©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Biologically Active Fusion Genes Encoding the Common Subunit and the Follicle-stimulating Hormone Subunit
ROLE OF A LINKER SEQUENCE (*)

(Received for publication, February 21, 1996; and in revised form, March 11, 1996)

Tadashi Sugahara Asomi Sato Masataka Kudo (§) David Ben-Menahem (¶) Mary R. Pixley Aaron J. W. Hsueh (§) Irving Boime (**)

From the Department of Molecular Biology & Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The gonadotropin/thyrotropin hormone family is characterized by a heterodimeric structure composed of a common alpha subunit noncovalently linked to a hormone-specific beta 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 beta subunit to the amino end of the alpha 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.


INTRODUCTION

One of the hallmarks of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common alpha subunit and a unique beta 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 beta subunit. Previously, we constructed a chimera composed of the human chorionic gonadotropin (hCG) (^1)beta subunit genetically fused to the alpha 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 alpha 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 alpha subunits are not the same in all the gonadotropin dimers (7, 8, 9) and the receptor contact sites on the alpha 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 beta subunits, bridges a loop (cysteine 26-110 in LHbeta/CGbeta and 20-104 in FSHbeta) that is important for stability of the heterodimer(1, 23, 24, 25, 26) . This is especially critical for the FSHbeta subunit because it terminates at residue 111, which contrasts with the CGbeta subunit whose carboxyl terminus is residue 145. Moreover, the carboxyl-terminal region of the FSHbeta contains determinants associated with secretion/assembly (27) .

In the construction of CG single chain, the carboxyl terminus of CGbeta was fused directly to the amino terminus of the alpha subunit, without an added spacer sequence(2) . The CGbeta subunit, length of 145 amino acids, is distinguished among the other beta 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 alpha subunit. Moreover, deleting this sequence to residue 114 in the CGbeta 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 alpha subunit sequences in the single-chain molecule. Here we construct single FSH chains by fusing the carboxyl end of the FSHbeta subunit to the amino end of the alpha 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.


MATERIALS AND METHODS

Hormones

Tethered FSH in serum-free conditioned medium was obtained from CHO cells stably transfected with the expression vector pM^2HA 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 beta subunit fused to the amino end of the alpha subunit as described previously (Fig. 1; (2) ). The exon II sequence in the alpha 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 pM^2HA. For CGbetaDeltaTalpha, (CGbetaalpha tether minus the 31 carboxyl-terminal amino acids of the CGbeta subunit), the final PCR product was digested by SalI and ligated into pM^2HA containing CGbeta exons I and II as described previously(2) . All final constructs described were sequenced to verify that no misincorporation occurred during the PCR.


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 CGbeta 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 CGbeta 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.

FCalpha (FSHbeta-CTP-alpha)

The vector pM^2FSHbeta-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 FSHbeta 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 FSHbeta subunit, 84 bp (Ser-118Gln-145) of the CGbeta CTP sequence, and 12 nucleotides of the 5` end of exon II in the alpha subunit.

A fragment containing the alpha subunit sequence was constructed with primers 4 and 6 and the pM^2 alpha-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 alpha 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 pM^2HAFCalpha was used as a template DNA for the construction of F1/2Calpha (FSHbeta-1/2 CTP-alpha). A fragment containing the FSHbeta 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 alpha 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^2HA for transfection into CHO cells(9, 29) .

Falpha

The construction of the single FSH chain without the CTP sequence was similar to FCalpha except that the vector pM^2FSHbeta WT (27) was the template. Primers 1 and 8 were used to synthesize the entire FSHbeta coding sequence and a part of the alpha exon II sequence (Fig. 1). The pM^2alpha minigene (29) was a template for primers 7 and 6 to generate a product containing the 3` end of the FSHbeta exon III and the alpha-minigene. These fragments were incubated with primers 1 and 6 to synthesize the Falpha construct containing an intron between FSHbeta exons II and III.

CGbetaDeltaTalpha

To construct the CGbetaalpha tether lacking the carboxyl-terminal 31 amino acids of the CGbeta 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.

The CGbetaWT 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 alpha exon II and CGbeta exon III without CTP. Primers 12 and 6 were used with the pM^2alpha minigene to synthesize a fragment containing the 3` end of CGbeta exon III without CTP and the alpha 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 CGbeta exon 3/alpha 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) .


RESULTS

Secretion of the Single Chain

When the hCGbetaalpha 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 CGbeta subunit (CTP) would provide the flexibility to permit the appropriate interactions between the alpha and beta subunit domains. However, for the FSHbeta subunit, a linker sequence could be critical for the correct orientation of the alpha/beta determinants. Previous studies showed that when the CTP is placed on either the carboxyl-terminal end of FSHbeta subunit or in the amino-terminal region of the alpha 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 beta subunit was fused directly to the amino terminus of the alpha 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).


Figure 2: Expression of single-chain FSH from transfected CHO cells. Cells expressing FSH variants constructed in the presence (FCalpha; A) or absence (Falpha; 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 alpha subunit and subjected to SDS-polyacrylamide gel electrophoresis.



It was demonstrated previously that the secretion kinetics of the CGbetaalpha 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 CGbetaalpha (Fig. 3). These data show that efficient secretion of the FSH single chain requires a linker between the carboxyl terminus of the FSHbeta subunit and the amino terminus of the alpha subunit.


Figure 3: Synthesis of single-chain hCG variants. The entire CGbeta sequence was fused directly to the alpha without an exogenously inserted linker sequence (CGbetaalpha; A). A construct bearing the CGbeta subunit truncated at amino acid 114 was also fused to the alpha subunit (CGbetaDeltaTalpha; 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 alpha 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).


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 (bullet-bullet) 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.


DISCUSSION

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 CGbeta subunit is secreted quantitatively, less than 10% of the uncombined pituitary beta 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 beta and alpha 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 CGbeta 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 FSHbeta subunit by the adjacent alpha subunit. This conclusion is also supported by the CG crystallography data and by models showing that the linkage between the carboxyl terminus CGbeta114, the CGbeta subunit devoid of the CTP sequence, to the alpha subunit would not support an alignment of the beta-alpha domains as seen in the heterodimer (25) . (^2)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 alpha/beta 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.


FOOTNOTES

*
This work was supported in part by grants from the Organon Company and National Institutes of Health Contract NO1-HD92922. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Div. of Reproductive Biology, Dept. of Gyn/Ob, Stanford University Medical Center, Stanford, CA 94305-5317.

Supported by a fellowship from the Lalor Foundation.

**
To whom correspondence should be addressed: Dept. of Molecular Biology & Pharmacology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.

(^1)
The abbreviations used are: hCG, human chorionic gonadotropin; FSH, follicle-stimulating hormone; LH, luteinizing hormone; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; bp, base pair(s); RIA, radioimmunoassay.

(^2)
P. D. J. Grootenhuis, personal communication.


ACKNOWLEDGEMENTS

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.


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