From the Eppley Institute for Research in Cancer and
Allied Diseases, § Department of Pharmacology,
¶ Department of Biochemistry and Molecular Biology, and the
§§ Department of Obstetrics and Gynecology,
University of Nebraska Medical Center, Omaha, Nebraska 68198
Received for publication, November 8, 2000
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ABSTRACT |
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Human chorionic gonadotropin (hCG) is a
heterodimeric member of a family of cystine knot-containing proteins
that contain the consensus sequences
Cys-X1-Gly-X2-Cys and
Cys-X3-Cys. Previously, we characterized the
contributions that cystine residues of the hCG subunit cystine knots
make in folding, assembly, and bioactivity. Here, we determined the
contributions that noncysteine residues make in hCG folding, secretion,
and assembly. When the X1,
X2, and X3 residues of
hCG- The cystine knot motif defines a superfamily of dimeric proteins
and appears to function as a structural scaffold that stabilizes the
3-loop structures of the individual subunits (1). As shown in Fig.
1, the cystine knot consists of three
disulfide (S-S)1 bonds; two
of these bonds bridge adjacent polypeptide strands, creating a ring
that includes the intervening polypeptide backbone, and the third bond
penetrates this ring (1, 2). The cystine knot is common to a
biologically diverse set of dimeric proteins including transforming
growth factor- and -
were substituted by swapping their respective cystine
knot motifs, the resulting chimeras appeared to fold correctly and were
efficiently secreted. However, assembly of the chimeras with their wild
type partner was almost completely abrogated. No single amino acid
substitution completely accounted for the assembly inhibition, although
the X2 residue made the greatest individual
contribution. Analysis by tryptic mapping, high performance liquid
chromatography, and SDS-polyacrylamide gel electrophoresis
revealed that substitution of the central Gly in the
Cys-X1-Gly-X2-Cys
sequence of either the
- or
-subunit cystine knot resulted in
non-native disulfide bond formation and subunit misfolding. This
occurred even when the most conservative change possible (Gly
Ala)
was made. From these studies we conclude that all three
"X" residues within the hCG cystine knots are
collectively, but not individually, required for the formation of
assembly-competent hCG subunits and that the invariant Gly residue is
required for efficient cystine knot formation and subunit folding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, vascular endothelial growth factor, platelet-derived
growth factor, and human chorionic gonadotropin (hCG) (1-4).
Additionally, more than 30 other proteins are predicted to contain this
motif (2). The functional importance of the cystine knots of hCG
(5-9), transforming growth factor-
1 (10), and platelet-derived
growth factor (11) is evident from studies where cysteine residues
within the knot were mutated, thus preventing a particular S-S bond
from forming. Disruption of the cystine knot disulfides results in the
synthesis of nonfunctional proteins that are usually degraded
intracellularly. Thus, a more detailed understanding of the functional
components of cystine knots will help to understand how members of this
emerging protein family fold and assemble into biologically active
molecules.
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Fig. 1.
Schematic diagram of hCG subunits.
GPH- and hCG-
each contain 3 loops (labeled L1,
L2, and L3) and are arranged in a head-to-tail
orientation in the hCG dimer (17, 18). Both subunits also contain a
cystine knot formed by three S-S bonds (displayed as lines
connecting the solid circles; numbers indicate
the cysteine residue number). Noncystine knot S-S bonds are represented
by the lines connecting open circles. The noncysteine
residues of the cystine knots are labeled X1,
X2, and X3. A
particularly interesting feature of the hCG dimer is the C-terminal
region of hCG-
that wraps around the GPH-
L2, creating the
so-called "seat-belt."
The subunits of hCG are prototypes for the cystine knot growth factor
family (1). Heterodimeric hCG forms when hCG- assembles with the
common glycoprotein hormone
-subunit (GPH-
). GPH-
also
assembles with luteinizing hormone
-subunit, thyroid-stimulating hormone
-subunit (TSH-
), and follicle-stimulating hormone
-subunit to form luteinizing hormone, TSH, and follicle-stimulating
hormone
-subunit, respectively (12). Luteinizing hormone-
,
TSH-
, and follicle-stimulating hormone
also contain the
consensus residues required to form a cystine knot, but the actual
presence of the knot has not yet been confirmed by structural studies.
Previously, we described the folding pathways of both hCG- (9,
13-15) and GPH-
(6, 16) using S-S bond formation as an index of
folding. The methods established in our laboratory for the folding of
these two subunits now allow us to determine the importance of
noncysteine residues of the cystine knot in hCG subunit folding,
secretion, and assembly. Fig. 1 illustrates the location of the cystine
knot motifs for both subunits of hCG, as well as the noncystine knot
disulfides (17, 18). The 8-residue ring of the cystine knot consists of
four cysteines that form two S-S bridges, a Gly residue common to all
8-membered cystine knot rings, and three nonconserved residues (termed
X1, X2, and X3). Thus, the consensus sequences for this
motif can be defined as
C-X1-G-X2-C and
C-X3-C (3, 19).
The residues at the X1,
X2, and X3 positions vary
among cystine knot-containing proteins and their functional importance
is largely unknown. The sequence containing X1
and X2 in all four glycoprotein -subunits is
CAGYC. The equivalent sequence in GPH-
is CMGCC, where the Cys at
the X2 position forms a noncystine knot S-S bond with
Cys7 (Fig. 1). In this report, we investigated the
contribution of hCG X1,
X2, and X3 residues in
folding, secretion, and assembly by employing a chimeric strategy where
the residues within the hCG-
and GPH-
cystine knots were
interchanged individually or collectively. Furthermore, the role of the
intervening Gly residue in both cystine knots was determined using
various amino acid substitutions. We report that: (i) there is a
subunit-specific complement of three "X" residues, all of which are
needed for efficient assembly and (ii) the presence of the central
invariant Gly is an absolute requirement for efficient folding, cystine knot formation, and hCG assembly.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- 293T cells (20) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, and penicillin (100 units/ml)/streptomycin (100 µg/ml) (Life Technologies, Inc.).
Site-directed Mutagenesis--
Mutations were made using a
"megaprimer" polymerase chain reaction methodology (21) with
Pfu polymerase (Stratagene). The GPH- polymerase chain
reaction products were cloned into pcDNA3 (Invitrogen) and the
hCG-
polymerase chain reaction products were cloned into pGS (9).
DNA sequencing confirmed incorporation of desired mutations. Plasmid
DNA was purified using the Maxi Plasmid Kit (Qiagen) according to the
manufacturer's protocol and used for transient transfection as
described below.
Transient Transfection--
293T cells (1.9 × 106) were plated into 60-mm plastic dishes and grown
overnight to 70-80% confluency. Plasmid DNA was precipitated as
described previously (6). For coexpression of hCG- and GPH-
, both
plasmids were included in the precipitation. To obtain comparable
expression levels in coexpression studies, a 40:1 GPH-
to hCG-
ratio of plasmid was used. The resulting precipitate was added dropwise
to the dishes and agitated gently to mix. To ensure a uniform
precipitate exposure, one large-scale precipitation was distributed
equally among all dishes. Cells were incubated for 2 days at 37 °C
and used for metabolic labeling.
Metabolic Labeling with
[35S]Cysteine--
Transiently transfected 293T cells
were pulse-labeled for the times indicated in the text with
L-[35S]cysteine (~1100 Ci/mmol; PerkinElmer
Life Sciences), at a concentration of 100-150 µCi/ml, in serum-free
medium lacking cysteine (9). For experiments using dithiothreitol
(DTT), the DTT was added with the [35S]cysteine at a
final concentration of 2.0 mM. Pulse incubations were
carried out as described previously (13) and cells were incubated for
the chase times indicated; the chase medium was saved for secretion
studies. Cells were harvested by rinsing with cold phosphate-buffered
saline and immediately lysed in 2.5 ml of phosphate-buffered saline
containing detergents (1.0% Triton X-100, 0.5% sodium deoxycholate,
and 0.1% SDS), protease inhibitors (20 mM EDTA and 2 mM phenylmethanesulfonyl fluoride), and 5 mM N-ethylmaleimide (NEM), pH 7.0, or 50 mM
iodoacetate, pH 8.0, to trap free thiols in folding intermediates that
contained unformed S-S bonds. NEM was used for GPH- because it
results in efficient alkylation of GPH-
thiols and better separation
of folding intermediates by HPLC (6). Similarly, iodoacetate was used
for hCG-
because it efficiently alkylates
-subunit thiols and
facilitates mapping of hCG-
tryptic peptides (13, 14). Cell lysates
were incubated for 10-20 min at room temperature in the dark, followed
by disruption through a 22-gauge needle (5 times) and centrifuged for
1 h at 100,000 × g. The collected chase medium
was also clarified by centrifugation.
Immunoprecipitation of hCG Subunits from Cell Lysates and Chase
Media--
Immunoreactive forms of GPH- or hCG-
were
immunoprecipitated with polyclonal antibodies specific for each
respective subunit (6, 22). Immunoprecipitations were carried out at
4 °C for 16-20 h with rotation in the dark. Immune complexes were
precipitated with protein A-Sepharose (Sigma) and prepared for
SDS-polyacrylamide gel electrophoresis (PAGE) or reversed-phase HPLC as
described below.
SDS-PAGE and Quantitation of [35S]Cysteine-labeled
Subunits--
Radiolabeled folding forms that adsorbed to protein
A-Sepharose beads were prepared as described previously (13). Briefly, protein A-Sepharose beads containing immunopurified subunits were resuspended in twice concentrated SDS gel sample buffer (125 mM Tris-HCl, pH 6.8, containing 2% SDS, 20% glycerol, and
40 µg/ml bromphenol blue). For reducing conditions,
-mercaptoethanol was included at a final concentration of 2%.
Samples were boiled for 5 min, loaded on polyacrylamide gradient slab
gels (5-20%), and run by the method of Laemmli (23). Gels were dried
in vacuo on filter paper and exposed to a phosphorscreen
(Molecular Dynamics). The phosphorscreen was scanned on a Molecular
Dynamics Storm 860 and bands were quantitated using the Molecular
Dynamics ImageQuant (version 5.0) volume report. To determine the
percentage of GPH-
that had combined with hCG-
in Figs. 3 and 7,
the following formula was used: [10/12 (amount of hCG-
that
coimmunoprecipitated with GPH-
) + (amount of GPH-
in anti-
immunoprecipitation)]/[total GPH-
]. For the
-C31Y mutant, 8/12
was used instead of 10/12 because this mutant has only 8 cysteine residues.
Reversed-phase HPLC Analysis--
Radiolabeled folding
intermediates that adsorbed to protein A-Sepharose beads were prepared
as described previously (13). Briefly, protein A-Sepharose
beads/antibody-antigen immunocomplexes were washed three times with
phosphate-buffered saline containing detergents (1% Triton X-100,
0.5% sodium deoxycholate, 0.1% SDS) followed by four washes with
phosphate-buffered saline lacking detergents. Immunocomplexes were
pelleted between washes by centrifugation for 1 min at 2000 × g. To dissociate the Sepharose/antibody/antigen interactions, immunocomplexes were treated with 6 M
guanidine HCl, pH 3.0 (sequenal grade; Pierce), for 16-20 h while
rotating at room temperature. 100 µg of myoglobin was added as a
carrier. The guanidine eluates were injected onto a Vydac 300-Å
C4 reversed-phase column equilibrated with 0.1%
trifluoroacetic acid and eluted using an acetonitrile gradient as
described previously (14). Fractions were collected in 1-min intervals
and quantitated by scintillation counting. Samples were stored at
20 °C until further characterization.
Tryptic Digestion and Reversed-phase HPLC Analysis of Tryptic
Peptides--
HPLC fractions from C4 reversed-phase HPLC
representing hCG- folding intermediates p
1 or p
2 were pooled,
concentrated, and digested for 16-20 h in silanized polypropylene
tubes containing 100-200 µg of myoglobin, 0.03% trypsin (Sigma), 5 mM CaCl2, and 50-150 mM Tris-HCl,
pH 8. Digestions were continued for 2 h with two additional
aliquots of 25 µg of trypsin (0.06% final concentration) (13, 14).
hCG-
tryptic peptides were separated on a Vydac C18
reversed-phase column as described previously (14). Amino acid
sequencing was used previously to identity the peptide(s) in each peak
(14).
Amino Acid Analysis Procedure to Determine S-S Bond
Content--
A modified protocol, similar to the one used in
determining the S-S folding pathways for potato carboxypeptidase
inhibitor and human epidermal growth factor (24, 25) was used.
[35S]Cysteine-containing folding forms isolated from
reversed-phase HPLC were dried in vacuo and hydrolyzed as
described previously (6). Quantitation of [35S]cystine
and succinyl-[35S]cysteine (hydrolysis product of
NEM-Cys) was performed using a modification of the method described by
Cohen and Michaud (26). Hydrolysates were resuspended in 10 µl of 10 mM HCl. To this, 70 µl of 0.2 N borate
buffer, pH 8.8, was added. Derivatization of amino acids was performed
by adding 30 µl of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (8 mg/ml in anhydrous acetonitrile). Samples were dried in vacuo. Before injection, samples were resuspended in 110 µl of buffer A (140 mM sodium acetate, 17 mM
triethylamine, pH 4.9). Derivatized amino acids were separated by HPLC
as described previously (6) using buffer A and buffer B (60%
acetonitrile in water). The column was eluted at 1.0 ml/min at 30 °C
and 1-ml fractions were collected and quantitated by scintillation
counting. Recovery of [35S]cystine represented S-S bonded
cysteine residues, whereas succinyl-[35S]cysteine
represented cysteine residues of unformed S-S bonds. The percentage of
[35S]cystine and succinyl-[35S]cysteine was
calculated by dividing the counts/min recovered for each species by the
total counts/min recovered. Fully folded [35S]cysteine-labeled hCG- was used as a positive
control for [35S]cystine content.
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RESULTS |
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Swapping of hCG- and GPH-
Cystine Knot Motifs--
The only
conserved sequences in cystine knot-containing proteins that have an
8-membered ring are the
C-X1-G-X2-C and
C-X3-C sequences (3, 19). The contribution that
the X residues make toward attaining a native conformation
is unknown. Furthermore, it is not known whether cystine knot sequences
are protein-specific, or whether the residues of the knot motif are
functionally interchangeable. To address this, we used a chimeric
strategy wherein cystine knot residues of GPH-
and hCG-
were
swapped singly or collectively.
Swapping of hCG cystine knot motifs was accomplished by site-directed
mutagenesis at the X1,
X2, and X3 positions to
match the residue(s) of the other subunit. Three single GPH- mutants (
-M29A,
-C31Y, and
-H83Q) and a GPH-
mutant containing all three mutations (termed
knot) were
constructed (Table I). Thus, the amino
acid sequence of the
knot cystine knot matched that of hCG-
. Cys7 (which normally pairs with
Cys31) was also changed to Ala in the
-C31Y mutant to
prevent a free thiol at Cys7 from causing S-S
rearrangements or retention of the subunit in the endoplasmic reticulum
(27). Importantly, removal of S-S bond 7-31 does not affect GPH-
folding, secretion, or hCG biological activity (6, 7). In addition, the
hCG-
cystine knot residues were replaced with those of GPH-
to
give
-A35M,
-Y37A,
-Q89H single mutants and a
knot triple mutant (Table I).
-Y37 was replaced with Ala instead of Cys to avoid introduction of a free
thiol for the reasons alluded to above. Ala was selected because its
side chain properties closely resemble those of Cys, and because Ala
and Ser substitutions have similar effects on hCG-
folding and
assembly when they are substituted for Cys (8).
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The six single mutants (three GPH- and three hCG-
), two triple
mutants (
knot and
knot), and wild type (WT) subunits were analyzed for proper
folding as described previously (6, 13, 14, 16). Briefly, GPH-
folding was monitored using S-S bond formation and HPLC elution times
(6, 16), while hCG-
folding was monitored by shifts in migration on
nonreducing SDS-PAGE (14, 22). No significant differences in folding
were observed for any of the mutants (data not shown), suggesting that these cystine knots may be interchangeable. To test this further, we
determined the efficiency of subunit secretion. Secretion was assayed
by pulse labeling transiently transfected 293T cells for 10 min,
followed by a 10-min or 8-h chase. The immunopurified cell lysates and
media were analyzed by SDS-PAGE (Fig.
2A), and the bands were
quantitated as described under "Experimental Procedures." The
percent secretion for the GPH-
and hCG-
chimeras are shown in
Fig. 2, B and C. Consistent with previous
studies, about 80% of WT GPH-
and hCG-
were secreted by 8 h. Furthermore, swapping of single residues or the entire cystine knot
did not significantly affect subunit secretion of GPH-
or hCG-
(Fig. 2). This efficient secretion is another indicator that these
subunits folded to a native or native-like conformation, since
misfolded or incompletely folded hCG subunits are generally retained
intracellularly and degraded (5, 6).
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Finally, we assayed for the ability of the GPH- cystine knot
chimeras to assemble with
-WT and, conversely, for the ability of
hCG-
cystine knot chimeras to assemble with
-WT. To do this, 293T
cells coexpressing both subunits were pulse-labeled and chased for
8 h. Unassembled GPH-
and intact hCG
/
dimer were first precipitated from the collected medium with a polyclonal
-antiserum. This step was followed by a second precipitation with a polyclonal
-antiserum to recover any excess, unassembled hCG-
. There was a
dramatic decrease in the ability of
knot
and
knot to combine with
-WT and
-WT, respectively (Fig. 3,
A and B). Furthermore, this decreased combination
was not due to altering any one residue, as all of the single mutations
were significantly less deleterious than the triple mutations. However,
the single mutations at the X2 position
(
-C31Y and
-Y37A) did decrease assembly to a level intermediate
to that of WT and the triple mutants. Co-transfection of
knot and
knot together resulted in <5% assembly (data not shown).
Taken together, these data indicate that the noncysteine residues
within the GPH-
and hCG-
cystine knots contribute to an
assembly-competent conformation in a subunit-specific manner.
Furthermore, this contribution appears to be a function of the set of
all noncysteine residues, as opposed to arising from the contribution
of a single residue.
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The Central Gly Residue in the hCG- Cystine Knot Is Critical for
Folding, Secretion, and Assembly--
As alluded to above, the central
Gly residue in hCG-
is conserved among all known cystine knots that
contain an 8-membered ring structure (1, 2). A mutation resulting in
the conversion of the central Gly residue to Arg in TSH-
causes
congenital isolated TSH deficiency, wherein the mutant TSH-
fails to
be secreted and assemble with GPH-
(28). This demonstrates that the
central Gly of the CAGYC region is critical for TSH activity and
suggests that it may also be essential to the function of the other
glycoprotein hormones.
Knowledge of the hCG- folding pathway (13, 14) provides a novel
system to determine the contribution that residues of the cystine knot
make in attaining an assembly-competent conformation. To determine
whether substitution of the Gly of the CAGYC region of hCG-
alters
folding, secretion, and/or assembly, we created and analyzed three Gly
mutants:
-G36A,
-G36N, and
-G36R. Mutation to Arg was chosen
because this mutation is observed in the naturally occurring TSH-
mutant (28), G36N was chosen because Asn has a smaller neutral side
chain in comparison with the positively charged Arg, and G36A was
chosen because it is the most conservative change possible; however,
Ala in most cases can adopt the required positive
torsion angle
only under conditions of unfavorable steric hindrance (29). 293T cells
expressing
-WT,
-G36N,
-G36R, or
-G36A were pulse-labeled
with [35S]cysteine and chased for 0, 5, 15, 30, 60, 120, or 480 min. Fig. 4A shows the
progression of
-WT from p
1 (the earliest detectable folding
intermediate) to p
2 to mature
. At chase times
60 min,
-WT
was detectable in the media as mature, secreted
. Fig. 4B shows that most of
-G36A did not progress beyond p
1 and was not
secreted. However, a p
2-like species of
-G36A was isolated by
reversed-phase HPLC (Fig. 4C). This species was termed
"p
2-like" since it eluted from HPLC at a similar time to that
of WT p
2 (13, 14). Folding and secretion data for
-G36N and
-G36R subunits yielded similar results to those shown in Fig. 4 (not
shown).
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Tryptic digestion of fully folded native hCG- (i.e. all
native S-S bonds formed) produces [35S]cysteine-labeled
peptides, all of which are linked by S-S bonds (13, 14). If a
particular S-S bond has not yet formed in a given intermediate, then
digestion with trypsin results in the release of a
[35S]cysteine-containing peptide from the S-S-linked core
material (13, 14). Thus, HPLC separation of released peptides from the
S-S-linked peptides identifies the S-S bonds of a given hCG-
folding
intermediate that are unformed. Fig. 5,
A and C, show the respective reversed-phase HPLC
profiles of trypsin-treated WT hCG-
that eluted from HPLC at the
positions of p
1 and p
2. As defined in previous studies (13, 14),
the release of peptides 9-20, 69-74, 87-94, 96-104, and 105-114
from G36A p
1 (Fig. 5B) indicates that Cys10,
Cys72, Cys90, Cys93,
Cys100, and Cys110, respectively, were not part
of a S-S bond. Additionally, unidentified peaks were present (labeled
with an asterisk in Fig. 5, B and D),
which suggest the presence of non-native S-S bonds. Peptides 9-20 and
87-94 were not present in the WT hCG-
p
2 tryptic profile (Fig.
5C), consistent with our previous report that the cysteines in these peptides are involved in S-S bonds in the p
2 intermediate (14). The tryptic profile of
-WT p
2 clearly differed from that of
-G36A p
2-like (Fig. 5, compare C and D),
suggesting that non-native S-S bonds had formed in
-G36A.
Furthermore, unlike WT-
, the 87-94 and 9-20 peptides (Fig.
5D, peaks 3 and 4, respectively) were
recovered in G36A p
2-like digested material, indicating that S-S
bonds 38-90 and 9-57 had not formed completely. The tryptic map of
G36A p
2-like material was similar to G36A p
1 (Fig. 5, compare
B and D), demonstrating that the material that
eluted from HPLC at the p
2 locus (i.e. p
2-like) more
closely resembled p
1 than the more folded p
2. Tryptic maps of
-G36N and
-G36R revealed that non-native S-S bonds had similarly
formed (data not shown).
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Taken together, these data indicate that the central Gly of the hCG-
CAGYC region is critical for the proper formation of S-S bonds and
thus, is important for its folding and secretion. This also implies
that the Gly
Arg mutation observed in congenital isolated TSH
deficiency (28) results from improper folding of TSH-
, which
prevents its assembly with GPH-
.
Mutation of the Invariant GPH- Cystine Knot Gly
Residue--
The previous section demonstrated that the Gly residue in
the CAGYC sequence of hCG-
is critical for proper folding. This implies that this Gly may be critical for the folding of other cystine
knot-containing proteins as well. To test this, we made the equivalent
G30A mutation in the CMGCC sequence of GPH-
.
Unlike hCG- folding intermediates, GPH-
intermediates do not
migrate differently on SDS-PAGE (6). However, GPH-
folding can be
monitored by changes in reversed-phase HPLC elution times; unfolded
GPH-
containing no S-S bonds and being more hydrophobic, elutes
later than the native conformation and folding intermediates (6, 16).
Moreover, HPLC elution position correlates with the formation of S-S
bonds as GPH-
folds to its less hydrophobic conformation
(i.e. earlier eluting species contain more S-S bonds than
the later eluting, less folded species) (6, 16). Shown in Fig.
6 are the HPLC profiles generated for
-G30A after a 10-min pulse in the presence of DTT, followed by 0-, 5-, and 30-min chases after DTT removal. DTT was used in the pulse to
delay the formation of S-S bonds until its removal during the chase
(6).
-G30A folding did not generate a species that eluted at the
position of native
-WT (Fig. 6, vertical dotted line).
Additionally, a late eluting peak (Fig. 6, peak *) was
observed at all chase times. This peak migrated at a relative molecular
weight of about twice that of
-WT, G30A-
1, and
G30A-
2 when analyzed by nonreducing SDS-PAGE (data not
shown). This suggests that some of
-G30A had formed a homodimer. In
contrast, reduction of S-S bonds before SDS-PAGE resulted in migration
at the molecular weight of monomeric GPH-
. These data suggest that a
significant proportion of
-G30A forms dimers that arise from
non-native, intermolecular S-S bonds.
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Amino acid analysis of -G30A
1 and
2
was used to quantitate the number of S-S bonds that had formed in each
species. Recovery of 97% of the 35S label as cystine and
3% as succinyl-cysteine (hydrolysis product of NEM-alkylated cysteine)
indicates that
2 contained five intact S-S bonds (Table
II). As expected, the later eluting
species,
1, contained less than five S-S bonds (3.7 out
of 5). Although it is not obvious whether the bonds present in these
two species are native, the differences in HPLC elution times compared
with native WT GPH-
(Fig. 6) nonetheless implies that these folding forms have non-native conformations.
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To determine the efficiency of secretion for -G30A, 293T cells
expressing
-G30A or
-WT were pulse-labeled for 10 min with [35S]cysteine and chased for 10 min or 8 h. The
immunopurified cell lysates and media containing GPH-
were analyzed
by reducing SDS-PAGE and bands quantitated as described under
"Experimental Procedures." About 80% of
-WT present at 10 min
was secreted into the medium by 8 h (Fig.
7A). In contrast, only about
40% of
-G30A was secreted (Fig. 7A). Furthermore, <10%
of
-G30A remained in the cell after 8 h (data not shown),
indicating that about 50% had been degraded. This is consistent with
our previous reports (6, 16) demonstrating that mutant forms of GPH-
that do not migrate at the position of
-WT on HPLC (i.e.
they contain a non-native conformation) are readily degraded.
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To determine whether -G30A could assemble with hCG-
, both
subunits were coexpressed in 293T cells, pulse-labeled with
[35S]cysteine for 20 min, and chased for 8 h.
Immunopurified subunits were analyzed by SDS-PAGE, the bands were
quantitated, and the percentage of GPH-
that had combined with
hCG-
was calculated (see "Experimental Procedures"). Results of
this analysis are shown in Fig. 7B. Only 30% of the
secreted
-G30A combined with WT hCG-
, compared with 80% for
-WT. These results further demonstrate the importance of the central
cystine knot Gly residue in protein folding and heterodimer formation.
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DISCUSSION |
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The growth factor cystine knot superfamily of dimeric proteins contain similar structural topologies but lack significant sequence similarity other than the spacing of the six cysteine residues that form the three cystine residues of the knot (1). The importance of the cystine residues in producing functional proteins has been well documented (6, 7, 9-11, 16), but the role of the noncysteine residues located within these cystine knots is largely unknown.
All known growth factors containing a cystine knot motif have an
8-amino acid ring structure, with the exception of nerve growth factor,
which contains a 14-membered ring. For the 8-residue rings, such as
those found in GPH- and hCG-
, the two sides of the ring contain
5- and 3-residues and are linked by two S-S bridges. Other than the
cysteine residues, the only conserved residue of the ring is a Gly
located at the central position of the 5-residue stretch, such that
this sequence is termed
C-X1-G-X2-C. Studies showing that this Gly residue is essential for producing functional TSH
(28, 30) have been repeated in hCG-
using identical (Gly
Arg)
and similar (Gly
Asn) mutations (31, 32). However, a lack of
structural data and knowledge of the folding mechanisms for these
subunits at the time of these observations failed to define why this
Gly is critical. In light of more recent findings, including the hCG
crystal structure (17, 18) and knowledge of GPH-
and hCG-
folding
(6, 9, 14, 16), we can now address the mechanism by which specific
residues within the hCG cystine knots contribute to hormone function.
In particular, results from mutational analyses can be more precisely
interpreted because we can distinguish between two general consequences
of these mutations: (i) the mutation removes a key residue important
for a direct subunit interaction; or (ii) the mutation causes global
misfolding such that the protein cannot attain native structure.
The central Gly residue located between X1 and
X2 is thought to be necessary because, in
contrast with other amino acids, Gly can readily adopt a positive torsion angle, which allows it to avoid steric hindrance with the
penetrating S-S bond of the cystine knot (1). The biological importance
of this Gly can be inferred from several observations. First, a
naturally occurring Gly
Arg mutation in TSH-
causes congenital
isolated TSH deficiency (28). Second, mutation of the equivalent Gly in
GPH-
prevents the production of functional hCG (31) and TSH (30).
Third, mutation of this Gly to Arg or Asp in hCG-
results in
undetectable levels of heterodimeric hCG being secreted from
Xenopus laevis oocytes (32).
In this report, we investigated the role of the invariant cystine knot
Gly residue in the folding, secretion, and assembly of GPH- and
hCG-
, two prototypes of the growth factor cystine knot superfamily
(1). Even the most conservative substitution possible, Gly
Ala,
resulted in nearly 100% of hCG-
being misfolded and degraded
intracellularly. The misfolding was evident from the non-native S-S
bonds that had formed (Fig. 5), as well as the failure to efficiently
convert to the p
2 folding intermediate (Fig. 4B). The
resulting non-native S-S bond formation and misfolding provides an
explanation for why the central Gly residue is essential for hCG function.
Mutation of the equivalent Gly in GPH- (
-G30A) gave similar
results, although, the deleterious effects were less pronounced; 40%
of
-G30A was secreted, 30% of the which assembled with WT hCG-
.
This result is consistent with a study that detected immunoreactive hCG
when
-G30A and WT hCG-
were coexpressed in X. laevis
oocytes (31). However, the mutant hCG heterodimer displayed no
bioactivity in a murine testosterone production-based Leydig cells
bioassay, suggesting that native hCG conformation was not attained
(31).
A notable effect of the G30A mutation on GPH- folding was that it
slowed folding significantly. Following a 30-min chase, less than half
of the
-G30A synthesized converted to the most folded form that
contained five S-S bonds. This compares with a t1/2
of about 90 s for WT GPH-
folding. Previously, we reported that
disruption of the GPH-
cystine knot S-S bonds results in inefficient
folding and secretion (6). The observation that
-G30A was
inefficiently folded and secreted implies that the
-G30A mutation
also interfered with formation of the GPH-
cystine knot.
There are several possible explanations for why mutation of the
invariant Gly was more detrimental to hCG- folding than that of
GPH-
. First, the inherent flexibility of the GPH-
loop 2 (residues 33-58) (33) may allow for greater perturbation of the
GPH-
cystine knot and permit closure of the cystine knot ring in a
portion of the molecules by adopting the positive
torsion angle
needed at residue 30. Second, WT hCG-
folds at a much slower rate
compared with GPH-
(t1/2 = 5 min
versus t1/2 = 90 s, respectively) and, therefore, when the rate of hCG-
folding is slowed even further
(as noted above for
-G30A),
-G36A may be more readily degraded
before the subunit has time to fold. The latter possibility is based
upon a recently proposed model (34, 35) that suggests that
glycoproteins only have a limited amount of time to fold and exit the
endoplasmic reticulum before being degraded.
The other three noncysteine residues of the hCG cystine knots are
X1, X2, and
X3 of the
C-X1-G-X2-C and
C-X3-C sequences. To understand the role(s) of
these residues, we constructed chimeras in which the X
residues of GPH- and hCG-
were swapped individually or in
combination, while leaving the central Gly unchanged. Folding and
subunit secretion was not significantly affected in either the single
or the triple mutants (
knot and
knot) (Fig. 2). However, assembly of
knot and
knot with their WT partners was decreased by about 90%
(Fig. 3). The decrease in assembly was not due to any one particular
substitution at the X1,
X2, or X3 positions but,
rather, was due to the combination of all three substitutions,
suggesting that the set of all three X residues act in a
subunit-specific manner.
The observation that knot and
knot are efficiently secreted but do not
assemble adds to a growing body of evidence that suggests that
determinants necessary for assembly and secretion of hCG subunits are
different. Recently, we reported two examples of modified GPH-
subunits that are efficiently secreted but do not assemble with hCG-
(6); one modification changed residues in loop 2 necessary for
combination while the other modification simultaneously removed both
7-31 and 59-87 S-S bonds. In addition, hCG-
mutants lacking the
93-100 or 26-110 S-S bonds are also efficiently secreted but do not
assemble with GPH-
(5, 9). Thus, structural determinants needed for
hCG assembly are not necessarily required for subunit secretion.
Our data suggest that the noncysteine residues within the hCG- and
GPH-
cystine knots are critical for intersubunit interactions that
are necessary to form a stable dimer. This finding is further supported
by an important feature of the hCG structure (17, 18). At the core of
the dimer interface is a series of interchain
-sheets. Intimately
involved in this
-sheet are the regions encompassing the
C-X1-G-X2-C sequences of
both subunits (residues 25-39 of GPH-
and 27-40 of hCG-
), which
form a significant number of intersubunit hydrogen bonds. Thus, the
simultaneous alteration of multiple X residues, as was done
in
knot and
knot, could interfere with formation of this intersubunit
-sheet, whereas single changes might be less disruptive because most
other intersubunit interactions remain intact.
The set of all three X residues within both hCG cystine
knots are required for biological activity since they are necessary for
dimer formation (Fig. 3) and only the hCG heterodimer is functional (12). Whether or not this region is important for receptor binding and
signal transduction is unknown. A single-chain model that tethers
assembly-incompetent subunits to their WT partner has been used
successfully to address similar questions. GPH- subunits containing
cysteine mutations that disrupt the cystine knot, such that the free
subunits alone cannot assemble with hCG-
, maintain in
vitro biological activity when tethered to hCG-
(36, 37). These
data suggest that the cystine knot region is necessary for heterodimer
formation, but not for receptor binding and signal transduction. Thus,
it seems likely that the intervening X residues may also not
be directly involved in receptor binding and signal transduction.
A complete understanding of the common cystine knot motif must take
into account all residues of the knot. Previous studies have focused
primarily on the S-S bonds and some aspects of the central Gly residue
(6, 8, 9, 28, 30-32). The data presented in this report provides
evidence that the intervening X residues located between the
cystine knot also make important contributions to hCG biosynthesis.
Specifically, these residues appear to play a critical role in dimer
formation rather than directly influencing individual subunit folding
or secretion. Thus, most noncysteine residues within cystine knots may
not be interchangeable because they appear to have subunit-specific
functions. Future studies aimed at elucidating the role of analogous
residues in other cystine knot proteins may determine how universal a
role these residues play in the function of other members of the
cystine knot superfamily.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant CA32949 (to E. B.), NCI, National Institutes of Health Cancer Center Support Grant P 30 CA36727 to the Eppley Institute, a National Science Foundation Graduate Fellowship (to R. J. D.), and an Emley Fellowship (to A. K. M.-L.).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.
Current address: Transgenomic Inc., 12325 Emmet St., Omaha, NE 68164.
** Current address: LI-COR Inc., 4308 Progressive Avenue, Lincoln, NE 68504.
Current address: Corporate Office of Science and Technology,
Johnson & Johnson, 410 George St., New Brunswick, NJ 08901.
Current address: Eli Lilly and Co., Lilly Corporate
Center, Indianapolis, IN 46295.
¶¶ To whom correspondence should be addressed: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Tel.: 402-559-6074; Fax: 402-559-4651; E-mail: ebedows@unmc.edu.
Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M010168200
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ABBREVIATIONS |
---|
The abbreviations used are:
S-S, disulfide;
hCG, human chorionic gonadotropin;
GPH-, glycoprotein hormone
-subunit;
TSH, thyroid-stimulating hormone;
DTT, dithiothreitol;
NEM, N-ethylmaleimide;
PAGE, polyacrylamide gel
electrophoresis;
WT, wild type;
HPLC, high performance liquid
chromatography.
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