Endocrinology Vol. 141, No. 12 4751-4756
Copyright © 2000 by The Endocrine Society
Disulfide Bond Mutations in Follicle-Stimulating Hormone Result in Uncoupling of Biological Activity from Intracellular Behavior1
Takashi Hiro’oka,
Danyal Maassen,
Peter Berger2 and
Irving Boime
Department of Molecular Biology and Pharmacology (T.H., D.M.,
I.B.), Washington University School of Medicine, St. Louis, Missouri
63110; Institute for Biomedical Aging Research (P.B.), Austrian Academy
of Sciences, A-6020, Innsbruck, Austria
Address all correspondence and requests for reprints to: Irving Boime, Ph.D., Washington University Medical School, Department of Pharmacology, 660 South Euclid, St. Louis, Missouri 63110-1010. E-mail:
iboime{at}pcg.wustl.edu
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Abstract
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The crystal structure of human CG reveals that each subunit is a member
of the superfamily of cystine-knot growth factors. Although the
distribution of the cysteine residues in all the ß-subunits is
conserved, the conformation of the human FSH dimer differs from that of
the CG/LH dimers. This suggests that the function of the cystine bonded
loops in the human FSHß-subunit may differ from that in the
CGß-subunit. To address this issue, we deleted two disulfide bonds in
the FSHß domain: cys 20–104 and cys 28–82, which correspond to the
disulfide bonds 26–110 and 34–88, respectively, in the CGß-subunit.
The cys 26–110 bond is associated with the "seat-belt" region and
cys 34–88 is a bond in the cystine knot. Coexpression of the wild-type
-subunit with the FSHß cysteine mutants in CHO cells revealed no
detectable heterodimer. The FSHß mutants were then incorporated into
a single chain where the ß-subunit is genetically fused to the
-subunit. In such a model, the rate-limiting subunit assembly step
is by-passed and mutations that otherwise block heterodimer formation
can be evaluated in terms of biological activity. Compared with the
nonmutated single chain, the single-chain 28–82 mutant is secreted
more slowly and its recovery is substantially reduced, whereas
secretion and recovery of the 20–104 mutant was not significantly
affected. The receptor binding affinity of the cys 28–82 mutant did
not differ from wild-type and binding of the cys 20–104 mutant was
decreased only 2-fold. The signal transduction data parallel the
binding affinities, although the maximal accumulation of cAMP is less
for the cys 20–104 mutant than that seen for cys 28–82 and nonmutated
single-chains variants. These data support the hypothesis that the
determinants for intracellular behavior and bioactivity of the
gonadotropins are not the same, and that the cystine knot is a critical
determinant for the formation of a stable, assembly-competent subunit.
In addition, the data imply that the "seat-belt" conformation does
not play a prominent role in the bioactivity of FSH.
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Introduction
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THE HUMAN GLYCOPROTEIN hormones,
FSH, LH, TSH, and CG, consist of two subunits, and
ß. The amino acid sequence of the -subunit is identical for all
hormones, and it is also highly conserved among the species
(1). The sequence of the ß-subunit is hormone specific,
and the biological activity of each hormone requires both subunits. The
crystal structure of CG shows that it is a member of the superfamily of
cystine-knot growth factors, which include transforming growth factor
(TGF)-ß, activins, nerve-and platelet-derived growth factors
(2, 3). The cystine-knot is a ring structure comprised of
two disulfide bonds through which the third disulfide bond penetrates
(Fig. 1
; Ref. 4), and
disruption of either of these three bonds in the CGß-subunit
dramatically reduces assembly and recovery of the dimer (5, 6). Based on these data, it was proposed that, at least for the
glycoprotein hormones, the cystine knot is the basic scaffold for the
subunits and represents a critical determinant for assembly and
secretion of a functional heterodimer (5, 6, 7, 8).
Although the distribution of the cysteine residues in all the
ß-subunits is conserved, the conformation of the FSH dimer differs
from that of CG/LH (9, 10, 11), and some of the sequences in
the -subunit, which form contact sites with the ß-subunits are not
identical (12, 13, 14). Thus, the function of the cystine knot
in the FSHß-subunit may differ from that in the CGß-subunit. To
address this issue, we engineered a single-chain gonadotropin where the
FSHß-subunit is genetically fused to the -subunit
(15). In such a model, the rate-limiting assembly step is
by-passed and mutations that otherwise block heterodimer formation can
be evaluated. Here, we delete two disulfide bonds in the FSHß domain:
Cys 20–104 and cys 28–82, which correspond to bonds cys 26–110 and
cys 34–88, respectively, in the CGß-subunit; the 28–82 bond is a
component of the cystine knot. We evaluated the efficiency of
heterodimer formation by the mutated subunit and the behavior of single
chains with the same mutations. Although disrupting either bond
prevents heterodimer formation, the FSH single chain bearing the
mutations are secreted at different rates compared with the nonmutated
single chain. We next evaluated the bioactivity of these mutants. The
receptor binding affinity of the cys 28–82 single chain was comparable
and the cys 20–104 single chain was 2-fold less than the wild-type.
The data support the hypothesis that the determinants for the
intracellular behavior of the gonadotropins are uncoupled from those
required for biological activity, and the cystine knot is a critical
determinant for the formation of a stable assembly-competent
FSHß-subunit.
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Materials and Methods
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The DNA vector pBluescript II KS(+) was purchased from
Strategene (La Jolla, CA). Oligonucleotides were prepared by the
Washington University Nucleic Acid Chemistry Laboratory (St. Louis,
MO). Cell culture media and reagents were prepared by the Washington
University Tissue Culture Support Center (St. Louis, MO) and were
obtained from Sigma (St. Louis, MO). FBS, dialyzed FBS,
the neomycin analog, G418 and immunoprecipitin were purchased from
Life Technologies, Inc. (Gaithersburg, MD).
[35S] Cysteine-methionine (Promix) (>1000
Ci/mmol) was purchased from Amersham Pharmacia Biotech
(Arlington Heights, IL). CentriPrep concentrators were purchased from
Amicon, Inc. (Beverly, MA).
Mutagenesis and vector construction
The construction of the FSHß- single chain with linker and
PCR mutagenesis was described previously (15). The linker
is comprised of the last 28 amino acids of the CGß-subunit,
designated carboxyl-terminal peptide or CTP (15). The
cysteine residues at positions 28 and 82, 20 and 104 were mutated to
alanine. All final constructs described were sequenced to verify that
no misincorporations occurred during the PCR.
DNA transfection and cell culture
All variants were inserted into the mammalian expression vector
pM2HA (16, 17), transfected into CHO cells,
and stable clones were selected approximately 11 days after
transfection with G418 (250 µg/ml) (16, 17). The clones
were maintained in Ham’s F-12 medium [supplemented with penicillin
(100 U/ml), streptomycin (100 µg/ml), and 2 mM
glutamine] containing 5% FBS and G418 (125 µg/ml) at 37 C in a
humidified atmosphere of 5% CO2/95% air.
Metabolic labeling
Cells were labeled overnight in F-12 based medium, containing
dialyzed calf serum contianing 25 µCi/ml
[35S] cysteine or mixture of
[35S] cysteine and methionine (Promix) as
described (8, 16). Aliquotes of cell lysate and medium
were immunoprecipitated with polyclonal antiserum directed against the
common -subunit. The reduced proteins were resolved on 15%
SDS-polyacrylamide gels.
Western blot analysis
Media samples were resolved on 12.5 or 15% SDS-polyacrylamide
gels under nonreduced conditions and blotted onto nitrocellulose. The
blots were probed as described in the figure legends. Recombinant (r)
human FSH was obtained from Organon (Oss, The
Netherlands). The antiserum to the -subunit was raised in the lab.
The mAb 4B which recognizes all forms of FSHß, was obtained form
Organon, and mAb INN-hFSH-117 (designated 117), which has
a 100-fold greater affinity for dimeric FSH than to the monomeric
FSHß-subunit (18), and the free -subunit-specific mAb
(Inn-hCG-72) (designated 72, Ref. 19) were also used. The
blots were visualized with the Western Light detection system (Tropix
Inc., Bedford, MA) following the manufacturer’s protocol.
RRA
Conditioned media were concentrated using either a Centricon
concentrator (Amicon) or an ultra-free concentrator (Millipore Corp.). Subsequently, the samples were washed in PBS and
quantitated using double polyclonal-based RIA (Diagnostic Products Inc., Los Angeles, CA), which includes antiserum which
was raised against the FSHß-subunit.
Receptor binding and cAMP production were determined using a stably
transfected CHO cell line expressing the human FSH receptor
(20). The accumulation of cAMP was determined using the
NEN Life Science Products Flashplate (Boston, MA) assay as
per manufacturer’s instructions. Briefly, 5 x
104 CHO cells were incubated for 2 h at room
temperature with ligands followed by the addition of
125I cAMP and then incubated for 17 h at
room temperature. The Flashplate was then read in Packard top counter.
Total binding was 15% and nonspecific binding–in the presence of 5 IU
of recombinant human rFSH–was 1.5% of total counts (
100,000
cpm).
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Results
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Assembly of FSHß mutants with -subunit
We initiated our studies by examining the effects of deleting
either the cys 20–104 or 28–82 FSHß disulfide bonds on assembly
with the -subunit. The wild-type and mutant FSHß-subunits were
cotransfected with -subunit and clones were isolated synthesizing
the excess -subunit (Fig. 2). The
media were Western blotted under nonreduced conditions and heterodimer
formation was assessed with either polyclonal antiserum (panel A)
or a monoclonal FSHß antibody (panel B), which recognizes FSH dimer
and uncombined FSHß-subunit. As expected wild-type FSH was detected
with both probes (A, B, lane 1), and uncombined -subunit was
detected by antiserum (A, lower arrow). In the case of
the cys 20–104 mutant, no heterodimer was seen (A, B, lane 2) despite
the presence of both the - (panel A) and FSHß- (panel B) subunits.
Only a trace of heterodimer bearing the cys 28–82 mutant was observed
(panel B, lane 3). However, although appearance of the -subunit was
evident (A, lane 3), the cys 28–82 ß-subunit was not observed in the
medium (B, lane 2) or in the corresponding lysates (data not shown).
This result was seen in several different transfections. These data
suggest that the FSHß-subunit containing the 28–82 mutation is
unstable and degraded rapidly. By contrast the cys 20–104 mutated
subunit accumulates and is secreted, but it nevertheless cannot
assemble with the -subunit.
Secretion kinetics of the FSH single-chain mutants
As shown above, absence of FSH dimer formation precluded studies
on the role of the above disulfide bonds on receptor binding/signal
transduction. However, if the mutated FSHß-subunit is covalently
linked to the -subunit, the rate-limiting assembly step is
by-passed, which could lead to accumulation of sufficient protein to
investigate receptor binding/signal transduction. The wild-type, cys
20–104, and cys 28–82 mutated FSHß-subunits, respectively, were
tethered to the wild-type -subunit via a CTP linker (see
Materials and Methods), transfected in CHO cells, and rates
of secretion examined by pulse-chase analyses (Fig. 3 and Table 1). As observed previously
(15), the nonmutated FSH single chain (FßC) is
secreted with a t1/2 of 140 min and 60% of the protein is
recovered from the media (Fig. 3A, Table 1). The secretion rate and
recovery of the single chain bearing the cys 20–104 mutation is
comparable to FßC. By contrast, the variant containing the cys
28–82 mutation is secreted less efficiently (t1/2 = 300 min)
and only 26% is recovered. These results show that disrupting the cys
28–82 bond decreases both secretion and recovery, whereas deleting cys
20–104 did not significantly alter the intracellular kinetics of this
mutein (Fig. 3).
Conformation of single-chain mutants
Because no detectable heterodimer is formed with the mutated
subunits, we suspected that the conformation of the single chains would
be modified. If the conformation is altered by breaking the disulfide
bonds, we should detect differences in the electrophoretic mobility and
immunoreactivity on SDS-gels. To address this issue, conditioned media
were examined by Western blotting under nonreduced conditions using
free -subunit- and FSHß-subunit-specific monoclonal antibodies
(mAb), and polyclonal antiserum (Fig. 4). The unmutated FßC migrates with
an apparent higher molecular weight than recombinant FSH due to the
addition of the linker (A, B; compare lanes 1 and 2). It is also
evident that the migration of both mutants differs from FßC (A, B;
lanes 3 and 4). In addition, molecular heterogeneity is detected for
Fß20–104C with the probes, including discrete higher molecular
weight species. It is likely that these forms are aggregates because
they are not observed when the proteins are resolved under reduced
conditions (data not shown). Their identity are not clear but in
previous studies of tethered mutants there was no correlation between
the proportion of aggregates formed by a particular mutant and the
in vitro biological activity (6).
It is intriguing that the -subunit-specific mAb (#72) reacts with
the Fß20–104C but not with Fß28–82C (C); as expected the
recombinant FSH heterodimer and the nonmutated single chain did not
exhibit significant reactivity with this mAb. This result suggests that
the -ß interaction at this epitope region in Fß20–104C is
disrupted by the mutation leading to a more open configuration of the
two domains.
Biological activity of the FSH single-chain mutants
To study the effect of deleting the disulfide bonds cys 20–104
and 28–82 on the biological activity, conditioned media containing the
nonmutated or cys-mutated FSH single chains were tested using stably
transfected CHO cells expressing the human FSH receptor. As shown
previously, receptor binding affinity of the nonmutated FSH single
chain (Fig. 5A) is comparable to that
observed for the recombinant FSH heterodimer (14). The
binding affinities of the Fß28–82C(IC50 = 877 ±
60) and Fß20–104C(IC50 = 735 ± 47) are within
2- to 3-fold of the nonmutated single chain (IC50 = 365 ±
40) (Fig. 5A). The signal transduction data show that the extent of
cAMP accumulation parallels the binding affinity of the variants (Fig. 5B). These data indicate that although an intact cystine knot in the
FSHß-subunit is required for intracellular stability, and the cys
20–104 bond is important for heterodimer formation, the overall
ability of the mutants to bind and activate the receptor is not
significantly impaired.
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Figure 5. Bioactivity of FSH single-chain mutants. A,
Binding assay: Stably transfected CHO cells expressing the human FSH
receptor were incubated for 16–18 h at room temperature with varying
concentrations of single-chain mutants. Concentrated conditioned media
were quantitated by an FSH RIA, and equal amounts of analog were added.
Data are the mean ± SEM of three experiments. The
IC50 (mIU) are (±SEM): recombinant FSH,
440 ± 28; FßC, 365 ± 40; FßC28–82, 877 ±
62 and for Fß20–104C, 737 ± 47. B, Signal transduction:
Using these CHO cells, the mutants were incubated for 16–18 h at room
temperature, and the total amount of cAMP (extra and intracellular) was
quantitated as described in Materials and Methods. Data
are the mean ± SEM of three experiments. The
EC50 values (mIU) are (±SEM): recombinant FSH,
26 ± 5; FßC, 11 ± 1.5; Fß28–82C, 24 ± 9,
and Fß20–104C, 20 ± 11.
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Discussion
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Crystal structure analyses reveal that CG is a member of the
superfamily of cystine-knot growth factors that include TGF-ß,
activins, nerve-and platelet-derived growth factors (2, 3, 21). It has been suggested that the cystine-knot motif is the
basic scaffold for the glycoprotein hormone subunits and represents a
critical determinant for secretion and assembly of functional ligand
(5, 6, 7, 8). Recent crystallographic analysis of the FSH dimer,
show that the FSHß disulfide bonds are at cysteine at residues 3–51,
17–66, 28–82, 32–84, 87–94, and 20–104. The corresponding
disulfide bonds in the CGß-subunit are at positions 9–57, 34–88,
38–90, 23–72, 93–100, and 26–110.
The monomeric FSHß-subunit containing the cys 20–104 mutation is
secreted but does not combine with the -subunit. The behavior of
this mutant is analogous to that observed for the monomeric
CGß-subunit mutated at cys 26–110 (5). This is
consistent with the hypothesis that the cys 26–110 bond is critical
for stabilizing the heterodimer through a "seat-belt"-like
configuration (See Fig. 1
; Refs. 2, 3) and thus the cys
20–104 bond apparently serves the same function. The secretion of the
CG and FSH single chains bearing the cys 26–110 and 20–104 mutations,
respectively, are comparable to the corresponding nonmutated tethers.
The relatively normal secretory behavior of these mutants is not
surprising because the "seat-belt" motif is required for stability
of the heterodimer (by embracing one of the -subunit loops) and thus
single chain construction by-passes the assembly step. In contrast to
the results seen with Fß20–104C, secretion and recovery of the
single chain containing the cys 28–82 mutation was substantially
reduced. The cys 28–82 bond is a component of the cystine knot. This
suggests that the configuration of the cystine knot in the ß-subunit
(and presumably the -subunit) is critical for mutual recognition and
subsequent interaction of the subunits. The seat-belt determinant is
required after these initial steps. That no uncombined cys 28–82
subunit was detected in the lysate and the recovery of Fß-28–82
C in the medium was decreased also shows that the cystine knot is
critical for maximal stability of the subunit. These results are
consistent with the genetic variants identified in patients bearing
frame-shift mutations resulting in FSHß-subunits truncated at the
carboxyl end of the molecule (23, 24, 25). In one naturally
occurring mutation in the FSHß gene, the cysteine residue at position
51 was mutated to glycine (24). This disrupts a disulfide
bond in the cystine knot, and, in transfection experiments using this
mutated FSHß gene, no FSH dimer was detected. Thus, taken together,
despite the dramatic effects of the 28–82 and 20–104 cysteine
mutations on the secretion and assembly of the monomeric ß-subunit,
the corresponding single-chain variants are still secreted and bind to
the receptor and activate adenylate cyclase.
It is intriguing that the free -subunit-specific mAb was
immunoreactive with Fß20–104C single chain but not
Fß28–82C. It has been proposed that the epitope for this mAb is
associated with the dimer interface sequence (amino acids 33–41) of
the -subunit (18, 26). Thus, this region is apparently
involved in the seat belt contacts with the -subunit, which is
exposed by disrupting the cys 20–104 bond but not when one of the
bonds in the cysteine knot is broken. These data imply that the
heterodimeric-like contacts encompassed by the seat belt differ from
those created by the cystine knot. The results support earlier
suggestions for CG that the native quaternary configuration of the
/ß heterodimer is critical for efficient secretion and
intracellular stability but not bioactivity, and that gonadotropins
with different conformations distinct from the native heterodimer are
bioactive (6, 7, 27).
It is not surprising that the CG and FSH single chains bearing cystine
knot mutations manifest similar intracellular behavior because the
relative positions of the cysteine residues are conserved in all the
ß-subunits (1). We would suggest that the major epitopes
for the bioactivity of the CG and FSH dimers are not encoded in the
tertiary structure of the cysteine knot. Given that the conformation of
the -subunit is not the same in gonadotropin dimers
(9, 10, 11), it is apparent that the structural constraints
generated outside the cystine knot are critical for the biological
specificity of the heterodimer. This does not exclude the participation
of hormone-specific contact sites at the hormone receptor interface
established by small clusters of amino acid residues in the ß-subunit
(12, 13, 28, 29, 30). It has been demonstrated that within the
same region in the ß-subunits, mutating sequences critical for
receptor binding of CG and TSH have less effect on FSH binding
(28, 29).
In summary, the disulfide bonds have different roles in the maturation
of a gonadotropin subunit. The cystine knot is necessary for
intracellular stability of the subunit, whereas at least one bond
outside the knot is required to maintain the integrity of the
heterodimer after the initial /ß contact. Both of the single-chain
mutants exhibited high receptor binding affinity. The data imply a
site-specific function for at least two disulfide bonded loops on the
FSHß-subunit and, in addition, these results support the hypothesis
that the determinants required the intracellular behavior is uncoupled
from those essential for receptor activation.
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Acknowledgments
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The authors are grateful to Dr. Raj Kumar for his comments
regarding the manuscript and to Ms. Mary Wingate for the preparation of
the manuscript.
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Footnotes
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1 This work was supported by a grant from Organon NV. 
2 Supported by a grant from the Austrian Science Funds
(P-13652-GEN).
Received May 5, 2000.
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A. Jablonka-Shariff, T. R. Kumar, J. Eklund, A. Comstock, and I. Boime
Single-Chain, Triple-Domain Gonadotropin Analogs with Disulfide Bond Mutations in the {alpha}-Subunit Elicit Dual Follitropin and Lutropin Activities in Vivo
Mol. Endocrinol.,
June 1, 2006;
20(6):
1437 - 1446.
[Abstract]
[Full Text]
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N. A. Horn, G. B. Hurst, A. Mayasundari, N. A. Whittemore, E. H. Serpersu, and C. B. Peterson
Assignment of the Four Disulfides in the N-terminal Somatomedin B Domain of Native Vitronectin Isolated from Human Plasma
J. Biol. Chem.,
August 20, 2004;
279(34):
35867 - 35878.
[Abstract]
[Full Text]
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G. B. Fralish, P. Narayan, and D. Puett
Consequences of Single-Chain Translation on the Structures of Two Chorionic Gonadotropin Yoked Analogs in {alpha}-{beta} and {beta}-{alpha} Configurations
Mol. Endocrinol.,
April 1, 2003;
17(4):
757 - 767.
[Abstract]
[Full Text]
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M. W. Szkudlinski, V. Fremont, C. Ronin, and B. D. Weintraub
Thyroid-Stimulating Hormone and Thyroid-Stimulating Hormone Receptor Structure-Function Relationships
Physiol Rev,
April 1, 2002;
82(2):
473 - 502.
[Abstract]
[Full Text]
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Top
Abstract
Introduction
