domain was the VL domain
of FAB D44.1; a V-type Ig-like fold comprised of nine
The MODELLER program works in such a way that the calculated model
follows most closely the best template (50). Therefore, our models for
the tandem FN III modules of sIL-6R are most closely aligned to the
recently published crystal structure for the CBD of gp130 (41). Models
A and B of the sIL-6R were shown to be of good quality as assessed by
the structural quality checks described under "Experimental
Procedures." The ProsaII Z-scores of the CBD of models A and B were
Knowledge of the cysteine connectivity pattern and carbohydrate
attachment sites of sIL-6R provides a foundation for the determination of the three-dimensional structure of this molecule and, ultimately, of
the IL-6·IL-6R·gp130 complex. Mass spectrometric and
NH2-terminal sequence analysis of a tryptic digest of the
sIL-6R identified four disulfide linkages
(Cys28-Cys77,
Cys102-Cys113,
Cys146-Cys157, and
Cys6-Cys174), two "free" cysteines
(Cys192 and Cys258), and three
N-linked glycosylation sites (Asn36,
Asn74, and Asn202).
The two consecutive disulfide linkages in the NH2-terminal
FN III domain of the sIL-6R (Cys102-Cys113 and
Cys146-Cys157) correspond with those reported
for other class I cytokine receptors, namely, granulocyte-colony
stimulating factor receptor (51, 52), gp130
(41)2 GHR (42, 53), EPOR
(43), and PRLR (44). Similarly, the Cys28-Cys77
connectivity in the Ig-like domain that links the two The Cys6-Cys174 disulfide is novel among the
cytokine receptors. Sequence alignment of the IL-6R with GHR, PRLR,
EPOR, and gp130, whose CBD structures are known (Fig. 6), predicts that
Cys174 is located on Cys192 and Cys258 do not form intramolecular
disulfide bonds and occur as either free or in a cysteinylated form.
These residues are predicted to be located at the COOH-terminal end of
Three N-linked glycosylation sites in the sIL-6R were
identified at Asn36, Asn74, and
Asn202. Asn36 and Asn74 are
predicted to be located on the loop region connecting Implications of the cysteine connectivities and carbohydrate
attachments on the overall topology of the IL-6R extracellular region
are modeled in Fig. 5. The inter-domain disulfide bond which links the
Ig-like and NH2-terminal FN III domains is distant from the
hinge region connecting these two modules. This causes the Ig-like
domain to fold back onto the NH2-terminal FN III domain either in the plane (Fig. 5A) or across the plane of the CBD
(Fig. 5B). A different type of inter-domain disulfide bond
has been reported for the extracellular region of the interferon- A model of the sIL-6R (Fig. 5A) complexed with the crystal
structure of IL-6 (48) and CBD of gp130 (41), superimposed over the
crystal structure of the growth hormone receptor complex (42), is shown
in Fig. 7. This model is consistent with
our previously proposed hexameric IL-6 receptor complex model which is
based upon the association of two GH/GHR-like trimers (2). While other
IL-6/IL-6R/sgp130 trimer models have been reported (69-71), these were
generated prior to the publication of the IL-6 and gp130 CBD
three-dimensional structures and most likely contain significant errors
in the sequence alignments for gp130 and IL-6R. For instance, Kalai
et al. (71) align Cys174 of the IL-6R with
Ile109 of the GHR, thereby directing the side chain of
Cys174 toward the core of the NH2-terminal FN
III domain which would prevent the formation of an inter-domain
disulfide bond with Cys6. In our model, Cys174
is aligned with Thr112 of the GHR and Glu177 of
gp130 (Fig. 6). This orientates the side chain of Cys174
outwards from the core of the protein, which is consistent with a
Cys6-Cys174 connectivity. The formation of a
disulfide bond between Cys6 and Cys174
restrains the Ig-like domain away from the IL-6-binding site (Fig. 7).
This suggests that the Ig-like domain is unlikely to be involved in
ligand binding.
INTRODUCTION
Top
Abstract
Introduction
References
-subunit receptor (IL-6R) and the signal transducing
-subunit, gp130. Gp130 also forms part of the receptor complexes of
leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M,
cardiotrophin-1, and IL-11 (6) which, in part, provides a molecular
basis for the functional redundancy of these cytokines. IL-6 first
binds the IL-6R with an affinity of ~1 nM and the
IL-6·IL-6R complex then binds gp130 with a resulting affinity of
~10 pM (7). The ternary complex of the IL-6 receptor
system is a hexamer, comprising two molecules each of IL-6, IL-6R, and
gp130 (8, 9).
-sheet folding topology called a Greek Key (15),
whereby neighboring
-strands form hydrogen bonds in an anti-parallel
fashion to form a
-pleated sheet. Two
-sheets are then packed
against each other to produce a hydrophobic core. Similarly, the two
COOH-terminal domains of the IL-6R are classified as fibronectin type
III-like (FN III) modules, a subclass of the
-sandwich fold (14).
The topology of these domains is similar to those of Ig-like modules,
with the notable exception of the "sheet switching" of
-strand D
from the first
-sheet of an Ig-like domain to form
-strand C' on
the second
-sheet of FN III domains. Together, the two FN III
domains form a cytokine-binding domain (CBD) which is characteristic of
class I cytokine receptors (16) (e.g. receptors for
interleukins-3, -5, -6, -11, gp130, erythropoietin (EPO), ciliary
neurotrophic factor, granulocyte-colony stimulating factor, growth
hormone (GH), and prolactin (PRL)). Generally, these receptors are
characterized by two conserved disulfide bonds located in the
NH2-terminal FN III domain and a conserved WSXWS
motif located in the COOH-terminal FN III domain.
, nerve growth factor, leukemia
inhibitory factor, granulocyte-stimulating factor and granulocyte
macrophage-colony stimulating factor) (for reviews, see Refs. 21 and
22), the sIL-6R acts as an agonist of IL-6 activity (17). It is not
clear whether sIL-6R is generated by proteolytic shedding of
membrane-bound IL-6R (23), or from an alternatively spliced mRNA
species (24, 25), or both. In certain disease states, for example,
patients with human immunodeficiency virus infection or multiple
myeloma, increased levels of sIL-6R have been reported (26, 27).
Therefore, inhibition of the IL-6·sIL-6R complex has been labeled as
a key target to antagonize the in vivo action of IL-6
(28).
EXPERIMENTAL PROCEDURES
-N-acetylglucosidase F (endo F)
activities (F1, F2, and F3) as well as peptide-N-glycosidase
(33, 34) was a kind gift from Dr. G. E. Norris (Massey University,
New Zealand). Tris-(2-carboxyethyl)-phosphine (TCEP) was obtained from
Pierce. 4-Vinylpyridine was purchased from Sigma. All other chemicals
were HPLC grade.
-sheets of the known
CBD structures were superimposed to provide the basis of the alignment.
The remaining sequences of unknown structure were manually aligned with
these structures, conserving the disulfide patterns, WSXWS
motif, and hydrophobic patterns of the
-sheets.
RESULTS
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Fig. 1.
Electrophoretic analysis of glycosylated and
deglycosylated sIL-6R. A, reducing SDS-PAGE. Lane
1, affinity-purified sIL-6R; lane 2,
neuraminidase-treated sIL-6R; lane 3, neuraminidase and
endoglycosidase mixture-treated sIL-6R. 10 µg of protein was applied
to each lane. B, IEF-PAGE (pH 3-10). Samples were the same
as for A, except that 30 µg of protein was applied to each
lane. Both gels were stained with Coomassie Brilliant Blue R-250.
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Fig. 2.
RP-HPLC/ESI-IT-MS analysis of the sIL-6R
tryptic digest. A, total ion current profile of
nonreduced sIL-6R tryptic digest. B, total ion current
profile of sIL-6R tryptic digest following reduction with TCEP.
C, source CID/SIM scan of nonreduced sIL-6R tryptic digest
for S-pyridylethyl fragment ions (m/z 106).
D, MS spectrum of tryptic peptide fraction T1a,b from
panel A. Multiply charged ions of tryptic peptide T1a ( );
calculated mass 2007.3 Da, and tryptic peptide T1b (
); calculated
mass, 1992.5 Da. E, CID spectrum of the (M + 2H)2+ ion of peptide T1a (m/z 1003.8) from
panel D identified this peptide as residues
Asp253-Arg268 of sIL-6R (see inset).
The mass difference between ions y10 and
y11 (m/z 221.9) is consistent with
cysteinyl-cysteine at position 258.
Calculated and observed masses of nonreduced and reduced tryptic
peptides of the deglycosylated extracellular domain of the human
interleukin-6 receptor
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Fig. 3.
CID mass spectrometric identification of
cysteine connectivities in disulfide-containing tryptic peptide
fractions T3, T4, and T5. A, CID spectrum of the +5 ion
(m/z 1309.6) of tryptic peptide T3 (observed mass, 6544.0 Da) from Fig. 2A. B, CID spectrum of the +4
charged ion (m/z 1224.3) of tryptic peptide T4 (observed
mass, 4893.6 Da) from Fig. 2A. C, CID spectrum of
the +4 charged ion (m/z 1065.9) of tryptic peptide T5
(observed mass, 4259.9 Da) from Fig. 2A.
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Fig. 4.
CID spectrum of the +2 charged parent ion
(m/z 815.9) of tryptic peptide T7 (observed mass, 1629.6 Da) from
Fig. 2B. The m/z difference
of 115.2 between ions y5 and
y6, and not 114.1 as expected from the amino
acid sequence, is indicative of the conversion Asn74 to Asp
caused by endoglycosidase F removal of the N-linked
carbohydrate chain. The m/z difference between the ions
either side of y5 and y6
(y4-y5, 163.0 (Tyr), and
y6-y7, 56.8 (Gly))
supports this conclusion.
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Fig. 5.
Homology models of the three-dimensional
structure of the sIL-6R. The Cys6-Cys174
connectivity causes the Ig-like domain to fold back onto the
NH2-terminal FN III domain either in the plane
(A) or across the plane (B) of the CBD. Cysteine
residues are shown in yellow. Disulfide bonds link
Cys6-Cys174,
Cys28-Cys77,
Cys102-Cys113, and
Cys146-Cys157. Cys192 and
Cys258 do not form intramolecular disulfide bonds.
Glycosylated asparagine residues are shown in pink.
Asn226 (non-glycosylated) is colored blue. The
Ig-like domain is modeled on a mouse antibody structure; the CBD
follows most closely the structure of the gp130 CBD. Model coordinates
are available at http://www.liba.ludwig.edu.au.
-strands (39).
The amino acid sequence of FAB D44.1 aligned well with the IL-6R
Ig-like domain in
-strands B, C, D, and F (Fig.
6A). These strands form the
hydrophobic core of the domain and contain a number of conserved
hydrophobic and aromatic residues, including the disulfide bond linking
-strands B and F. In contrast, the peripheral strands A, E, and G
give less favorable alignments. The absence of
-strands C' and C''
of the FAB D44.1 VL domain in the IL-6R suggests that the
Ig-modeled domain forms a C1-type rather than a V-type fold (for
review, see Ref. 14). Regardless of the assignment of the particular
Ig-like fold, the overall
-sandwich structure is retained. In
particular, the relative position of Cys6 will not vary.
The second and third domains of the IL-6R are classified as FN III
domains. Sequence analysis of the sIL-6R revealed 25% sequence
identity to the CBD of gp130, which is higher than any other template
in the data base (Fig. 6B).
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Fig. 6.
A, sequence alignment of the IL-6R
Ig-like domain with the mouse antibody FAB D44.1 VL domain
(Protein Data Bank code 1MLB, chain A). This sequence alignment forms
the basis of the Ig-like model of the IL-6R. Sequences shaded in
gray represent -sheets, while the conserved cysteines of
Ig-like domains are colored pink. B, sequence
alignment of IL-6R with selected class I cytokine receptors. The
Ig-like domains have been manually aligned and a structure-based
alignment of the CBD is presented.
-Sheets of known CBD structures
are highlighted gray and labeled 1A-1G for the
NH2-terminal FN III domain and 2A-2G for the
COOH-terminal FN III domain. Sequence numbering corresponds to the
mature form of the receptors, apart from hIL-11R (italicized
numbering), where the length of the signal peptide is unknown and
the numbering is of the expressed sequence. Cysteine residues forming a
disulfide between
-strands B and F of the Ig-like domain are
highlighted pink, and conserved disulfides of class I
cytokine receptors in the NH2-terminal FN III domain are
blue. The disulfide between the Ig-like and FN III domain is
highlighted green and the free cysteines are
yellow. Potential glycosylation sites are red,
although Asn226 is not glycosylated.
4.8 and
5.3, respectively, comparing favorably with a score of
4.8 for the CBD of gp130. The ProsaII plot of the IL-6R Ig-like
domain is less favorable than the profile of the template, FAB D44.1, a
general feature observed in homology model building with low sequence identity.
DISCUSSION
-sheets, thus
forming the
-sandwich fold, is characteristic of the Ig superfamily
(13, 14).
-strand F of the
NH2-terminal FN III domain, while Cys6 is
situated at the NH2-terminal end of
-strand A of the
Ig-like domain. Therefore, the disulfide bond formed between these two residues provides an additional link between the Ig-like and FN III
domains. Cys6 is conserved in the IL-11R (54, 55), however,
the IL-11R has no corresponding cysteine partner in the
NH2-terminal FN III domain with which to form an
inter-domain disulfide bond. No other known class I cytokine receptors
contain a cysteine arrangement which could facilitate the same
disulfide bond as observed for the IL-6R. Interestingly, substitution
of Cys174 with Asp in a soluble form of the CBD resulted in
a complete loss of ligand binding (17). The significance of this
observation is not yet known. Truncation experiments of the IL-6R have
shown that the Ig-like module is not required for IL-6 binding (17, 56), although the affinity of the truncated receptor for IL-6 was not
determined. Recently, it has been suggested that the Ig-like domain may
be required for receptor internalization (57). Together, these
observations indicate that further studies are needed to elucidate the
role of the inter-domain disulfide bond and Ig-like module in IL-6
receptor complex formation.
-strand G of the NH2-terminal FN III domain and
-strand E of the COOH-terminal FN III domain, respectively. The
three-dimensional models of the sIL-6R shown in Fig. 5 predict that
Cys192 and Cys258 are spatially distant from
each other and are unable to form a disulfide bond. Cys258
is conserved among human, mouse, and rat IL-6 receptor species, while
Cys192 is replaced by a leucine in the mouse and rat
sequences (Fig. 6). Cys258 is also conserved in the EPOR
(58) and cytokine-like factor-1, a recently cloned soluble member of
the class I cytokine receptor family whose function is presently
unknown (59). The presence of free cysteine residues in the
membrane-proximal region of the IL-6R extracellular domain suggests
that the IL-6R may homodimerize via intermolecular disulfide bond
formation, in a manner similar to that of gp130 (60), or EPOR (61).
Moreover, disulfide linkages between specific ligand-binding
(
-subunit) and signaling (
-subunit) receptors have been reported
for the IL-3/IL-5/granulocyte macrophage-colony stimulating factor
subfamily of cytokine receptors (62-64). These observations indicate
that the potential for disulfide-linked oligomerization of the IL-6R
warrants further investigation.
-strands B-C
and on
-strand F of the Ig-like domain, respectively, while Asn202 is expected to be on
-strand A of the
COOH-terminal FN III domain. These carbohydrate moieties do not
directly affect ligand binding as shown by the expression of functional
sIL-6R in Escherichia coli (65). However, the presence of
carbohydrate chains in these regions almost certainly excludes them
from protein-protein interaction sites within the IL-6R complex. The
absence of glycosylation at Asn226 is consistent with the
known involvement of neighboring residues Ser228-Leu232 in ligand binding (17, 66).
receptor, a class II cytokine receptor (67, 68). Here, the inter-domain disulfide bond is located in the hinge region of the CBD, close to the
linker sequence connecting the two FN III domains. This maintains a
hinge angle of approximately 120°, whereas the inter-domain disulfide
bond found in the IL-6R imposes a hinge angle approaching 0° between
the Ig-like and NH2-terminal FN III domains.
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Fig. 7.
Ribbon model of a trimeric
IL-6·sIL-6R·sgp130 (CBD) receptor complex. This model is based
on the growth hormone receptor complex (42). A, top view,
and B, side view of the receptor complex. IL-6 is colored
green, sIL-6R (model A) orange, with the Ig-like
domain of the IL-6R yellow, and the CBD of gp130
magenta. Residues of IL-6 known to be involved in binding to
IL-6R and gp130 are colored green, with binding site III,
involved in binding to a second gp130 receptor colored cyan.
IL-6R residues involved in binding to IL-6 are colored blue.
The disulfide between the Ig-like and FN III domains of the sIL-6R
(Cys6-Cys174), Cys192 and
Cys258 are colored yellow. Potential
N-linked glycosylation sites are colored pink.
(Coordinates of this model are available at
http://www.liba.ludwig.edu.au.)
Our model predicts that cysteinylated Cys192 is located
within the IL-6-binding site of the IL-6R. However, this modification seemingly does not interfere with ligand binding since the modified receptor was purified by affinity chromatography using IL-6-Sepharose. This finding is in accord with a previous study, which demonstrated that mutation of Cys192 to alanine did not inhibit IL-6
binding (17). It is unlikely that the IL-6R forms an inter-molecular
disulfide bond with IL-6 upon binding, since biophysical experiments on
both human (72) and mouse (73) IL-6 have shown that it does not contain
free cysteines. These observations suggest that despite its location, Cys192 is not involved in ligand binding. However, the
potential for disulfide exchange has not yet been addressed.
Asn226 is also located inside the predicted ligand-binding
site of the IL-6R which is consistent with this residue not being
glycosylated, since carbohydrate chains in this region would be
expected to sterically inhibit ligand binding. N-Linked
carbohydrate chains located on Asn36, Asn74,
and Asn202 do not interfere with any of the protein-protein
interaction sites of the receptor complex predicted in our model. The
role of the Ig-like domain and the free cysteine residues in the
extracellular domain of the IL-6R must await further studies.
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ACKNOWLEDGEMENTS |
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We thank J. Bravo and W. Somers for the coordinates of the CBD of gp130 and PRLR, respectively, prior to release into the Protein Data Bank.
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FOOTNOTES |
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* This work was supported by a grant from the National Health and Medical Research Council of Australia (to R. J. S.) and a Cooperative Research Center grant from the Federal Government of Australia (to N. E. H. and H. R. T.).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: Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, P. O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia. Tel.: 61-3-9341-3155; Fax: 61-3-9341-3192; E-mail: Richard.simpson{at}ludwig.edu.au.
2 R. L. Moritz and R. J. Simpson, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: IL, interleukin; sIL-6R, extracellular or soluble domain of the IL-6 receptor; FN III, fibronectin-type III; Ig, immunoglobulin; CBD, cytokine-binding domain; s, soluble; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel; GlcNAc, N-acetylglucosamine; TCEP, tris-(2-carboxyethyl)-phosphine; MS, mass spectrometry; CID, collision-induced dissociation; RP-HPLC, reversed-phase high performance liquid chromatography; EPO, erythropoietin; GH, growth hormone; PRL, prolactin; ESI, electrospray ionization; IT-MS, ion trap mass spectrometry.
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REFERENCES |
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