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(Received for publication, February 21, 1996; and in revised form, March 11, 1996) From the
The gonadotropin/thyrotropin hormone family is characterized by
a heterodimeric structure composed of a common One of the hallmarks of the gonadotropin and thyrotropin hormone
family is their heterodimeric structure, consisting of a common Although
the gonadotropin dimers have similar structural features, they are
nevertheless unique. The Tethering a variety of multisubunit complexes into single chains has
been performed by several laboratories to increase protein stability or
activity(11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) .
In such studies, a linker sequence was designed to give optimal
alignment of determinants. In the case of the glycoprotein hormones, we
presumed that a linker would be required for successful expression of
the corresponding single chains because the region of cysteine residue
110 which, in all of the In the construction of CG single
chain, the carboxyl terminus of CG
Figure 1:
Construction of the FSH and CG tethers
(see text). Single-chain FSH constructs were made in the absence and
presence of the 28 carboxy amino acids (CTP) of the CG
We engineered three forms of FSH without
and with intervening linkers comprised of the carboxyl-terminal amino
acids of the CG
A fragment containing the The primer
1/3 and 2/5 fragments were used as overlapping templates for an
additional PCR step with primers 1 and 5. The resulting product
contained the complete FSH-CTP sequence with 12 bp of the The vector pM
The CG
Figure 2:
Expression of single-chain FSH from
transfected CHO cells. Cells expressing FSH variants constructed in the
presence (FC
It was
demonstrated previously that the secretion kinetics of the CG
Figure 3:
Synthesis of single-chain hCG variants.
The entire CG
Figure 4:
In vitro biological activity of
FSH single chains. A, receptor binding. Binding activity was
determined using the 293 cell line stably transformed with the FSH
receptor and in the presence of conditioned media from CHO cells
expressing either heterodimeric FSH (
Taken together, these data show that both the FSH
and the hCG single chains exhibit a similar signal transducing response
to the corresponding heterodimer and in contrast to that observed for
secretion, the presence of spacers are not a prerequisite for the
biological activity in vitro. Converting multisubunit complexes into single chains was
previously attempted to increase stability/activity of the parent
compounds or to fuse complementary functional domains into a single
molecule. In those experiments, a linker sequence was often
used(18, 36) . When an artificial linker was used, it
was designed to give flexibility, hydrophilicity, and resistance to
proteases and, thus, stability to the single chain. (Detailed
thermodynamic analysis of artificial linkers is discussed in (36) .) The CTP motif used here contains several proline and
serine residues and thus lacks significant secondary structure. This
presumably permits flexibility between the two subunits and thus is an
appropriate candidate as a linker. Based on our previous studies of
the CG tether, we could not expect a priori that converting
FSH into a functional single chain would be successful. The
conformation of the heterodimers and the intracellular behavior of the
subunits differ substantially. For example, although the noncombined
CG One key question in constructing the FSH tether was whether the
linker was essential for secretion and biologic activity. When the CTP
sequence was inserted between the The availability of an FSH
single chain permits one to expand the repertory of gonadotropin
analogs for structure-function analysis. As one might predict, such
studies of the glycoprotein hormone family are often hampered by
mutagenesis-induced defects in subunit combination and secretion, e.g. hormone analogs lacking carbohydrates or deleted
disulfide bonds(22, 38) . Because of the presence of
the CTP sequence and the absence of dissociation, the single-chain
forms may represent excellent templates for generating long-acting
agonists and antagonists. Moreover, the single-chain approach offers
the prospect of introducing different functional domains within the
molecule, e.g. dual-acting gonadotropin analogs.
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10445-10448
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit and
the Follicle-stimulating Hormone 
Subunit
ROLE OF A LINKER SEQUENCE (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit
noncovalently linked to a hormone-specific 
subunit. The
conformation of the heterodimer is essential for controlling secretion,
hormone-specific post-translational modifications, and signal
transduction. Structure-function studies of follicle-stimulating
hormone (FSH) and the other glycoprotein hormones are often hampered by
mutagenesis-induced defects in subunit combination. Thus, the ability
to overcome the limitation of subunit assembly would expand the range
of structure-activity relationships that can be performed on these
hormones. Here we converted the FSH heterodimer to a single chain by
genetically fusing the carboxyl end of the FSH subunit to the
amino end of the subunit in the presence or absence of a linker
sequence. In the absence of the CTP linker, the secretion rate was
decreased over 3-fold. Unexpectedly, however, receptor binding/signal
transduction was unaffected by the absence of the linker. These data
show that the single-chain FSH was secreted efficiently and is
biologically active and that the conformation determinants required for
secretion and biologic activity are not the same.
subunit and a unique subunit(1) . Subunit assembly is
vital to the function of these hormones: (i) only the dimers are
bioactive, (ii) maturation of the hormone-specific oligosaccharides is
dependent on the formation of the heterodimer complex, and (iii) the
secretion efficiency of the dimer is determined by the subunit.
Previously, we constructed a chimera composed of the human chorionic
gonadotropin (hCG) (
) subunit genetically fused to the
subunit, and the resulting single polypeptide chain was
efficiently secreted and was biologically active(2) . Because
subunit dissociation would lead to inactivation of the heterodimer, a
single-chain form could have higher biological activity. subunit in the dimers has a different
conformation which is manifested by distinct immunological and spectral
characteristics(3, 4, 5, 6) . In
addition, the carbohydrates on the subunits are not the same in
all the gonadotropin dimers (7, 8, 9) and the
receptor contact sites on the subunit differ among the hormones (10, 39, 40) . Thus, a priori one
cannot predict based on the CG model that other members of the
glycoprotein hormone family can be converted to single chains. subunits, bridges a loop (cysteine
26-110 in LH/CG and 20-104 in FSH) that is
important for stability of the
heterodimer(1, 23, 24, 25, 26) .
This is especially critical for the FSH subunit because it
terminates at residue 111, which contrasts with the CG subunit
whose carboxyl terminus is residue 145. Moreover, the carboxyl-terminal
region of the FSH contains determinants associated with
secretion/assembly (27) . was fused directly to the amino
terminus of the subunit, without an added spacer
sequence(2) . The CG subunit, length of 145 amino acids,
is distinguished among the other subunits by the presence of a
carboxyl-terminal extension with four serine-linked oligosaccharides
(CTP). We considered this sequence as a natural linker, since it is
serine/proline-rich and thus lacks significant secondary structure and
would provide sufficient distance between Cys-110 and the first
disulfide bond in the linked subunit. Moreover, deleting this
sequence to residue 114 in the CG subunit did not significantly
affect secretion of the heterodimer(28) . Because the FSH
subunit contains a shorter carboxyl-terminal region, there could be
more interference between the cysteine 20-104 loop and the
adjacent subunit sequences in the single-chain molecule. Here we
construct single FSH chains by fusing the carboxyl end of the FSH
subunit to the amino end of the subunit in the absence or
presence of the CTP sequence(2, 27) . We show that the
single-chain variant of FSH is secreted efficiently and is biologically
active. However, the presence of the spacer sequence is important for
the maximal expression of a functional single chain but not for
receptor binding/signal transduction. Thus, the conformation around
this region is more critical for secretion than for the biologic
activity.
Hormones
Tethered FSH in serum-free conditioned
medium was obtained from CHO cells stably transfected with the
expression vector pM
HA containing the tethered FSH coding
gene. Recombinant human FSH and FSH-CTP were obtained from CHO cells
transfected with expression vectors as described
previously(2, 27) . hCG (CR127; 14,900 IU/mg) and
purified pituitary hFSH (FSH 1-3; 6,890 IU/mg) were obtained from
the National Hormone and Pituitary Distribution Program, NIDDK, NIH.Construction of Tethered Gonadotropins
The
tethered forms were constructed with the carboxyl end of the
subunit fused to the amino end of the subunit as described
previously (Fig. 1; (2) ). The exon II sequence in the
subunit encoding the signal peptide was deleted(2) . In
the case of FSH tethers, the final PCR products were digested by BamHI/SalI and ligated into pMHA. For
CG
T, (CG tether minus the 31 carboxyl-terminal
amino acids of the CG subunit), the final PCR product was digested
by SalI and ligated into pMHA containing CG
exons I and II as described previously(2) . All final
constructs described were sequenced to verify that no misincorporation
occurred during the PCR.
subunit. In
the case of CG, a tether was created where this CTP sequence was
detected.
subunit (CTP). The following primers were used (5`
3`): 1, CCC AAC GGA TCC ACA AGG TGT TAG TTG; 2, TGC TAC ACC AGG
GAT CTG GTG TAT AAG GAC; 3, ATA CAC CAG ATC CCT GGT GTA GCA GTA GCC; 4,
ATC CTC CCA CAA GCT CCT GAT GTG CAG; 5, CAC ATC AGG AGC TTG TGG GAG GAT
CGG; 6, TGA GTC GAC ATG ATA ATT CAG TGA TTG AAT; 7, GAA ATG AAA GAA GCT
CCT GAT GTG CAG GAT; 8, CAC ATC AGG AGC TTC TTT CAT TTC ACC AAA; 9, CTG
CAC ATC AGG AGC TGG ACT TGG AAG; 10, AGC CTT CCA AGT CCA GCT CCT GAT
GTG CAG.FC
The vector
pM (FSH-CTP-)FSH-CTP (27) was used as a template DNA for
the PCR. Primers 1 and 3 were used to generate a fragment which
contains exon I, intron I, exon II, and the 5` end of exon III of the
FSH subunit (Fig. 1). In a second reaction, primers 2 and 5
were used to synthesize a product containing the 3` end exon II and the
entire exon III of the FSH subunit, 84 bp (Ser-118
Gln-145) of
the CG CTP sequence, and 12 nucleotides of the 5` end of exon II
in the subunit. subunit
sequence was constructed with primers 4 and 6 and the pM -minigene(29) . This intermediate was also
engineered to contain a portion of the 3` end of the CTP. subunit
exon 2 to overlap with the sequence in the a minigene-CTP chimera.
These fragments were annealed and subjected to a final PCR with primers
1 and 6.HAFC was used as a template
DNA for the construction of F1/2C (FSH-1/2 CTP-). A
fragment containing the FSH subunit and the first 42 bp of the CTP
(Ser-118Pro-131) was generated using primers 1 and 9. Primers 6
and 10 were used to synthesize a fragment containing the subunit
sequence. Those two products were used as overlapping templates for an
additional PCR step with primers 1 and 6. Following BamHI/SalI digestion, the later fragment was ligated
into pMHA for transfection into CHO
cells(9, 29) .F
The construction of the single FSH chain
without the CTP sequence was similar to FC except that the vector
pMFSH WT (27) was the template. Primers 1 and
8 were used to synthesize the entire FSH coding sequence and a
part of the exon II sequence (Fig. 1). The
pM minigene (29) was a template for primers 7
and 6 to generate a product containing the 3` end of the FSH exon
III and the -minigene. These fragments were incubated with primers
1 and 6 to synthesize the F construct containing an intron between
FSH exons II and III.CG
To construct the CGT
tether lacking the carboxyl-terminal 31 amino acids of the CG
subunit, the following additional primers were used (Fig. 1) (5`
3`): 11, CAC ATC AGG AGC GCG GGG GTC ATC ACA GGT; 12, GAT GAC
CCC CGC GCT CCT GAT GTG CAG GAT; 13, GGA GGA AGG GTG GTC GAC CTC TCT
GGT.WT exon III was subcloned into M13mp19 at the SalI site. The replicative form was a template for primers 11
and 13 to synthesize a fragment containing 12 bp of exon II and
CG exon III without CTP. Primers 12 and 6 were used with the
pM minigene to synthesize a fragment containing the 3`
end of CG exon III without CTP and the gene sequence. The
products generated with primers 13/11 and 12/6 were used as overlapping
templates for primers 13 and 6 to form the CG exon 3/
chimera.Bioassay
For the in vitro bioassays,
conditioned media were concentrated using a Centricon concentrator
(Amicon). The FSH variants in conditioned media were quantitated by
using a dimer-specific RIA (Diagnostic Products, Los Angeles) or by
enzyme-linked immunosorbent assay (Organon, Oss, The Netherlands). Both
assays gave comparable data. The enzyme-linked immunosorbent sandwich
assay employs a monoclonal antibody to each subunit. Purified
recombinant FSH was used as a reference but calibrated against LER-907,
and the immunological activity was expressed as international units.
Binding was quantitated using a human fetal kidney cell line 293
expressing recombinant human LH receptors (30) or FSH
receptors(31) . The specific activity and maximum binding of 
I-labeled hCG (CR127: 14,900 IU/mg), as determined by
radioligand receptor assay (32) , were 53,000 cpm/ng and 40%,
respectively. For I-FSH (FSH I-3), the specific activity
and maximal binding were 100,000 cpm/ng and 7%, respectively.
Nonspecific binding was 10-12% of total I-labeled
hCG or FSH bound as determined by the addition of a 1,000-fold excess
of pregnyl (LH receptor assay) or pergonal (FSH receptor assay). The
production of cAMP was also assayed in 293 cells expressing human LH
receptors or FSH by RIA(33) .
Secretion of the Single Chain
When the
hCG single chain was constructed, no exogenous linker was
inserted between the subunits (2) . We reasoned that the
several proline and serine residues clustered in the last 28 amino
acids of the CG subunit (CTP) would provide the flexibility to
permit the appropriate interactions between the and subunit
domains. However, for the FSH subunit, a linker sequence could be
critical for the correct orientation of the / determinants.
Previous studies showed that when the CTP is placed on either the
carboxyl-terminal end of FSH subunit or in the amino-terminal
region of the subunit, assembly, secretion, and biological
activity of the component dimers were comparable to the wild type
hormones(27, 34) . To assess if the spacer between the
subunits affected the rate and/or extent of secretion of the
single-chain molecule, FSH tethers were constructed in which either the
carboxyl terminus (residue 111) of the subunit was fused directly
to the amino terminus of the subunit or connected through the CTP
sequence. The secretion kinetics of the tethered FSH forms were
determined by pulse-chase analysis (Fig. 2). Cells were labeled
with 
S-Pro-mix for 20 min, chased for up to 24 h, and the
lysate and media samples were immunoprecipitated. It is clear that both
single chains are released quantitatively, and the secretion kinetics
of the single chain bearing the linker is comparable to that for
heterodimeric FSH synthesized in transfected CHO
cells(27, 35) . However, the secretion rate of single
chain devoid of CTP is prolonged by 3-4-fold compared to the
tether molecule containing the CTP (t
=
360 min versus 100 min); its delayed secretion is reflected by
increases in the intracellular content. Although it appears that a
linker is required for efficient secretion, the entire CTP sequence is
not essential for this effect since an FSH tether containing only
residues 118-131 of the CTP was secreted at a rate (t = 95 min) similar to the FSH
heterodimer and the single-chain FSH (data not shown).
; A) or absence (F; B) of the
CTP linker were pulse-labeled with 100 µCi/ml S-Pro-mix for 20 min and chased for the indicated times.
Lysate (L) and medium (M) samples were
immunoprecipitated with antiserum against the subunit and
subjected to SDS-polyacrylamide gel
electrophoresis.
single chain was comparable to that of the CG heterodimer. We assumed
that the presence of the CTP in the native sequence was important for
its efficient secretion. The CTP sequence from the CG single chain was
eliminated to assess if secretion would be altered, analogous to that
seen in the FSH case. Similar to the FSH results, deleting this
sequence reduced the secretion rate 3-fold compared to intact
CG (Fig. 3). These data show that efficient secretion
of the FSH single chain requires a linker between the carboxyl terminus
of the FSH subunit and the amino terminus of the subunit.
sequence was fused directly to the without an
exogenously inserted linker sequence (CG; A). A
construct bearing the CG subunit truncated at amino acid 114 was
also fused to the subunit (CGT; B). The
transfected cells were subjected to a pulse-chase protocol as described
in Fig. 2, and the labeled products were precipitated with
antiserum against the subunit.
Biological Activity of Single Chains
Human kidney
cells expressing the human FSH receptor were incubated with conditioned
medium from CHO cells expressing the FSH heterodimer or single chain
containing the CTP linker. Both the binding affinity and the potency of
adenylate cyclase activation for single-chain FSH are similar to that
seen with recombinant FSH heterodimer (Fig. 4). We examined
whether or not the linker is required for biological activity.
Unexpectedly, we observed that the FSH variant lacking the CTP sequence
exhibited similar receptor binding and signal transduction activity
when compared to the linker-containing FSH single chain (Fig. 4). Consistent with this observation, the receptor binding
and signal transduction dose-response curves for the CG tether devoid
of the CTP sequence were comparable to those seen for wild-type CG
(data not shown).
-) or the
FSH single chains with CTP linker (
-), or no
linker (
-). The FSH activity was quantitated
as described under ``Materials and Methods.'' I-Labeled FSH was incubated in the absence or presence of
different concentrations of unlabeled wild-type FSH or single-chain
variants. B, signal transduction. Adenylate cyclase activation
by the FSH analogs was also determined with the 293 cell line. Assays
were conducted overnight at 37 °C, and total cAMP production was
measured by RIA. The standard derivation is less than 5% of the
indicated values.
subunit is secreted quantitatively, less than 10% of the
uncombined pituitary subunits are
secreted(9, 37) . Thus, it was not clear that the FSH
single chain would readily fold appropriately into a functional form.
Here we show that FSH was converted from its native heterodimeric form
to a single chain without significant changes in the biologic activity. and subunits, the single
chain was secreted four times faster than the single chain lacking the
linker. Consistent with this observation, secretion rate of the
tethered form of CG was reduced when the CTP segment was deleted. The
complete CG and the FSH single chains were secreted at rates comparable
to the heterodimers, and, thus, maximal secretion of tethered forms
requires a spacer. The entire CTP segment was not necessary for this
effect since secretion of the FSH tether containing only 14 amino acids
of the CTP domain was comparable to the intact FSH tether. Although it
is not clear why deleting the entire spacer sequence slows secretion,
we demonstrated previously that the loop bridged by 26-110
disulfide bond in the CG subunit was critical for
secretion(23) . Absence of the linker sequence in the single
chain may lead to perturbation of the conserved 20-104 disulfide
loop in the FSH subunit by the adjacent subunit. This
conclusion is also supported by the CG crystallography data and by
models showing that the linkage between the carboxyl terminus
CG114, the CG subunit devoid of the CTP sequence, to the
subunit would not support an alignment of the - domains
as seen in the heterodimer (25) . ()This also
implies that a tight interaction between subunits is not a prerequisite
for biologic activity but rather the essential feature for receptor
recognition is the presence of the / domains in the same
complex. Thus, it is apparent that the tertiary structure created by
this loop is more important for the intracellular behavior than for the in vitro biological activity.
))
We thank Dr. P. D. J. Grootenhuis (N.V. Organon) for
his advice on the modeling of the single chain. We are also grateful
for the technical assistance of Ricia Hyde and to Susan Carnes for the
preparation of this manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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