From the Joint Protein Structure Laboratory, Ludwig
Institute for Cancer Research (Melbourne Tumor Biology Branch) and the
Walter and Eliza Hall Institute of Medical Research, Parkville,
Victoria 3050, Australia and § Ludwig Institute for Cancer
Research (Melbourne Tumour Biology Branch) and the Cooperative Research
Centre for Cellular Growth Factors, Parkville,
Victoria 3050, Australia
Received for publication, November 2, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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gp130 is the common signal transducing receptor
subunit for the interleukin-6-type family of cytokines. Its
extracellular region (sgp130) is predicted to consist of five
fibronectin type III-like domains and an NH2-terminal
Ig-like domain. Domains 2 and 3 constitute the cytokine-binding
region defined by a set of four conserved cysteines and a
WSXWS motif, respectively. Here we determine the disulfide
structure of human sgp130 by peptide mapping, in the absence and
presence of reducing agent, in combination with Edman degradation and
mass spectrometry. Of the 13 cysteines present, 10 form disulfide
bonds, two are present as free cysteines (Cys279 and
Cys469), and one (Cys397) is modified by
S-cysteinylation. Of the 11 potential
N-glycosylation sites, Asn21,
Asn61, Asn109, Asn135,
Asn205, Asn357, Asn361,
Asn531, and Asn542 are glycosylated but not
Asn224 and Asn368. The disulfide bonds,
Cys112-Cys122 and
Cys150-Cys160, are consistent with known
cytokine-binding region motifs. Unlike granulocyte colony-stimulating
factor receptor, the connectivities of the four cysteines in the
NH2-terminal domain of gp130
(Cys6-Cys32 and
Cys26-Cys81) are consistent with known
superfamily of Ig-like domains. An eight-residue loop in domain 5 is
tethered by Cys436-Cys444. We have created a
model predicting that this loop maintains Cys469 in a
reduced form, available for ligand-induced intramolecular disulfide
bond formation. Furthermore, we postulate that domain 5 may play a role
in the disulfide-linked homodimerization and activation process of gp130.
The family of cytokines that signal through the common receptor
subunit gp130 (referred to as the gp130 cytokines) comprises interleukin (IL)1-6, leukemia
inhibitory factor (LIF), ciliary neurotrophic factor, oncostatin
M, cardiotrophin-1, herpesvirus IL-6, interleukin-11 (IL-11), and
neurotrophin-1/B cell-stimulatory factor-3/cardiotrophin-like cytokine.
These cytokines play a pivotal role in the immune, hematopoietic and
nervous systems, as well as in bone metabolism, inflammation, and the
acute-phase response (1-4). The gp130 cytokine family are
characterized by a four- Intracellular signaling by the gp130 cytokines is initiated by the
ligand first making low affinity (~1 nM) contact with its cognate receptor The cDNA of human gp130 encodes a protein of 918 amino acids (11),
including a signal peptide of 22 amino acids, an extracellular domain
of 597 amino acids (sgp130), a transmembrane domain of 22 amino acids,
and an intracellular domain of 277 amino acids. The amino acid sequence
of murine (23) and rat gp130 (24) share an overall sequence identity of
85 and 88%, respectively, with that of human gp130. There are 11 potential N-linked glycosylation sequons in the
extracellular domain of the predicted 101-kDa human gp130 (11),
suggesting that the mature ~130-kDa gp130 (11) is highly
glycosylated. The extracellular region of gp130 has a modular structure
consisting of six domains of ~100 amino acids each. The
NH2-terminal domain (domain 1) is predicted to be a member
of the immunoglobulin superfamily (Ig-like) (25, 26), which has
"Greek key" The functional anatomy of the extracellular region of gp130 is still
poorly understood. A truncated form of gp130 lacking the
membrane-proximal FN III modules and the cytoplasmic and transmembrane domains has been shown to bind a complex of either IL-6·sIL-6R or
LIF·sLIF receptor (30, 31). Val252 in the BC loop of the
COOH-terminal domain of the CBR (domain 3 of gp130) has been implicated
in the interaction of gp130 with IL-6·IL-6R (32) and IL-11·IL-11
receptor (33). The location of this residue corresponds to a tryptophan
residue in the growth hormone receptor that has been shown to be
critical for ligand binding (34), suggesting a conserved mode of ligand
binding among the cytokine receptor superfamily. The Ig-like module of gp130 (domain 1) has been shown to be involved in the interaction of
gp130 with ligands that induce homodimerization of gp130 (IL-6 (35, 36)
and IL-11 (37)) and essential for the formation of high affinity
hexameric complexes (15, 35). This domain is thought to bind site III
in IL-6 and IL-11 (35), and Ig-like modules in the GCSFR and LIF
receptor are also thought to make contact with their cognate ligands
(38, 39). The three membrane-proximal FN III modules of gp130, although
not directly involved in ligand binding, may be required for gp130
dimerization by ligands such as IL-6 (40) and IL-11 (41) or
transmembrane signaling events such as stabilization and/or orientation
of transmembrane receptor dimers (5).
The structure of the COOH-terminal domain of the gp130 CBR (domain 3)
has been determined by NMR (42), and the complete CBR (i.e.
domains 2 and 3) by x-ray crystallography (43); however, there is no
complete structure for gp130 or any member of the gp130 family of
receptors. To elucidate the tertiary structure of the gp130
extracellular region, we have purified human sgp130 using a Chinese
hamster ovary (CHO) cell expression system (13, 44). This form of human
sgp130 contains 11 potential N-linked glycosylation sites
and 13 cysteine residues. Four cysteines are located in the Ig-like
domain (domain 1), four are in the NH2-terminal FN III
domain of the CBR (domain 2), one is in the COOH-terminal FN III domain
of the CBR (domain 3), one is in domain 4, and three are in domain 5. Previously, we have shown that affinity purified sgp130 bound a binary
complex of IL-6·sIL-6R with a 2:2:2 stoichiometry (13). Here, reverse
phase HPLC peptide mapping under reducing and nonreducing conditions in
combination with mass spectrometric and NH2-terminal
sequence analysis was used to determine the cysteine connectivities and
carbohydrate attachment sites for sgp130.
Materials--
4-Vinylpyridine was purchased from Aldrich.
Trifluoroacetic acid (HPLC/Spectro grade) was from Pierce.
Dithiothreitol (DTT, Ultrol grade) was obtained from
Calbiochem/Novabiochem. Sequencing grade trypsin (EC 3.4.21.4),
chymotrypsin (EC 3.4.21.1), pepsin (EC 3.4.23.1), and neuraminidase (EC
3.2.1.18) were from Roche Molecular Biochemicals. An endoglycosidase
preparation from Flavobacterium meningosepticum (45)
containing three Chromatography--
Reverse phase (RP)-HPLC was performed using
either a Vydac 5-µm, 300 Å octadecyl silica column (inner diameter,
250 × 4.6 mm) (Vydac) or a Brownlee RP-300 7-µm, 300 Å octylsilica column (inner diameter, 100 × 4.6 mm) (Applied
Biosystems), operated at a flow rate of 0.5 ml/min. For capillary
RP-HPLC, a fused silica column (inner diameter, 50 × 0.2 mm) was
packed "in-house" with Brownlee RP-300 7-µm, 300 Å octylsilica
(49). For SEC, an analytical Superose 12 column (inner diameter,
300 × 10 mm) operated at 0.5 ml/min was employed. Rapid desalting
and buffer exchange of proteins was performed using a
Fast-desaltingTM column (Sephadex G25; inner diameter,
100 × 10 mm; Amersham Pharmacia Biotech). Chromatography was
performed using a HP-1090A Liquid Chromatograph (Agilent Technologies)
equipped with a manual injector fitted into the column compartment, and
a diode array UV detector was used for real time multiple wavelength
monitoring of the column eluent. Samples were collected manually in
polypropylene (Eppendorf) tubes.
Purification of the Extracellular or Soluble Domain of the
Recombinant IL-6 Signaling Receptor gp130 (sgp130)--
sgp130 was
purified from the conditioned medium of CHO (G16) cells stably
transfected with a plasmid (pECEdhfrgp620) that encodes the
extracellular domain of gp130 truncated at amino acid 621 (44).
Briefly, transfected CHO cells were grown in a large scale fermentation
apparatus with a working volume of 1.25 liters (New Brunswick Celligen
plus fermenter). Cell conditioned medium was concentrated (20-fold) by
ultrafiltration using a Sartocon Mini apparatus (Sartorius, Germany)
fitted with a 30,000 molecular weight cut-off filter. sgp130 was
purified from concentrated CHO cell medium by binding to a 10-ml column
of AM64-Sepharose and eluting sgp130 with 4 M
MgCl2. sgp130 was further purified by preparative SEC
(>95% pure, as judged by SDS-PAGE and Western blot analysis), and
sgp130-containing fractions were pooled, and buffer was exchanged using
a Fast-desaltingTM column prior to storage at SDS-Polyacrylamide Gel Electrophoresis and
Isoelectricfocusing--
Samples were analyzed by SDS-PAGE on precast
4-20% polyacrylamide gels (Novex) using nonreducing or reducing
(5.25% (v/v) Deglycosylation of sgp130--
sgp130 (400 µg, 1 mg/ml) in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.025 M EDTA was treated with 1 unit of neuraminidase and/or
endoglycosidase mixture (37 °C, 16 h).
Labeling of Free Cysteine Residues in sgp130--
Prior to
disulfide determination, free cysteine residues in sgp130 (400 µg)
were treated with a 5-fold molar excess of 4-vinylpyridine in 0.1 M Tris-HCl, pH 8.4, for 1 h at 25 °C in the dark.
The modified protein was buffer exchanged using a
Fast-desaltingTM column operated at 1 ml/min.
4-Vinylpyridine was chosen as the alkylating agent because
S- Peptide Mapping of sgp130--
sgp130 (400 µg) in 2 ml of 1%
(w/v) ammonium bicarbonate, containing 2 mM calcium
chloride, was digested at 37 °C for 16 h with either trypsin or
chymotrypsin at an enzyme to substrate ratio of 1:20. For pepsin
digestion, sgp130 (400 µg, ~4 nmol) in 5% formic acid was digested
for 1 h at 37 °C using an enzyme to substrate ratio of 1:20.
Resultant peptides were fractionated by RP-HPLC on either a Vydac C18
column or a Brownlee RP-300 column at 0.5 ml/min at 45 °C. The
column eluent was split (~1:160) post-detector, using a stainless
steel Tee-union (52), whereas the remainder (99.4%) was collected for
further analysis.
For disulfide-containing peptide identification, a portion of the
digest (25%) was reduced with 10 mM DTT at 45 °C for
1 h and rechromatographed under identical conditions. Peaks
whose retention times shifted upon reduction were subjected to
NH2-terminal sequence analysis.
NH2-terminal Sequence
Analysis--
NH2-terminal sequence analyses were
performed using a Hewlett-Packard biphasic NH2-terminal
protein sequencer (model G1005A, Hewlett-Packard) using version 3.0 chemistry as described (50).
Electrospray Mass Spectrometry--
On-line MS analysis of
peptide fractions was performed using either a Finnigan LCQ quadrupole
ion trap mass spectrometer or a TSQ-700 triple quadrupole mass
spectrometer, both equipped with an ESI source as described (50, 53).
S-Pyridylethylated peptides were identified by parent-ion
scanning using the TSQ mass spectrometer by monitoring all peptides for
the labile protonated S-pyridylethyl group
(m/z 106) following CID. Source CID/single ion
monitoring using the LCQ ion trap mass spectrometer was employed to
identify S-pyridylethyl cysteine-containing peptides as
described (52). Peptides were identified from their CID product ion
spectra using either the Finnigan Xcalibur-Biomass software or Protein
Prospector MS-Product algorithm (ProteinProspector Pacific-Rim mirror).
Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass
Spectrometry--
Peptides analyzed by MALDI-TOF mass spectrometry
(Kompact MALDI-IV fitted with a 337 nm laser, Kratos) were
co-crystallized with Homology Modeling--
Domain 5 of sgp130 was modeled using the
two FN III domains from the structure of the cytoplasmic tail of human
integrin
The MODELLER program (55) was used to generate a model of FN III domain
5. The quality of the model was assessed as described (56) in
particular using the ProsaII program (57). A disulfide restraint
between Cys436 and Cys444 in FN III domain 5 was introduced in accordance with our experimental results. Over 100 models of domain 5 were generated with the final model being chosen on
the basis of the quality checks described. The final model of gp130
domain 5 follows integrin Purification and Characterization of sgp130--
Purified sgp130
(yield, ~1.0 mg/liter of CHO cell conditioned medium) was homogeneous
as judged by SDS-PAGE (Fig.
1A, lane 2) and 20 cycles of Edman degradation, which yielded the unique NH2-terminal sequence ELLDPXGYISPESPVVQLHS (data
not shown). At cycle 6, no phenylthiohydantoin (PTH) amino acid was
observed, which is consistent with the predicted cysteine at this
position that would not be revealed by Edman degradation. This
preparation of sgp130 was monomeric (apparent molecular mass, ~120
kDa) as judged by analytical SEC (data not shown), a value that is in agreement with the mass of ~100-120 kDa determined by SDS-PAGE. This
value is significantly higher than 67,914.3 Da, which is calculated
from the amino acid composition (11). Upon treatment with either
neuraminidase or a combination of neuraminidase and an endoglycosidase
mixture, the mass of sgp130 was reduced to ~95 kDa and ~70-80 kDa,
respectively (Fig. 1A, lanes 3 and 4). These data suggest that the increased mass of sgp130 observed by both
SDS-PAGE and analytical SEC is due to N-glycosylation. Pronounced charge heterogeneity of the mature sgp130 was observed by
IEF (Fig. 1B, lane 2). Treatment of sgp130 with
neuraminidase (Fig. 1B, lane 3) reduced the
levels of the acidic forms of the molecule, whereas treatment with a
mixture of neuraminidase and endoglycosidase (Fig. 1B,
lane 4) further reduced the complexity. Taken together,
these data indicate that the heterogeneity of the sgp130 preparation is
primarily due to differential N-linked glycosylation
The immunological integrity of sgp130 was evaluated by Western blot
analysis using a panel of anti-sgp130 monoclonal antibodies (AM64,
GPX7, GPX22, and GPZ35; Ref. 48). Although monoclonal antibody AM64
recognized sgp130 under both nonreducing and reducing conditions, GPX7,
GPX22, and GPZ35 (35) only recognized conformational epitopes of sgp130
under nonreducing conditions (data not shown). The functional integrity
of the sgp130 used in this study was assessed by its ability to bind
the IL-6·sIL-6R binary complex as well as its ability to form a
stable IL-6·sIL-6R·sgp130 ternary complex (13). The mass of the
ternary IL-6 receptor complex observed by analytical SEC (~380 kDa)
(data not shown) was in good agreement with a value calculated for a
hexameric complex of two molecules each of IL-6, IL-6R, and gp130
(13).
Identification of Unpaired Cysteine(s) in sgp130--
Soluble
gp130 was alkylated with 4-vinylpyridine at pH 8.5, digested with
trypsin, and then chromatographed by RP-HPLC using a trifluoroacetic
acid/acetonitrile solvent system (Fig.
2). Using ESI-MS/MS, the parent-ion scan
of the total tryptic digest of the S-pyridylethylated sgp130
identified three Pec containing peptides with molecular masses of 914.6 Da (T1), 2314.6 Da (T2), and 2548.4 Da (T3) (data not shown). Two of
these Pec-containing peptides (T1 and T2) were also confirmed by their
UV absorbance at A254 nm (see tryptic peptide
fractions T1 and T2 in Fig. 2A, inset).
Edman degradation identified tryptic peptide T1 as residues
Cys279-Lys285, T2 as residues
Cys469-Lys489, and T3 as residues
Ser387-Lys409. Cysteines were observed as
PTH-Pec derivatives at positions 279, 469, and 397 in these peptides,
respectively (Table I). The yield of
PTH-Pec in peptide T3 (Cys397) was significantly lower than
that observed for the PTH-Pec derivatives in T1 and T2. Further
confirmation of the identity of these peptides was obtained by
rechromatography and subsequent ESI-MS/MS analysis (Table I). Automated
CID MS/MS analysis of the doubly charged ion of peptide T2
(m/z 1158.9) localized the difference in mass observed from the calculated mass to Cys469 (Fig.
3).
To evaluate the extent of S-pyridylethylation of peptide
fraction T3, this fraction (from Fig. 2A) was
rechromatographed using a capillary RP-HPLC system coupled on-line to
ESI-MS/MS (Fig. 4A). Although
Edman degradation of T3 revealed only one sequence (residues
Ser387-Lys409), examination of the
average mass spectrum of the major total ion current peak
(Fig. 4B) reveals two peptide ions with molecular masses of
2548.4 and 2562.7 Da that differed by 14 Da. The charge state
distribution patterns and relative ion intensities of these ions are
very similar. The parent-ion scan of the 2548.4-Da peptide ion (Fig.
4C) revealed that this peptide was
S-pyridylethylated (Figs. 4C and 4D). Because the
mass difference between S-pyridylethyl and
S-cysteinyl moieties is 14 Da, the possibility that
Cys397 in T3 may also be cysteinylated was further
examined.
To this end, peptide fraction T3 was reduced with DTT and immediately
reanalyzed by capillary RP-HPLC/ESI-MS. It can be seen from the total
ion current profile (Fig. 5A)
that the minor peak (fraction I) corresponds to the 2548.4-Da
Pec-modified form of tryptic peptide
Ser387-Lys409 (compare Figs. 5B and
4D). The major peak (Fig. 5A, fraction II), which was resolved from the Pec peptide-containing fraction, has a mass of 2443.0 Da (Fig. 5C), indicating a loss of
119.7 Da from the parent 2562.7-Da peptide upon reduction (compare
Figs. 5C and 4B). Further analysis of the doubly
charged ion of T3 (m/z 1282.3) by automated
CID-MS/MS identified the peptide as
Ser387-Lys409 containing the additional mass
of 118.9 Da located at Cys397 (Fig.
6). Taken together, these data indicate
that Cys397 is cysteinylated (119 Da) and that this
cysteine modification is readily removed upon reduction with DTT. A
comparison of the two forms of the tryptic peptide fraction T3 (2548.4- and 2562.7-Da peptide ions) in sgp130 indicated that the major cysteine
modification was S-cysteinylation (97%) with ~3% as the
free thiol (as revealed by S-pyridylethylation following
treatment with 4-vinylpyridine).
Determination of Disulfide Bonds of sgp130 by Proteolytic Digestion
and Peptide Mapping--
A portion (25%) of the tryptic digest of
S-pyridylethylated sgp130 was reduced with 10 mM
DTT at pH 8.0 and rechromatographed by RP-HPLC using chromatographic
conditions identical to those employed for the separation of the
nonreduced S-pyridylethylated tryptic digest of sgp130 (Fig.
2). A comparison of panels A and B in Fig. 2
revealed that, upon reduction, only peptide fraction T4, had an altered
retention time, suggesting that it is a disulfide-containing peptide.
Edman degradation of fraction T4 (Table I) identified two peptides, T4a
APXITDWQQEDGTVHR (residues 442-457) and T4b YILEWXVLSDK, (residues 431-441).
Further analysis of peptide fraction T4 by MALDI-TOF MS yielded an
average molecular mass of 3220.7 Da (Fig.
7), which is in good agreement with the
mass observed by ESI ion trap MS (3222.4 Da). The ion
(m/z 1611.9) observed by MALDI-TOF MS is the
doubly charged form of the parent peptide ion
(m/z 3221.7). In addition, two further peptide
ions of (m/z 1856.0) and
(m/z 1368.6) were observed (Fig. 7). Presumably,
these peptide ions are the pseudomolecular ions corresponding to
reduced forms of peptide T4a and T4b (Table I), respectively, that have
arisen by prompt fragmentation (58) of the disulfide linkage.
No peptides from the NH2-terminal region of sgp130,
including four potential disulfide-containing peptides, were obtained using this approach. Close inspection of the peptide map (Fig. 2A) revealed a shallow peak (peptide fraction T5-T6) eluting
late in the gradient. Edman degradation of these pooled fractions
yielded two peptide sequences in low yield, one corresponding to the
NH2 terminus of sgp130 and the other commencing at residue
63 (data not shown). Nonreducing SDS-PAGE analysis of peptide fraction T5-T6 revealed a single protein band of ~50 kDa (Fig. 2A,
inset), suggesting that this fraction contained at least
domains 1 and 2 of sgp130, held together by disulfide bonds, consistent
with a partially digested NH2-terminal "core" polypeptide.
To establish the cysteine connectivities of the Ig-like domain of the
molecule, S-pyridylethylated sgp130 was subjected to extensive digestion (48 h) using a high concentration of trypsin (enzyme to substrate ratio 1:5). Peptide mapping of this tryptic digest
under reducing and nonreducing conditions (data not shown) revealed the
identity of two disulfide-containing peptide fractions. One of these
(T7) was identified as Cys150-Cys160 (Table
I), whereas the other fraction (T8) contained cysteine residues
Cys6, Cys26, Cys32, and
Cys81 linked by disulfide bonds. To determine the
connectivities of Cys6, Cys26,
Cys32, and Cys81, tryptic peptide fraction T8
was subdigested with chymotrypsin. Using a combination of Edman
degradation and MS analysis of the resultant peptides, disulfide bond
linkages were established between Cys6-Cys32
and Cys26-Cys81 (Table I).
An additional cysteine connectivity in sgp130 was identified following
S-pyridylethylation, deglycosylation (see "Experimental Procedures"), and then digestion with pepsin. Peptide mapping of the
resultant peptic digest of S-pyridylethylated sgp130 (400 µg) under reducing and nonreducing conditions (data not shown) revealed the presence of a disulfide-containing peptide P1 (Table I).
Edman degradation and MS analysis established the identity of peptide
P1 as a linear peptide (residues Ile98-Leu132)
containing the disulfide linkage Cys112-Cys122
(Table I). NH2-terminal sequence analysis and MS analysis
of the peptic peptides of sgp130 reconfirmed the identity of the S-pyridylethylated Cys279 and Cys469
and S-cysteinylated Cys397.
N-linked Glycosylation Sites of sgp130--
Of the 11 possible
N-glycosylation sites (Asn-Xaa-(Ser/Thr) motifs) in the
extracellular domain of gp130, nine were found to be glycosylated as
determined by the nonappearance of asparagine residues at positions 21 (NFT), 61 (NRT), 109 (NLT), 135 (NYT), 205 (NLS), 357 (NYT), 361 (NAT),
531 (NYT), and 542 (NET) at the expected cycle during automated Edman
degradation of tryptic digest generated peptides (Table
II). Further confirmation of
N-glycosylated asparagine residues 61, 205, 357, 361, 531, and 542 was provided by Asn/Asp conversion in both tryptic and peptic
sgp130 peptides following endoglycosidase F treatment (59) (see Asn/Asp
conversion at position 109 in peptide P1 (Table I)). Asparagine
residues 224 (NPS) and 368 (NLT) were identified as PTH-asparagine
during Edman degradation indicating that these residues are not
N-glycosylated.
To date there is no complete three-dimensional structure for the
extracellular domain of gp130 or any member of the gp130 family of
receptors. The results presented here for the cysteine connectivity
pattern and carbohydrate attachment sites of sgp130 provide a
foundation for the determination of the tertiary structure of this
molecule and, ultimately, of the IL-6·IL-6R·gp130 complex. Mass
spectrometric and NH2-terminal sequence analysis of
proteolytic digests of sgp130 identified five disulfide linkages
(Cys6-Cys32,
Cys26-Cys81,
Cys112-Cys122,
Cys150-Cys160, and
Cys436-Cys444), two free cysteines
(Cys279 and Cys469), one cysteinylated cysteine
(Cys397), and nine N-linked glycosylation sites
(asparagines 21, 61, 109, 135, 205, 357, 361, 531, and 542), whereas
the two potential N-glycosylation sites at
Asn224 (NPS) and Asn368 (NLS) have no
carbohydrate moieties attached. Previous studies have shown that NPS is
not favorable for N-linked glycosylation, and the NLS is
only glycosylated in 40% of the instances studied (60). Fig
8 shows a schematic representation of the
disulfide bonding pattern and N-glycosylation sites of
gp130.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix bundle structure, the helices being
connected in an up-up-down-down arrangement by three polypeptide loops
(3, 5). These cytokines signal through their respective receptor
systems that comprise a ligand-specific
-subunit and the common
signal-transducing
-subunit, gp130. The shared use of gp130, in
part, provides a molecular basis for the functional redundancy of the
gp130 cytokines. The result of gp130-mediated signaling is the
regulation of a variety of complex cellular processes such as
proliferation, differentiation, and gene activation in a wide variety
of adult tissue systems, owing to the ubiquitous expression of gp130
(6). Insights into the possible in vivo role of gp130 have
come from transgenic and knock-out animal studies. Targeted
inactivation of the gp130 gene results in a prenatal lethal phenotype
that includes defects in the cardiac and hematopoietic systems (7-9).
Chronic activation of gp130 signaling in a transgenic mouse model
results in cardiac hypertrophy (10).
-subunit, which then recruits the signal
transducing gp130 subunit with a resulting ligand-binding affinity of
~10 pM. Importantly, receptor activation depends on
homodimerization or heterodimerization of gp130 (1, 3). gp130 was
initially cloned as a component of the IL-6 receptor complex (11). The binding of IL-6 to its cognate receptor subunit (IL-6R) induces the
dimerization of gp130 (12) and the formation of a hexameric complex
comprising two molecules each of IL-6, IL-6R, and gp130 (13, 14). The
IL-11 receptor complex is similar (15), whereas in the case of LIF
(16), oncostatin M (17, 18), cardiotrophin-1 (19), and ciliary
neurotrophic factor (16), ligand binding induces heterodimerization of
gp130 and the LIF receptor, another signal transducing subunit (20).
Soluble forms of many of the gp130 cytokine receptors, including gp130,
have been found in body fluids of different mammalian species (3).
Soluble gp130 (sgp130) has been detected in human serum (21) and is
most likely translated from alternatively spliced mRNA (22). sgp130
can neutralize IL-6·sIL-6R complexes, thereby acting as an antagonist (21).
-sheet topology (27). In the Ig-like fold,
neighboring
-strands form hydrogen bonds in an antiparallel fashion
to form a
-pleated sheet, and two
-sheets pack against each other
to produce a hydrophobic core. Domains 2-6 of the gp130 are classified
as fibronectin type III (FN III)-like modules, a subclass of the
-sandwich fold (26). The topology of these domains is similar to
those of the Ig-like modules, the notable exception being 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.
The two membrane distal FN III domains (domains 2 and 3) form the
cytokine-binding region (CBR) that is characteristic of class I
cytokine receptors (28, 29). CBRs are characterized by two conserved
disulfide bonds in their NH2-terminal domain and a
WSXWS motif in their COOH-terminal domain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-N-acetylglucosidase F (endo F)
activities (F1, F2, and F3) as well as peptide N-glycosidase (46, 47) was a kind gift from Dr. G. E. Norris (Massey University, New Zealand). HPLC grade solvents were from Mallinckrodt, and all other
buffers and reagents (Analar grade) were from BDH. Anti-human gp130 monoclonal antibodies AM64, GPZ35, GPX7, and GPX22 (11, 48) were
from Dr. K. Yasukawa (TOSOH, Tokyo, Japan). All buffers and solutions
were prepared with deionized water purified and polished by a tandem
Milli-RO and Milli-Q system (Millipore).
20 °C.
-mercaptoethanol) conditions and by isoelectrofocusing
(IEF-PAGE) on precast linear pH gradient (pH 3-10) 5% polyacrylamide
gels (Novex) according to the manufacturer's instructions. Protein bands were visualized by staining with Coomassie Brilliant Blue as
described (50).
-(4-pyridylethyl)-cysteine (Pec) containing peptides
can be identified by their characteristic absorption spectra at 254 nm
(51) and are also readily identified during ESI-MS by the presence of
an ion of m/z 106 following collision-induced dissociation (CID), characteristic of the protonated
S-pyridylethyl moiety (50).
-cyano 4-hydroxycinnaminic acid (16 mg of
matrix/ml aqueous 60% acetonitrile/0.1% (v/v) trifluoroacetic acid).
Matrix (0.5 µl) was deposited onto a clean sample slide immediately
followed by 0.5 µl of peptide fraction. Spectra were calibrated using
the external standards angiotensin (1297.5 [M+H]1+) and a
matrix-derived ion (173.17 [M+H]1+).
6
4 (54), which showed 19 and
16% amino acid sequence identity. These particular FN III domains were
chosen as templates to minimize the insertions and deletions in the
loop regions. The coordinates for the template were taken from the
Protein Data Bank entry 1qg3. Domain 5 of sgp130 was manually aligned
with the template of the cytoplasmic tail of integrin
6
crystal structure (54), conserving the
hydrophobic and sequence patterns of the FN III
-sheets.
6
4 domain 1 in
the B-C and C'-E loops, and follows domain 2 in the C-C' loop and G
strand. The ProsaII Z-score of the final model is
6.26, comparing
favorably with
4.70 and
6.39 for the templates of domains 1 and 2 of integrin
6
4.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (63K):
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Fig. 1.
SDS-PAGE and IEF of recombinant sgp130.
A, SDS-PAGE 4-20% acrylamide precast gel. Lane
1, protein molecular weight standards (SeeBlueTM, Novex);
lane 2, affinity-purified sgp130; lane 3,
neuraminidase-treated sgp130; lane 4, sgp130 treated with a
mixture of neuraminidase and endoglycosidase. The sample load was ~10
µg. B, IEF-PAGE (pH 3-10). Lane 1, protein IEF
standards (Broad pI calibration kit; Amersham Pharmacia Biotech).
Lanes 2-4 were the same as for A. Proteins were
visualized by Coomassie Blue staining (50).
View larger version (28K):
[in a new window]
Fig. 2.
Comparative peptide mapping of a tryptic
digest of sgp130. A, sgp130 was digested with trypsin
as described under "Experimental Procedures," and generated
peptides were fractionated by RP-HPLC using a Vydac octadecyl 5-µm,
300 Å silica column (inner diameter, 250 × 4.6 mm) with a
gradient from 0-70% B, where solvent A was 0.1% (v/v)
trifluoroacetic acid and solvent B was 0.09% trifluoroacetic acid
(v/v)/acetonitrile. Flow rate was 0.5 ml/min, and temperature was
45 °C. Peptides were monitored with four different wavelengths (215, 254, 280, and 290 nm), and spectral data were obtained on-the-fly by
diode array detection. Cysteine-containing peptide fractions identified
by NH2-terminal sequence analysis and MS analysis are
annotated by the letter T (see also Table I). All peptide fractions
were analyzed, and the obtained sequence information is in perfect
agreement with the published amino acid sequence (11) (data not shown).
B, chromatography of a portion (25%) of the tryptic digest
of sgp130 from A following reduction with DTT (see
"Experimental Procedures"). Chromatographic conditions are
identical to A. The arrow indicates the position
of T4 when chromatographed under nonreducing conditions (see
A). Left inset, diode array spectra of T1 and T2.
S-Pyridylethylated peptides (T1 and T2) were identified by
their characteristic spectral properties (maxima at
A254 nm) indicated by an arrow.
Right inset, SDS-PAGE analysis of tryptic peptide fraction
T5 and T6. Fractions T5-T6 from Fig. 2 (A) were pooled (10%
of fraction T5 (1 ml) and T6 (0.9 ml)) and concentrated to 10 µl by
centrifugal lyophilization, diluted 3:1 (v/v) with nonreducing SDS
sample buffer, and loaded onto a 4-20% polyacrylamide precast gel, as
described in Fig. 1. Protein was visualized by silver staining (76).
Lane 1, protein molecular weight standards (Mark12TM,
Novex); lane 2, undigested gp130; lane 3,
fraction T5-T6. The sample load was ~100 ng.
Cysteine-containing peptides generated from proteolytic cleavage of the
extracellular domain of gp130
View larger version (27K):
[in a new window]
Fig. 3.
RP-HPLC/ESI ion trap MS analysis of sgp130
tryptic peptide fraction T2. CID spectrum of the doubly charged
ion of peptide T2 (m/z 1158.9) from Fig.
2A identified this peptide as residues
Cys469-Lys489 with an NH2-terminal
S- -(4-pyridylethylcysteine.
View larger version (23K):
[in a new window]
Fig. 4.
Identification of an
S-Pec-containing peptide from a tryptic digest of
sgp130 by parent-ion scanning ESI-MS. A, total ion
current trace of peptide fraction T3 from Fig. 2. Ionizing plasticizers
from polypropylene collection tubes are indicated by an
asterisk. B, ESI-MS spectrum of the major peak
observed in A, indicated by horizontal bar.
C, parent-ion scan (m/z 106) of
peptide fraction T3 from Fig. 2. D, ESI-MS spectrum of the
parent ion identified in C.
View larger version (17K):
[in a new window]
Fig. 5.
Mass spectrometric analysis of reduced
peptide fraction T3 from Fig. 2. A, total ion current
of reduced peptide T3. B, ESI-MS spectrum of peak I from
A. C, ESI-MS spectrum of peak II from
A. Ionizing plasticizers from polypropylene collection tubes
are indicated by an asterisk. Reducing conditions: 5 µl
(1% of total fraction) of peptide T3 from Fig. 2 was reduced with 10 mM DTT in 0.1 M Tris-HCl, 1 mM
EDTA, pH 8.4, for 1 h.
View larger version (29K):
[in a new window]
Fig. 6.
RP-HPLC/ESI ion trap MS/MS analysis of
tryptic peptide fraction T3. CID spectrum of the doubly charged
ion of peptide T3 (m/z 1282.3) from Fig.
2A identified this peptide as residues
Ser387-Lys410 of sgp130. The mass difference
between ions y12 and y13
(m/z 222.0) is consistent with cysteinyl-cysteine
at position 397.
View larger version (15K):
[in a new window]
Fig. 7.
MALDI-TOF MS analysis of tryptic peptide
fraction T4. Peptide fraction T4 from Fig. 2A (0.5 µl) was co-crystallized with -cyano 4-hydroxycinnaminic acid (see
"Experimental Procedures"). The mass spectrum displayed is an
accumulated average of 50 profiles. Metastable and doubly charged ions
of disulfide peptide T4 (m/z 3221.7) are shown at
a 5-fold increased intensity.
Summary of N-glycosylation sites in human gp130
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
[in a new window]
Fig. 8.
Schematic view of the extracellular domains
of human gp130 and the GCSFR. Cysteine residues are indicated by
solid blue circles and numbered according to their position
in the polypeptide sequence. Cysteine connectivities determined
experimentally for sgp130 and soluble GCSFR (62) are indicated as a
blue line. Potential N-linked glycosylation sites
that contain a carbohydrate moeity are indicated by a green
ellipse, and nonglycosylated sites are indicated by a red
ellipse. The potential domain structure of sgp130 was obtained
from Hibi et al. (11) and that of GCSFR by homology with
gp130 (data not shown). Domain 1, Ig-like;
domains 2 and 3, cytokine-binding region;
domains 3-5, FN III. For illustration purposes, domain 1 is
shown as an I-type Ig-like domain.
The two disulfide linkages of the NH2-terminal Ig-like
domain of gp130 (Cys6-Cys32 and
Cys26-Cys81) (Fig.
9A) are found between the A
strand and B-C loop and between the B and F strands, respectively, with
the second disulfide linkage characteristic of a typical I-type Ig-like
fold (25). The amino acid sequences of the Ig-like domains of gp130 and
GCSFR are homologous, suggestive of an identical fold, yet their
disulfide patterns differ (Fig. 9A). The percentage amino
acid sequence identity is 19% over the whole Ig-like domain, with 30%
identity over the region encompassing the first three
cysteines (Fig. 9A).
|
The two consecutive disulfide linkages in the NH2-terminal
FN III domain of the CBR of gp130
(Cys112-Cys122 and
Cys150-Cys160) correspond to those
experimentally determined by a peptide mapping approach for the CBR of
other class I cytokine receptor families, namely GCSFR (61, 62), growth
hormone receptor (63), and leptin receptor (64). These cysteine
connectivities have also been confirmed by the x-ray crystal structure
analysis of the CBR of gp130 (43), the growth hormone-growth hormone
receptor complex (65), the GCSF·GCSFR complex (66), the growth
hormone receptor-prolactin receptor complex (67), the erythropoietin receptor-peptide agonist complex (68), the IL-4·IL-4R complex (69),
and IL-12R (70). An inspection of the gp130 CBR three-dimensional structure reveals that the N-linked carbohydrate moieties
found at Asn109 (A strand, domain 2), Asn135 (C
strand, domain 2), and Asn205 (A strand domain 3) are
consistent with these asparagines being solvent exposed, whereas the
potential N-glycosylation site at Asn224 would
not be expected to be glycosylated because this asparagine residue is
partially buried in the structure (43).
Cys279, Cys397, and Cys469 located in domains 3, 4, and 5, respectively, do not form disulfide bonds and occur as either a free thiol or in the case of Cys397, a cysteinylated form (Fig. 8). Previously, we have reported the presence of both free and cysteinylated cysteine residues in the human IL-6R (52). The occurrence of free cysteines has also been reported for GCSFR (62) and leptin receptor (64). Cys279, which is present as a free thiol in sgp130, is not conserved among the species variants of gp130 whose sequences have been determined to date (i.e. mouse (23), rat (24), chicken (71), and frog2). Analysis of the gp130 CBR crystal structure (43) indicates that Cys279 is situated on the B strand of domain 3 and is partially buried. Cys397, which is located at the end of the G strand of domain 4 and likely forms part of the hinge region with domain 5, was found to be S-cysteinylated. This finding is consistent with Cys397 being solvent exposed.
Cys469, located in domain 5, is the only free cysteine
conserved in all species of gp130 studied to date. As such,
Cys469 may be involved in the homodimerization related
intracellular signaling of this molecule that is reported to involve
disulfide-bond formation (12). The free thiol Cys469 is
located at the NH2 terminus of the F strand flanked by
conserved lysine and tyrosine residues and would be predicted to be
solvent exposed and hence modified. Our homology modeling data predict that although the free thiol Cys469 is orientated away from
the hydrophobic core of domain 5, it is shielded from the solvent by a
short eight-residue "protective loop" tethered by the disulfide
bond Cys436-Cys444 in strands C and C' of
domain 5 (Fig. 10). Thus, a
conformational change in gp130 would be required to reposition this
protective loop and expose Cys469 to possible disulfide
bond formation with another gp130 molecule. Müller-Newen and
colleagues (73) have recently demonstrated, using a panel of monoclonal
antibodies, that the enforcement of gp130 dimerization alone is not
sufficient for receptor activation and that an additional conformation
requirement is needed. A similar phenomenon has also been reported for
the erythropoietin receptor (74) and is also applicable to other types
of receptors from both eukaryotic and prokaryotic origins (75), where
dimerization alone is not sufficient for intracellular signaling but a
further conformational change within the receptor dimer induced by
ligand binding may be essential to propagate the signal across the cell membrane. It is tempting to speculate that the suggested conformational switch of the gp130 dimer required for activation of intracellular signaling (73) may involve the movement of the protective loop, thereby
exposing the buried thiol (Cys469) in such a way that it
can partake in disulfide bond formation with another gp130 molecule.
The role of FN III domain 5 and the conserved free Cys469
residue in gp130 homodimerization and intracellular signaling process
must await further studies.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. H. Ji for assistance in production of sgp130 used in this study, Dr. G. E. Reid, J. Eddes, and D. F. Frecklington for help in tandem MS/MS, and Dr. M. J. Layton and A. R. Cole for critical reading of the manuscript
![]() |
FOOTNOTES |
---|
* 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: Ludwig Inst. for Cancer Research, P.O. Box 2008, Royal Melbourne Hospital, Parkville, VIC 3050, Australia. Tel.: 61-3-9341-3155; Fax: 61-3-9341-3192; E-mail: richard.simpson@ludwig.edu.au.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M009979200
2 J. Chen, A. Grace, and K. R. Chien, NCBI accession number 29 05611.
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ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
GCSF, granulocyte colony-stimulating factor;
GCSFR, GCSF receptor;
FN
III, fibronectin type III;
CBR, cytokine-binding region;
s, soluble;
v, viral;
MS, mass spectrometry;
CID, collision-induced dissociation;
ESI, electrospray ionization;
PAGE, polyacrylamide gel electrophoresis;
RP, reverse phase;
HPLC, high-performance liquid chromatography;
SEC size
exclusion chromatography, Pec,
S--(4-pyridylethyl)-cysteine;
LIF, leukemia inhibitory
factor;
IEF, isoelectrofocusing;
IL-6R, IL-6 receptor;
CHO, Chinese
hamster ovary;
DTT, dithiothreitol;
MALDI, matrix-assisted laser
desorption ionization;
TOF, time-of-flight;
PTH, phenylthiohydantoin.
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