(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 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.
One of the hallmarks of the gonadotropin and thyrotropin hormone
family is their heterodimeric structure, consisting of a common
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.
Although
the gonadotropin dimers have similar structural features, they are
nevertheless unique. The 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.
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 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) .
In the construction of CG single
chain, the carboxyl terminus of CG 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.
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 subunit. In
the case of CG, a tether was created where this CTP sequence was
detected.
We engineered three forms of FSH without
and with intervening linkers comprised of the carboxyl-terminal amino
acids of the CG 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.
A fragment containing the 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.
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 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.
The vector pMHAFC
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-118
Pro-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 pM
HA for transfection into CHO
cells(9, 29) .
The CGWT 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.
Figure 2:
Expression of single-chain FSH from
transfected CHO cells. Cells expressing FSH variants constructed in the
presence (FC; 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.
It was
demonstrated previously that the secretion kinetics of the CG
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.
Figure 3:
Synthesis of single-chain hCG variants.
The entire CG 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 (CG
T
; 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.
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 (-
) 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.
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 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.
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 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
CG
114, 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.
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.