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Endocrinology, doi:10.1210/en.2006-1499
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Endocrinology Vol. 148, No. 8 3977-3986
Copyright © 2007 by The Endocrine Society

Translational Fusion of Two ß-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Satarupa Roy, Sunita Setlur, Rupali A. Gadkari, H. N. Krishnamurthy and Rajan R. Dighe

Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science, Bangalore 560012, India

Address all correspondence and requests for reprints to: Professor Rajan R. Dighe, Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science, Bangalore 560012, India. E-mail: rdighe{at}mrdg.iisc.ernet.in.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The strategy of translationally fusing the {alpha}- and ß-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCG{alpha}ß expressed using Pichia expression system. Using the same expression system, another analog, in which the {alpha}-subunit was replaced with the second ß-subunit, was expressed (hCGßß) and purified. hCGßß could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCG{alpha}ß and hCGßß using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGßß interacts with two LH receptor molecules. These studies demonstrate that the presence of the second ß-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of {alpha}-subunit, the molecule is unable to elicit response. The strategy of fusing two ß-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GLYCOPROTEIN HORMONE family members LH, FSH, TSH, and human chorionic gonadotropin (hCG) are heterodimers with a common {alpha}-subunit associated noncovalently with the hormone-specific ß-subunit (1, 2). Their receptors belong to the family of G protein-coupled receptors consisting of a large extracellular domain (ECD) followed by a hinge region connecting the ECD to the seven-transmembrane (TMD) helices and a cytosolic domain (3). To date, there is no consensus on the specific mechanisms by which the glycoprotein hormones dock onto their receptors leading to signal transduction (4). The recently resolved structure of FSH-FSH receptor (FSHR)-ECD complex has raised questions on the key contact points between the hormone and receptor necessary for signaling (5, 6, 7). The structure-function studies of multimeric proteins are often hindered by the mutagenesis-induced defects in subunit association leading to decreased biological activity. Genetically linking subunits to form single-chain analogs is one of the approaches to investigate the structure-function relationship of such proteins (8, 9, 10). Single-chain hormone analogs with the C terminus of the ß-subunit fused to the N terminus of the {alpha}-subunit have been shown to be biologically active (11, 12). Earlier studies from the laboratory demonstrated generation of a single-chain analog in which the C terminus of {alpha}-subunit was fused to the N terminus of the ß-subunit using a single glycine as a linker (hCG{alpha}ß). This analog could bind to LH receptor with an affinity comparable with that of the natural hormone and also elicit biological response (13).

In the present study, we report purification and characterization of two single-chain analogs with distinct biological properties, an agonist hCG{alpha}ß, and an antagonist hCGßß. Furthermore, we demonstrate that hCGßß mediates its antagonistic activity by binding to the receptor in a different orientation, compared with hCG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones and chemicals
hCG (CR127), hCGß (CR123), and human FSH (hFSH) were obtained from Dr. A. F. Parlow (National Hormone and Pituitary Program, Torrance, CA). [125I]NaI and [1,2,6,7,16,17-3H] testosterone were purchased from PerkinElmer Life Sciences (Boston, MA). All restriction and modifying enzymes were purchased from Boehringer-Mannheim GmbH (Mannheim, Germany), MBI Fermentas (Burlington, Ontario, Canada), and Roche Diagnostics GmbH (Mannheim, Germany). Phenyl-Sepharose and SP-Sepharose were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). All other reagents were obtained from Sigma Chemical Co. (St Louis, MO).

Antibodies
All hCG antibodies, both monoclonal (MAb) and polyclonal antibodies used in this study, were produced and characterized as described earlier (14). MAb B110 was a kind gift of Dr. W. R. Moyle (Rutgers University, Piscataway, NJ). cAMP antiserum was obtained from the National Hormone and Pituitary Program.

Cloning and expression vectors
Plasmid pBluescript SK+ was obtained from Stratagene (La Jolla, CA); Pichia expression vector, pPIC9K, and Pichia pastoris strain (GS115) used in this study were kind gifts from the Phillips Petroleum Co. (Bartlesville, OK).

Experimental animals
All animal experiments described were conducted according to the accepted standards of humane animal care, as outlined in ethical guidelines of the institute.

Construction of hCG single-chain analogs
Expression of hCG{alpha}ß (pPIC9KhCG{alpha}ß) has been described previously (13). To generate hCGßß, hCGß cDNA was amplified using the forward primer that had an EcoRI site followed by codons for the first six amino acids of the mature, secreted form of hCGß subunit and the reverse primer having BamH1 site and codons for six C-terminal amino acids without the termination codon. The amplified product was cloned into hCGß cDNA previously used for producing the hCG{alpha}ß construct, thus fusing the two hCGß subunits with a single glycine residue between the two subunits. Thus, the hCGßß has two ß-subunits plus eight amino acids from the yeast {alpha}-mating factor signal peptide at the N terminus contributed by the vector, pPIC9K (bold letters in the sequence given below) and six histidine residues (bold and underlined) added at the C terminus of the second ß-subunit as in case of hCG{alpha}ß construct (13) joined by glycine (bold and italic). The total numbers of amino acids in the expressed protein were 305 amino acids with calculated molecular mass of 32,947 Da. However, it is difficult to arrive at the precise molecular mass due to heterogeneity in the carbohydrate content (15) of the recombinant hormones expressed by the Pichia cells. The translated amino acid sequence of the full-length hCGßß protein is as follows:

MLEKRSKEPLRPRCRPINATLAVEKEGCSQCGAPNTTICAGYCPTMTRVLQGVLPALPQVVCNYRDVRFESIRLPGCVTVMGGFKSYAVALSCQCALCRRSTTDCGGPKDHPLTCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQGSKEPLRPRCRPINATLAVEKEGCSQCGAPNTTICAGYCPTMTRVLQGVLPALPQVVCNYRDVRFESIRLPGCVTVMGGFKSYAVALSCQCALCRRSTTDCGGPKDHPLTCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQHHHHHH

Expression of hCG{alpha}ß and hCGßß
The Pichia pastoris clone expressing hCG{alpha}ß was used to hyperexpress the protein in the fermenter. For expression of hCGßß, pPIC9KhCGßß linearized with BglII was used to transform P. pastoris (GS115) (16) and the transformants selected on a histidine-deficient medium were screened for their ability to express and secrete hCGß-like immunoactivity on induction with methanol (16, 17) by RIA using MAb B52/12, which does not distinguish between hCG and hCGß, and 125I hCG as the tracer. The clone secreting maximum level of hCGßß was selected to scale up the expression using fermenter.

Large-scale expression and purification of hCG{alpha}ß and hCGßß
Fermentations were carried out with clones expressing single-chain analogs of hCG as described previously (18). To purify the analogs, the fermentation medium was loaded onto Phenyl Sepharose column in presence of 1.6 M (NH4)2SO4, 50 mM phosphate buffer (pH 7.4) and sequentially eluted with 50 mM phosphate buffer and 50% acetonitrile. hCG{alpha}ß and hCGßß activities eluted in the phosphate buffer fractions were loaded onto SP-Sepharose previously equilibrated with 25 mM Tris-Cl buffer (pH 7.0) and eluted with a stepwise gradient of NaCl (18).

Immunological characterization of single chain hCG analogs
The purified single-chain proteins were analyzed by RIA using hCGß specific MAbs B52/12 and B110 and a polyclonal hCGß a/s with 125I hCG as the tracer using the procedures described previously (19) and by ELISA using a heterodimer-specific MAb E12. The ELISA was carried out by adsorbing increasing amounts of hCG or its single-chain analogs on ELISA plates (Nunc-Immuno-MaxiSorp), followed by incubation with the MAb (50 ng IgG per well) at 37 C for 1 h and finally with antimouse IgG horseradish peroxidase conjugate (18). The competition ELISA was performed by preincubating the MAb E12 with increasing concentrations of competing ligands for 1 h at 37 C in solution followed by addition of the mixture to hCG-coated (50 ng/well) ELISA plates and incubating at 37 C for 1 h. The antibody bound to hCG-coated plates was quantified using antimouse IgG horseradish peroxidase conjugate (18).

Radioreceptor assay
hCG and hFSH were radioiodinated as described earlier (14), whereas hCGßß was radioiodinated using the Iodogen method. Binding of 125I hCG to LH receptor was demonstrated as described earlier (18, 20). The same procedure was used to demonstrate binding of 125I hCGßß as well as 125I FSH using membrane receptor preparation from HEK293 cells expressing rat FSH receptor (21). The nonspecific binding in all these experiments was determined by incubating the radiolabeled hormone in presence of excess of the unlabeled ligand. The binding data obtained with displacement analysis were subjected to Scatchard analysis (22).

Effect of MAb B110 on hCG/hCGßß-receptor interaction was determined by incubating the 125I MAb B110 (radioiodinated by the Iodogen method) with hCG/hCGßß for 30 min at room temperature followed by addition of particulate rat LH receptor and continuing the incubation for additional 30 min. In an another experiment, unlabeled hCG/hCGßß was incubated with the particulate rat LH receptor first followed by addition of 125I MAb B110 and continuing incubation for another 30 min and 125I MAb B110 bound to the hormone-receptor complex was determined (17). The nonspecific binding of the labeled antibody was determined by carrying out binding experiments in presence of excess of the unlabeled antibody.

In vitro bioassays
The mouse Leydig cells were incubated with hCG/hCG{alpha}ß/hCGßß at 32 C for 4 h and testosterone secreted into the medium was determined by RIA (20, 23). To determine the effect of hCGßß on FSH-stimulated response, HEK293 cells expressing rat FSH receptors were incubated in DMEM containing 1 mM 3-isobutyl-1-methylxanthine with 1 nM hFSH in the presence and absence of hCGßß for 15 min at 37 C, and cellular cAMP produced was determined by RIA (24).

In vivo bioassay
hCG and hCGßß alone or together were administered ip to adult male rats, and the serum testosterone was determined after different time intervals as described earlier (25).

Gel electrophoresis and immunoblotting
Equal amounts of proteins were resolved on 12.5% SDS-PAGE under reducing conditions and either stained with Coomassie Brilliant Blue or transferred to polyvinyl difluoride membranes and subjected to Western blot analysis using hCGß-specific antiserum.

Cross-linking of 125I hCG/125I hCGßß with LH receptor
Rat LH receptor preparation was separately incubated with 150,000 cpm of either 125I hCG or 125I hCGßß in 0.05 M sodium phosphate buffer (pH 7.4) containing 150 mM NaCl for 1 h at room temperature. The unbound ligands were removed by centrifugation, and the resulting complexes obtained were incubated with 1 mM glutaraldehyde for 15 min at 4 C and electrophoresed on 8% low cross-linker SDS-PAGE under nonreducing conditions as described by Fan and Hendrickson (5). The gels were dried and subjected to autoradiography. The specificity of binding was demonstrated by incubating the labeled ligands with receptor in the presence of large excess of unlabeled ligands.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and purification of single-chain hormone analogs
The Pichia clone expressing hCGßß was identified by determining hCG-like activity secreted into the medium on methanol induction by RIA using hCGß-specific MAb B52/12 and 125I hCG as the tracer and used for further characterization.

The clones expressing hCG{alpha}ß and hCGßß were grown in a 10l fermenter vessel and induced with methanol as described previously (18), and purification of analogs from the fermentation medium was achieved by phenyl Sepharose chromatography followed by SP-Sepharose chromatography and yielded approximately 5–10 mg of hCG{alpha}ß/hCGßß per fermentation. As shown in Fig. 1AGo, the reducing SDS-PAGE analysis on Coomassie brilliant blue staining revealed one band with an apparent molecular mass of 60 kDa in case of hCGßß and approximately 45 kDa in case of hCG{alpha}ß. The Western blot analysis with hCGß subunit-specific antiserum confirmed the authenticity of the recombinant proteins (Fig. 1BGo). Deglycosylation of hCGßß with Endo-H resulted in decrease in the molecular mass to 35 kDa, which correlates with the calculated molecular mass of the entire protein sequence of hCGßß, which is 32.9 kDa (Fig. 1CGo).


Figure 1
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  		FIG. 1. SDS-PAGE and Western blot of hCG{alpha}ß and hCGßß. A, Equal amounts of proteins were electrophoresed under reducing conditions on 12% SDS-PAGE and stained with Coomassie Brilliant Blue. B, They were then transferred onto polyvinyl difluoride membrane and probed with polyclonal hCGß antiserum. The sizes of the molecular weight markers are indicated in the lanes marked as M. Highly purified urinary hCG was used as the control. C, Endo-H-digested mixtures of hCGßß and hCG were similarly electrophoresed under reducing conditions and probed with a polyclonal hCGß antiserum.

 
Although the carbohydrate portion of hCGßß has not been characterized, mass spectrometric analysis and nuclear magnetic resonance of the glycan moiety of the recombinant hCG produced by the same strain of Pichia revealed (15) predominant moiety having 9–10 mannose residues with and without an unusual phosphodiester linkage. The Pichia cells do not process the glycan part and hence do not add terminal galactose or sialic acid to the recombinant hormones. However, absence of processing the glycan moiety by the yeast cells does not affect the folding, receptor binding, and in vitro bioactivity of the heterodimeric hCG (13, 17, 18).

Immunological characterization of hCG{alpha}ß and hCGßß
The single-chain analogs were characterized further by RIA using monoclonal and polyclonal antibodies raised against hCG or its subunits. With MAb B52/12, which recognizes the epitope in the seatbelt region of the ß-subunit (26), no differences could be observed among the displacement curves of hCG, hCGß, and hCG{alpha}ß, suggesting that the epitope recognized by this antibody was not altered as a result of heterodimerization or fusion with the {alpha}-subunit, and hence, this antibody was used to quantify different preparations of the analogs (Fig. 2AGo). However, with hCGßß (Fig. 2BGo), there was a shift in the EC50 of the RIA displacement curve, suggesting that the epitope may have been altered as a result of the fusion of the two ß-subunits. Interestingly, with a highly specific hCGß-specific antibody, which has a relatively higher affinity for hCGß subunit, compared with that for hCG, both single-chain hCG{alpha}ß and hCGßß exhibited much lower EC50, suggesting that the subunit interactions in the single-chain analogs are not identical with that seen in hCG (Fig. 2CGo). However, the fact that the single-chain analogs were able to completely inhibit binding of 125I hCG to all these antibodies suggested that the conformation of hCG{alpha}ß and hCGßß were similar but not identical with that of the natural hormone.


Figure 2
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  		FIG. 2. Immunological characterization of single-chain analogs using RIA (A–C) and ELISA (D and E). RIAs of hCG{alpha}ß/hCGßß were carried out using ß-subunit-specific MAb B52/12 (A and B) and polyclonal hCGß antiserum (C), with 125I hCG as the tracer and hCG (CR127) and hCGß (CR123) as reference preparations. The data represent percent specific binding of 125I hCG after subtracting the nonspecific binding obtained in the absence of antibodies. D, Increasing quantities of hCG/hCG{alpha}ß/hCGßß were coated on ELISA plates and binding of MAb E12 (50 ng/well) was determined by ELISA. E, MAb E12 was preincubated with increasing concentrations of hCG/hCG{alpha}ß/hCGßß followed by addition of the mixture to hCG-coated ELISA plates and determining the antibody bound to the plates. The values presented are the means of duplicates, and each experiment was carried out two times.

 
Immunological characteristics of the single-chain hCG analogs were further explored using hCG MAb E12, which is a heterodimer-specific MAb with no cross-reactivity with either subunits of hCG as well as hLH. The epitope recognized by this MAb is contributed by both subunits and is located in the ß-loop 2 and {alpha}-loop 3 (23). As shown in Fig. 2Go, D and E, this MAb recognizes hCG{alpha}ß relatively weakly, compared with hCG, whereas it does not bind to hCGßß at all.

Receptor binding
hCG{alpha}ß and hCGßß were able to completely inhibit binding of 125I hCG to the receptor (Fig. 3Go), suggesting that the two analogs could bind to the receptor. Direct evidence for binding of hCGßß to LH receptor was obtained by demonstrating specific binding of 125I hCGßß to the receptor (Fig. 4AGo). The Scatchard analysis of the binding data confirmed that hCGßß binds to the receptor with an affinity 2.5 times lower than that of hCG. To demonstrate the specificity of hCGßß, the membrane preparation obtained from HEK293 cells expressing rat FSH receptor was incubated with 125I hFSH in presence and absence of hCGßß. As shown in Fig. 4BGo, hCGßß did not inhibit binding of 125I hFSH to FSH receptor.


Figure 3
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  		FIG. 3. Radioreceptor assay. 125I hCG was incubated with rat LH receptor preparation in the presence of increasing concentrations of hCG/hCGß{alpha}/hCGßß (as estimated by RIA using MAb B52/12), and the receptor bound radioactivity was determined. The nonspecific binding was determined by incubating the labeled hormone with the receptor preparation in the presence of a large excess of hCG (400 ng/ml). The data represent the percentage of specific hormone-receptor bound radioactivity. The values presented are the means of duplicates, and each experiment was carried out twice.

 

Figure 4
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  		FIG. 4. A, Direct binding of hCGßß to LH receptor. 125I hCGßß was incubated with rat LH receptor preparation in the presence of increasing concentrations of hCGßß/hCG, and the binding data were converted into a Scatchard plot, n = 3 (inset), using the computer program GraphPad Prism (version 4.0; GraphPad, San Diego, CA). The data represent the percentage of specific hormone-receptor bound radioactivity. The values presented are the means of duplicates, and each experiment was carried out two times. B, Effect of hCGßß on FSH receptor interaction. 125I hFSH was incubated with rat FSH receptor membrane preparation in the presence of increasing concentrations of hCGßß, and the receptor bound radioactivity was determined. The nonspecific binding was determined by incubating the labeled hormone with the receptor preparation in the presence of a large excess of hFSH (400 ng/ml). The data represent the specific hormone-receptor bound radioactivity. The values presented are the means of duplicates.

 
In vitro biological response
Ability of hCG{alpha}ß and hCGßß to elicit biological response was investigated by in vitro Leydig cell bioassay. hCG{alpha}ß stimulated testosterone production by the cells in a dose-dependent manner with an EC50 equal to that of hCG (Fig. 5AGo). However, hCGßß alone did not elicit any biological response, even at higher concentration, suggesting that hCGßß can bind to the receptor but is unable to elicit any biological response.


Figure 5
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  		FIG. 5. In vitro and in vivo antagonistic activity of hCGßß. A and B, In vitro Leydig cell bioassay. A, Equal quantities of hCG/hCG{alpha}ß/ßß (as estimated in RRA) were incubated with the mouse Leydig cells for 4 h at 32 C. B, Ability of hCGßß to inhibit hCG stimulated response was tested by incubating increasing concentrations of hCGßß and 1 ng/ml hCG with the Leydig cells for 4 h at 32 C. In both the cases, testosterone secreted into the medium was determined by RIA. Values presented are means ± SD of quadruplicates and are representative of three independent experiments. C and D, In vivo antagonistic activity of hCGßß. C, Adult male rats were administered 1 µg hCGßß/saline followed by hCG (100 ng/1.3 IU) 30 min later, and serum testosterone was determined by RIA. D, Effect of hCGßß on the basal serum testosterone levels was determined by administering 1 µg hCGßß to each animal and estimating serum testosterone after different time intervals. The 0 h time point represents the basal serum testosterone level at the pretreatment stage for the individual animal. The values presented are for five animals and are representative of two independent experiments.

 
To test whether hCGßß can inhibit response to the hormone, the cells were first incubated with hCGßß for 30 min and then hCG was added to the tubes, and incubation was continued for 4 h. Figure 5BGo demonstrates that hCGßß inhibits response to hCG in a dose-dependent manner, further confirming the antagonistic nature of hCGßß. hCGßß did not inhibit hFSH-stimulated response by the HEK293 cells expressing FSH receptor (Table 1Go), providing evidence for the specificity of hCGßß action.


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  		TABLE 1. Effect of hCGßß on FSH-stimulated response

 
Antagonistic effect of hCGßß in vivo
The antagonistic effect of hCGßß in vivo was demonstrated by determining the effect of hCGßß on hCG-stimulated increase in serum testosterone in the adult male rats. Administration of 100 ng hCG (1.3 IU) increased serum testosterone levels, which remained high for almost 24 h. Administration of 1 µg hCGßß 30 min before hCG administration blocked this increase in serum testosterone (Fig. 5CGo). In another experiment, the animals were administered hCGßß (1.0 µg) alone, and the serum testosterone was determined after different time intervals. The basal serum testosterone levels decreased significantly by 30 min and remained low for 3–6 h, further confirming the antagonistic nature of hCGßß (Fig. 5DGo).

Mechanism of hCGßß action
To determine the stoichiometry of hCGßß binding to LH receptor, cross-linking experiments were performed as described in Materials and Methods. The LH receptor-125I-hCG complex exhibited a band of molecular mass of 135 kDa, suggesting a ratio of hCG to receptor of 1:1. 125I-hCGßß-LH receptor showed a complex of 230 kDa, suggesting that hCGßß was complexed with two molecules of LH receptor in 1:2 ratio (Fig. 6Go).


Figure 6
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  		FIG. 6. Cross-linking of 125I hCG and 125I hCGßß to LH receptor. 125I hCG or 125I hCGßß was incubated with rat LH receptor preparation, and the cross-linked complexes were electrophoresed on 8% SDS-PAGE under nonreducing conditions and autoradiographed. To determine the nonspecific binding, cross-linking was carried out with the receptor in presence of 100-fold excess of hCG/hCGßß. The molecular mass estimates of the radioactive bands and the apparent composition of band materials are indicated with arrows. Lane 1, 125I hCGßß; lane 2, 125I hCGßß + unlabeled hCGßß + receptor (nonspecific); lanes 3 and 4, 125I hCGßß + receptor; lanes 5 and 6, 125I hCG + receptor; lane 7, 125I hCG + unlabeled hCG + receptor (nonspecific); lane 8, 125I hCG. The data presented are representative of three independent experiments.

 
The orientation of hCGßß in hCGßß-receptor complex was further investigated by determining the accessibility of an epitope in the hCGßß when it is complexed with the receptor. As shown in Fig. 7AGo, hCGß-specific MAb B110, which recognizes the epitope present in the tip of L3 loop of hCGß subunit, was able to bind 125I hCGßß with much higher affinity. 125I MAb B110 recognizes the hormone in hCG-receptor complex (Fig. 7BGo). Conversely, the receptor can also bind to antibody-hCG complex, suggesting that this antibody epitope is not involved in receptor interaction directly. Although this MAb binds 125I hCGßß directly, 125I MAb B110 does not bind to hCGßß-receptor complex, suggesting that the epitope recognized by this antibody is not accessible to the antibody in the hCGßß-receptor complex.


Figure 7
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  		FIG. 7. Orientation of hCGßß in hCGßß-receptor complex. A, Binding of hCG and hCGßß to MAb B110. 125I hCG was incubated with MAb B110 (0.025 mg/ml) in the presence of increasing concentrations of hCG/hCGßß, and binding of the labeled probe to the MAb was determined. The data are presented as the percentage of specific 125I hCG bound radioactivity. The nonspecific binding was separately determined by incubating the tracer without the MAb and subtracting from the total binding. B, Binding of 125I MAb B110 to the receptor bound hCG and hCGßß. hCG/hCGßß was preincubated with the receptor (R) for 30 min followed by removal of the unbound ligand by centrifugation. 125I MAb B110 was then added, incubated for another 30 min, and the radioactivity bound to the receptor determined. Conversely, hCG/hCGßß was preincubated with 125I MAb B110 followed by addition of the receptor, and at the end of additional incubation for 30 min binding of the labeled MAb was determined. The nonspecific binding in each case was determined by adding a large excess of unlabeled MAb B110 along with 125I MAb B110. Values represented here are mean ± SD, n = 3, and are representative of two independent experiments.

 
To investigate the mechanism of hCGßß action, the effect of hCGßß on the preformed hCG-receptor complex was investigated in terms of both binding and response. The hormone-receptor interaction was initiated by mixing 125I hCG with the receptors and incubating for 30 min when 90% of maximum binding was observed. To the mixture either buffer, saturating concentrations of hCG or hCGßß was added, and incubation was continued for 1 more hour, and 125I hCG bound to the receptor was determined. As shown in Fig. 8Go, hCG could not dissociate the preformed hCG-receptor complex, whereas hCGßß was able to dissociate the complex significantly.


Figure 8
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  		FIG. 8. Effect of hCGßß on preformed hCG-receptor complex. 125I hCG was preincubated with rat LH receptor preparation for 30 min at room temperature and then centrifuged to separate the hCG-receptor complex. The complex was then resuspended with hCG (200 ng/ml)/hCGßß (200 ng/ml)/buffer, and incubation was continued for another 30 min. At the end of the incubation, receptor bound radioactivity was determined. The nonspecific binding was determined by incubating 125I hCG with the receptor preparation in the presence of a large excess of unlabeled hCG (400 ng/ml).

 
To investigate the effect of hCGßß on response to the hormone, the mouse Leydig cells were exposed to hCG (1 ng/ml), and hCGßß (20 ng/ml) was added to the cell suspension after various time intervals, incubated at 32 C for 4 h, and testosterone secreted into the medium was estimated. The time course of response to hCG was determined separately by stopping the incubation of cells with hCG at different time points. Table 2Go shows time-dependent increase in testosterone secretion. Addition of hCGßß any time after formation of hCG-receptor complex resulted in almost immediate termination of response suggesting that hCGßß can dissociate the preformed hormone receptor complex.


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  		TABLE 2. Effect of hCGß ß on preformed hCG-receptor complex

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The strategy of translationally fusing the subunits of multimeric proteins into a single-chain analog has been used to overcome the mutagenesis-induced defects in subunit interactions. Several reports have shown that single-chain variants of glycoprotein hormones with C terminus of the ß-subunit fused to the N terminus of the {alpha}-subunit display bioactivities comparable with those of the corresponding heterodimers (27, 28, 29, 30, 31). However, the relative position of the two subunit does not appear to be important for generation of a biologically active molecule (13). These results also suggested that the C terminus of the {alpha}-subunit need not be free for hormone binding and signal transduction. Furthermore, a deletion of five C-terminal residues of the {alpha}-subunit affected signaling without affecting the binding, indicating the importance of the C terminus of the {alpha}-subunit in signaling rather than in binding. The {alpha}-subunit in hCG{alpha}ß was replaced with another ß-subunit by linking the C terminus of one ß-subunit to the N terminus of second ß-subunit keeping a single glycine residue in between the two subunits to generate hCGßß. Both single-chain analogs bind to the receptor with an affinity slightly lower than that of urinary hCG. Whereas hCG{alpha}ß elicits response, hCGßß is unable to do so. The effect of hCGßß is very specific because it was unable to inhibit FSH binding to FSH receptor and unable to inhibit FSH-stimulated response.

High affinity binding of hCGßß to the receptor is rather intriguing because the isolated natural hCGß subunit on its own can bind very weakly to the receptor (32, 33). However, Butler et al. (34) showed that homodimeric hCGß secreted by certain epithelial tumors has receptor binding and uncontrolled growth-promoting activities. It appears that the proximity of two ß-subunits in hCGßß has resulted in a conformation equivalent to that seen in the heterodimeric hormone necessary for receptor binding. However, it is evident from the immunological characterization of the single-chain molecules that there are subtle changes in the conformation of some of the epitopes, whereas some epitopes appear to be intact. The MAb E12, which recognizes a unique epitope present in the heterodimeric hCG contributed by ßL2 and a region close to the {alpha}L3 loops, which lie at the interface of the two subunits in hCG heterodimer, appears to be altered in hCG{alpha}ß and is completely absent in hCGßß. Involvement of this region in the receptor binding has been debated over the years, and conflicting theories have been put forth regarding the functionality of this region (35). Our earlier data suggest that this epitope is critical for signal transduction (20, 26), and its complete absence in hCGßß is at least partly responsible for its inability to elicit response.

The subunits in the single-chain analogs, on separation by low pH treatment, do not reassociate at neutral pH as seen in the case of heterodimeric molecule, further confirming that subunit interactions in single-chain hCG analogs are not exactly identical with that seen in hCG (data not shown). This indicated that folding of hCG{alpha}ß and hCGßß during biosynthesis in Pichia cells has the advantage of the presence of several factors necessary for assisted protein folding. Despite all these changes in the conformations, the single-chain molecules attain the overall folding necessary for receptor binding. This also points out that there is a tremendous degree of flexibility in the hormone-receptor interactions and the receptor has the ability to accommodate analogs with altered conformations. The ß-subunits in hCGßß were also unable to interact with the {alpha}-subunit as indicated by lack of bioactivity generated on incubation of the hCGßß with hCG{alpha} subunit (data not shown). This is in contrast to the similar molecule reported by Lobel et al. (36) in which the ß-subunit homodimers were able to interact with two {alpha}-subunits to produce a biologically active analog. However, lack of biological response on interaction with hCG{alpha} does not mean that the {alpha}-subunit is not interacting with hCGßß. Interestingly, Lobel et al. (36) also reported that hCGßß elicited biological response in vitro but at much higher concentration. It also appeared to have much lower affinity for LH receptor. Whether the relatively higher affinity exhibited by Pichia expressed hCGßß, which is expected to have nine to 12 mannose residues and no sialic acid, is due to different structure of the glycan moiety needs to be investigated (15).

The mechanism by which hCGßß brings about inhibition of hormone action is not clearly understood. hCGßß could inhibit response to the preformed hormone receptor complex in vitro. Similarly, hCGßß administered to the adult rats after hCG treatment inhibited increase in the serum testosterone levels (data not shown), suggesting that the analog could dissociate the hormone-receptor complex. This is in contrast to the data obtained with the polyclonal and monoclonal antibodies against hCG (20, 26), which could not terminate response to the preformed hCG-receptor complex, probably due to lack of accessibility of the epitope to the antibodies in the hormone-receptor complex.

Binding of hCGßß to the receptor appears to be different from that of hCG. The ßL3 of hCG is accessible to MAb B110 in the hCG-receptor complex (4, 37), whereas it appears to be buried inside the hCGßß-receptor complex, clearly indicating that the orientation of hCGßß in the analog-receptor complex is different. Our cross-linking and immunomapping studies showed that hCGßß can bind to two receptor molecules, further confirming the differences in binding of hCGßß and the hormone. We also observed that hCGßß, but not hCG, was able to dissociate 125I hCG from the preformed hormone-receptor complex, confirming the differences in interaction of the two with the receptor and may involve two sites displaying negative cooperativity (38).

The above data demonstrating interactions between hCGßß and the receptor lead to an intriguing question of how fusion of two ß-subunits results in a structure that can be recognized by the receptor with a relatively high affinity. Modeling studies by Lobel et al. (36) suggested that the loop 2 of the ß-subunit can substitute for the loop 2 of the {alpha}-subunit in the structure of hCG and can still maintain a stable structure with low receptor affinity. It can be hypothesized that hCGßß molecule adopts a conformation in which the loops 1 and 3 of the first ß-subunit are in close proximity with the loop 2 of the second ß-subunit in the fusion protein and vice versa, thus mimicking the heterodimeric hCG structure and resulting in receptor binding but interacting with two independent receptor molecules. This difference in receptor binding may be the key factor in determining the success or failure of signal transduction and activation of adenylyl cyclase. Earlier studies have indicated that the initial interaction between the hormone and receptor occurs mainly through the hormone-specific ß-subunit, and then the {alpha}-subunit is brought into contact with the TMD of the receptor (20, 39, 40). Remy et al. (41) showed that the peptide mimicking the second extracellular loop of the TMD inhibited binding of a MAb directed to site {alpha}L3 loop, suggesting that this region of the endodomain may be interacting with the {alpha}-tip. Furthermore, photoaffinity labeling studies using peptides of exoloops have revealed close interactions between the {alpha}-subunit and the exoloop 3 (42). Recently we also demonstrated similarity of the complementarity determining region of the MAb C10 that recognizes the {alpha}L3 loop to the extracellular loops of the TMD of LH receptor, further confirming the interaction between this loop and the TMD (26). Importance of the {alpha}-subunit (43, 44, 45) and the TMD of all G protein-coupled receptors in signal transduction is well documented (3, 46). The crystal structure of FSH complexed with the ECD of the FSH receptor suggested binding of the hormone to the FSH receptor-ECD establishes the contact between the tips of loops 1 and 3 of {alpha}-subunit and the TMD leading to signaling (6, 7). These data suggest that all the critical determinants required for hormone binding reside in the ß-subunit, and it probably interacts with the ECD. But in the absence of the {alpha}-subunit, hCGßß, although binding tightly to the receptor, is incapable of transducing the signal. The data presented here provide strong evidence for the model proposed in the past for glycoprotein hormones-receptor interactions (47, 48, 49, 50, 51).

Preliminary studies with single-chain FSHßß produced using a similar strategy have shown that such an analog is an inhibitor of FSH action, whereas hCGßß in the same experiment has no effect, demonstrating the specificity of the ßß analogs (manuscript in preparation). Whether the approach of fusing two ß-subunits of glycoprotein hormones can lead to novel antagonists of the hormones, and newer contraceptive strategies remain to be established.


   Footnotes
 
This work was supported by grants from the Department of Biotechnology, the Indian Council of Medical Research, and the University Grants Commission, New Delhi. S.R. and S.S. were supported by the fellowship from the Council for Scientific and Industrial Research, New Delhi.

Part of the work presented in this manuscript was presented at the 87th Annual Meeting of The Endocrine Society, San Diego, CA, June 4–7, 2005 (Abstract 04538).

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online May 3, 2007

Abbreviations: ECD, Extracellular domain; FSHR, FSH receptor; hFSH, human FSH; hCG, human chorionic gonadotropin; MAb, monoclonal antibody; TMD, transmembrane domain.

Received November 9, 2006.

Accepted for publication April 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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