Consequences of Single-Chain Translation on the Structures of Two Chorionic Gonadotropin Yoked Analogs in {alpha}-ß and ß-{alpha} Configurations

Gregory B. Fralish, Prema Narayan and David Puett

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229

Address all correspondence and requests for reprints to: Dr. David Puett, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229. E-mail: puett{at}bmb.uga.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human chorionic gonadotropin (hCG) is a placental-derived heterodimeric glycoprotein hormone, which, through the binding and activation of the LH receptor, rescues the corpus luteum and maintains pregnancy. The three-dimensional structure of hCG is known; however, the relevance of its fold to bioactivity is unclear. Although both subunits ({alpha} and ß) are required for activity, recent data with single-chain analogs have suggested a diminished role for the cystine knot and an intact heterodimeric interface in binding and receptor activation in vitro. Herein, we report the purification and structural characterization of two yoked (Y) hCG analogs, YhCG1 (ß-{alpha}) and YhCG3 ({alpha}-ß). The fusion proteins yielded higher IC50s and EC50s than those of hCG; the maximal hCG-mediated cAMP production, however, was the same. Circular dichroic spectroscopy revealed that the three proteins exhibit distinct far UV circular dichroic spectra, with YhCG1 containing somewhat more secondary structure than YhCG3 and hCG. Limited proteolysis with proteinase K indicated that heterodimeric hCG was much more resistant to cleavage than the single-chain analogs. YhCG1 was more susceptible to proteolysis than YhCG3, and the fragmentation patterns were different in the two proteins. Taken together, the data presented herein provide direct structural evidence for altered three-dimensional conformations in the two single-chain hCG analogs. Thus, the cognate G protein-coupled receptor can recognize and functionally respond to multiple ligand conformations.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
REPRODUCTION IN PRIMATES is governed by the actions of the pituitary-derived gonadotropins, LH and FSH, and the placental-derived chorionic gonadotropin (CG). LH and FSH promote gonadal steroidogenesis and the growth and maturation of follicles, whereas a midcycle surge in LH levels induces ovulation. The implanted, fertilized egg leads to the production of CG by the placenta, which in turn promotes the production of progesterone, thus maintaining pregnancy.

TSH, FSH, LH, and CG form the family of glycoprotein hormones, which are characterized by their heterodimeric quaternary structure consisting of a common {alpha}-subunit and a hormone-specific ß-subunit (1). Biological activity of the glycoprotein hormones is conferred through their respective G protein-coupled receptors. TSH and FSH bind to specific receptors (TSH receptor and FSH receptor, respectively), whereas LH and CG bind to a common receptor [LH receptor (LHR)]. This is not surprising given the high degree of sequence homology (85%) shared between CG and LH ß-subunits, with most of their dissimilarity occurring from the C-terminal 30-amino-acid residue extension (CTP, C-terminal peptide) of human (h)CG-ß (1). The CTP, which contains four O-linked glycosylation sites, is important for the relatively long circulatory half-life of hCG (2). LHR binds hCG and LH with high affinity [dissociation constant (Kd) = 0.1–1.0 nM] primarily through its large, N-terminal extracellular domain (3).

The crystal structure of hCG (4, 5) revealed that, surprisingly, the {alpha}- and ß-subunits have nearly identical folds despite having very little sequence homology. Both subunits form a three-looped structure maintained by a central growth factor-like cystine knot. The heterodimer is not constrained by a hydrophobic core and is devoid of significant helical structure. However, it is stabilized by a significant number of contacts at the dimer interface and by the ß-subunit seat-belt loop, which wraps around a portion of {alpha} and forms an intramolecular disulfide bond during the later stages of hCG folding in the endoplasmic reticulum (6). Based upon the structure of hCG and the conservation of the key cysteine residues, the other glycoprotein hormones were postulated to fold in a similar manner. This was recently confirmed with the structure of hFSH (7).

The role of the three-dimensional structure in the binding and signal-transducing activity of the hormone is unclear. It is evident that heterodimerization is a prerequisite for biological activity, and the individual subunits cannot activate LHR (8). The seat-belt loop is intimately involved in conferring specificity (9), and the C terminus of {alpha} is important in high-affinity receptor binding and activation (10, 11, 12, 13). These critical regions are located on one face of the hormone (4, 5), which carries a net positive surface charge that is complementary to the receptor’s net negative surface charge reported in several homology models of the extracellular domain (14, 15). These data, coupled with the evidence of only subtle conformational changes during binding (16), strongly suggest the importance of the three-dimensional structure of CG for its biological activity.

We and others have generated a single-chain hCG, consisting of the C terminus of the ß-subunit yoked (Y) or fused to the N terminus of the {alpha}-subunit, a configuration chosen primarily to maintain the free {alpha}-carboxy terminus, as well as having the endogenous CTP as a flexible linker (17, 18). The N-ß-{alpha}-C-fused hCG (YhCG1) was biologically active, displaying in vitro and in vivo bioactivity similar to that of native hCG. It was suggested that the N-ß-{alpha}-C configuration, with the 30-amino-acid residue CTP linker, permitted native-like folding of the fused hormone (17). Our laboratory and others have also reversed the order of subunit tether to N-{alpha}-CTP-ß-C (YhCG3; Refs. 19, 20, 21). This analog bound LHR poorly, but activated the receptor efficiently, thus uncoupling the two activities (19).

Single-chain hCGs have enabled novel mutagenic and chimeric approaches to better understand glycoprotein hormone structure-function relationships since the obligatory dimerization step in secretion is bypassed, thus permitting expression of the variants. It was shown with a single-chain hCG that an intact cystine knot in either subunit is not required for biological activity (22, 23). Furthermore, multiple ß-subunits, when fused to a single {alpha}-subunit, could form bifunctional hormones (24, 25). Using monoclonal antibodies, single-chain hCG (N-ß-{alpha}-C) was shown to retain biological activity when some of the native quaternary interactions at the interface between the subunits were disrupted by mutation (26). However, complete disruption of subunit interactions, as in the single chain N-{alpha}-ß-C (without linker), causes nearly complete loss of activity (21). From these elegant studies, it is apparent that native tertiary and quaternary structure are not a prerequisite to biological activity. Indeed, these structural characteristics have ascribed more importance to intracellular behavior (21, 22, 23, 26).

While the studies discussed above were critical in detecting changes in conformation, the nature and extent of the conformational differences between heterodimeric and single-chain hCG are still unclear. Is the conformation affected locally at the dimer interface only or are there global changes to structure? Does translation as a single polypeptide affect the folding of the individual subunits and how does the configuration of the subunits in the polypeptide affect their conformation? To address these questions, we have purified two single-chain analogs, YhCG1 and YhCG3. The structural character of the two yoked hormones and native heterodimeric hCG were compared using the complementary techniques of circular dichroism (CD) and limited proteolysis. The CD spectra for the three proteins were all distinct, revealing considerable changes in conformational character. Furthermore, when subjected to limited proteolysis, the hormones displayed proteolytic fragmentation patterns that were distinct from one another. The native heterodimeric hCG was far more resistant to proteinase than the fused analogs, with only the ß-subunit showing limited sensitivity. YhCG1 was more sensitive to proteolysis than YhCG3, but both yoked analogs yielded stable products consisting of the {alpha}-subunits or a major portion thereof, as well as portions of both the subunits. These results suggest that global conformational differences may be occurring in the single-chain glycoprotein hormones. Interestingly, conformational differences occur not only between hormones that display altered bioactivities to native hCG (YhCG3), but also between proteins with similar bioactivity to hCG (YhCG1). Thus, it is apparent that LHR can productively recognize and respond to multiple protein conformers.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, we used two previously characterized yoked hCGs (YhCG1 and YhCG3) to assess the possible differences in global conformation of hormones, since differences in bioactivity have been noted between the two and between each and hCG (17, 18, 19, 20, 21). The two proteins differ in the linear arrangement of the subunits (Fig. 1Go). YhCG1 contains the N-ß-{alpha}-C configuration, with the ß-subunit’s CTP fulfilling the role of flexible linker between the two subunits; YhCG3 contains the reverse configuration, N-{alpha}-CTP-ß-C, with an additional CTP inserted after {alpha} to allow for more conformational freedom for hormone folding (19). Both hormones have a Flag tag (DYKDDDDK) on their C termini for purification purposes. The tag did not affect the binding and signaling activity of the crude YhCG1 and YhCG3 (data not shown). YhCG1 (17) and other similar constructs retain native-like in vitro and in vivo (18) activity, whereas YhCG3 (19) and similarly conceived fusion hCGs (20, 21) display significant reduction in binding affinity while retaining the ability to transduce signal effectively.



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Figure 1. Schematic Representations of Native hCG, YhCG1, and YhCG3

The C terminus of the ß-subunit is fused to the N terminus of the {alpha}-subunit in YhCG1 as previously described (17 ). The flexible 30-amino-acid residue CTP of the ß-subunit was used as a endogenous linker. YhCG3 consists of the C terminus of the {alpha}-subunit fused through a CTP to the N terminus of the ß-subunit. Thus, YhCG3 contains two CTPs (19 ).

 
Both YhCG1 and YhCG3 were purified to a high level using immunoaffinity chromatography against the Flag tags (Fig. 2Go, A and B). YhCG3, with its additional CTP and four additional O-glycosylation sites, migrates at a significantly higher apparent molecular mass (43 kDa) than YhCG1 (36 kDa). Silver-stained gels display relatively homogeneous samples that were routinely achieved using the described purification protocol.



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Figure 2. Purification of YhCG1 and YhCG3

Immunoaffinity chromatography was used to purify YhCG1 (A) and YhCG3 (B). Purity was assessed by SDS-PAGE and silver staining of 50–100 ng protein. Molecular mass standards are indicated.

 
Heterodimeric hCG was analyzed for its purity using SDS-PAGE and silver staining (Fig. 3Go). hCG that was not boiled or reduced retained its heterodimeric configuration (Fig. 3AGo). Silver staining, which can detect as little as 1 ng of protein, shows the high level of purity of the sample. A minor band was apparent at a molecular mass lower than that of the intact dimer that is too large to be a nicked or ß-core fragment (27). Boiling the hCG sample results in the dissociation of the subunits into two bands with approximate molecular masses of 20 kDa and 40 kDa, representing the {alpha}- and ß-subunits, respectively, as confirmed by Western blot analysis (Fig. 3BGo). The {alpha}-subunit stains with a lower intensity than the ß-subunit under the experimental conditions used. The {alpha}-subunit may absorb less silver than ß.



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Figure 3. Characterization of Heterodimeric hCG

A, Protein (50–100 ng) was analyzed by SDS-PAGE under nonreducing conditions (with and without boiling) and silver staining. B, Western blot analysis of 5–10 ng of purified hCG using antibodies specific to either the {alpha}-subunit (left panel) or the ß-subunit (right panel). The sizes of the molecular mass standards (kDa) are indicated.

 
The in vitro bioactivities of the purified hormones were assessed using standard competitive binding and cAMP assays. The three hormones displayed similar cAMP dose-response profiles (Fig. 4AGo), and the EC50s of the three hormones were in relatively good agreement with those originally reported (19). Although the potencies of YhCG1 and YhCG3 were less than that of hCG (2-fold and 6-fold, respectively), the maximum cAMP values at saturating hormone levels were similar (Fig. 4AGo and Table 1Go). In competitive binding assays, the IC50 of YhCG1and YhCG 3 were 4-fold and 7-fold greater than that of native hCG, respectively (Fig. 4BGo and Table 1Go). Differences in absolute values from those previously reported (19, 20, 21) can be attributed to the variation when assaying crude and purified proteins with disparate RIAs. Nonetheless, the same trend is apparent, with YhCG1 displaying more native-like binding activity than YhCG3.



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Figure 4. In Vitro Biological Activity of Heterodimeric hCG and Purified Single-Chain Analogs YhCG1 and YhCG3

A, Cells stably expressing LHR were stimulated with increasing amounts of hormone and assayed for the production of cAMP (picomoles/milliliter). B, Hormones were assayed for their ability to compete with 125I-hCG for binding sites on cells stably expressing LHR. The data shown represent mean ± SEM of three experiments, each performed in duplicate.

 

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Table 1. Summary of in Vitro Bioactivities of Purified Heterodimer and Yoked hCGs

 
CD spectroscopy is highly sensitive to conformation, particularly the secondary structure of proteins given the spectral properties of the peptide bond in the far-UV (170–250 nm; Ref. 28). Accordingly, we have used CD spectra to compare the overall structures of the three hormones. Interestingly, each of the hormones displays a unique CD spectrum (Fig. 5Go), indicating differences in their solution conformations. Heterodimeric hCG exhibits two prominent (negative) CD bands at 207 nm and 196 nm, in agreement with published spectra (29, 30). The CD spectra for YhCG1 and YhCG3 also exhibit a similar negative band at 207 nm, but there are significant differences between the two fusion proteins, and each differs from that of hCG. The mean residue ellipticity of YhCG1 is slightly (but reproducibly) more negative above 219 nm and significantly more positive below 219 nm. YhCG3 displays less negative ellipticity than hCG and YhCG1 below 225 nm and exhibits two shoulders at 230 nm and 220 nm. These spectral differences are consistent with immunogenic data published on a similar single-chain analog, where the N-{alpha}-CTP-ß-C orientation displayed a dramatic reduction in immunoreactivity to antibodies recognizing the individual subunits (21). Taken together, these results suggest a loss of the epitopes and reduction in secondary structure when the subunits are tethered in this configuration.



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Figure 5. Far UV CD Spectroscopy of hCG, YhCG1, and YhCG3

The CD spectra for 5–10 µM purified hormone samples were recorded using a Jasco 710 CD spectrometer. Each spectrum represents the mean ± SEM of at least two separate experiments, of five scans each, with different protein preparations. Data are presented as [{theta}], mean residue ellipticity vs. wavelength.

 
Secondary structure estimates can be extracted using a variety of algorithms, with {alpha}-helical values being by far the most accurate (31). Three commonly employed methods were used to obtain the values in Table 2Go (32, 33, 34). These methods are based upon relatively large reference sets of standard proteins and are capable of detecting low levels of helicity and ß-structure. An earlier technique, based on curve resolution into Gaussian bands (29), did not detect the low level of {alpha}-helicity in hCG. YhCG1 shows a slight increase in helix content over native hCG, whereas helical estimates for YhCG3 are less than those obtained in hCG and YhCG1. Correcting for the differences in the number of amino acid residues in hCG, YhCG1, and YhCG3, the average percent {alpha}-helix of each from Table 2Go yields 11–12, 16–17, and 9–10 helical residues, respectively. That for hCG is an overestimate based on the crystal structure (4, 5), which gives five helical residues. This discrepancy may arise from the inability of the existing programs to correct for glycosylation, aromatic amino acid residues, and disulfides, all which can make minor contributions to the far-UV CD spectrum of hCG (29).


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Table 2. Secondary Structure Analysis of the CD Spectra of hCG and Fused Analogs, YhCG1 and YhCG3

 
To further test the conformational differences suggested in the CD experiments, limited proteolysis experiments were performed with the proteins. Proteases are sensitive probes of conformation, particularly tertiary structure, as cleavage depends not only on the amino acid sequence of the protein, but also on the accessibility of the cleavage site. The general specificity protease, proteinase K (aliphatic or aromatic at the P2 site and Ala at the P1 site), was chosen based upon the presence of approximately 90 potential cleavage sites throughout the entire sequences of the hormones. Thus, subtle variations in conformational states should be discernable with this enzyme.

A combination of silver-stained gels (Fig. 6Go) and Western analyses, using anti-{alpha} (Fig. 7Go) and anti-ß (Fig. 8Go) polyclonal antibodies, showed dramatic differences in the susceptibility of the single-chain analogs, compared with heterodimeric hCG, to proteinase K. For these studies, obtained under nonreducing conditions, different standards were used in the gels that were silver stained and those for Western analysis; thus, there are variations in apparent molecular masses of the resulting fragments. The ranges obtained for {alpha}, ß, YhCG1, and YhCG3 are 18–22, 36–38, 36–42, and 43–53 kDa, respectively. Moreover, several of the average bands resulting from proteinase K digestion are composed of closely spaced discrete bands, perhaps reflecting heterogeneity in carbohydrate components, N and C termini, and internal peptide bond cleavages resulting in peptides held together by disulfide bonds. Small peptides and amino acids, of course, would not be detected in the systems used. Densitometric scans were made and analyzed of all the gels, but are shown only for the silver-stained gels.



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Figure 6. SDS-PAGE and Silver Staining of Limited Proteolysis (Proteinase K) Reactions with hCG, YhCG1, and YhCG3

Hormones were digested with a 1:100 enzyme-substrate (wt/wt) ratio for various times at room temperature. The reaction was stopped by the addition of sample buffer and boiling. Samples were analyzed by gel electrophoresis (15% acrylamide) and visualized by silver staining. Proteolytic fragments are labeled with molecular mass estimates (kDa) based upon the migration of standards. Reactions containing hormone only (0) or protease only (not shown) were incubated at room temperature for the entire 80 min. Densitometric scans of the major bands are shown in the panels to the right. For these, the number of pixels was normalized to the bands at t = 0 for {alpha}, ß, YhCG1, and YhCG3 and t = 80 for proteolytic products.

 


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Figure 7. Proteolysis of the {alpha}-Subunit of hCG, YhCG1, and YhCG3

Proteinase K was used to digest the hormones for the various times listed. The reactions were subjected to electrophoresis in 15% polyacrylamide gels, and fragments specific to the {alpha}-subunit were detected using Western blot analysis. Reactions containing only hormone (0) and only protease (P) were incubated at room temperature for the entire 80 min. Molecular mass estimates of the products were made based upon the migration of standards.

 


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Figure 8. Proteolysis of the {alpha}-Subunit by Proteinase K in the Hormones hCG, YhCG1, and YhCG3

Hormones were subjected to proteolysis for the listed times and loaded onto 15% polyacrylamide gels for Western blot analysis with a ß-subunit-specific antiserum. Estimates of the molecular mass (kDa) of proteolytic products were made based on the migration of standards. Reactions containing only hormone (0) or only protease (P) were incubated at room temperature for the entire 80 min.

 
In heterodimeric hCG, the {alpha}-subunit is remarkably stable to proteolysis (Figs. 6Go and 7Go), while the ß-subunit is cleaved to one major (25–29 kDa) and one minor (21 kDa) product (Figs. 6Go and 8Go) with an apparent t1/2 of about 25–30 min. The minor product of ß comigrates with that of the {alpha}-subunit (Fig. 6Go).

YhCG1 and YhCG3 are rapidly cleaved by proteinase K with an apparent t1/2 of about 2–4 min (Figs. 6–8GoGoGo). The fragmentation patterns, however, are distinct. YhCG3 first undergoes a cleavage(s) to yield a 47- to 48-kDa band intermediate that gives rise to the resulting products ({alpha} epitope-containing bands at 16–18 and 24–26 kDa and ß-epitope-containing bands at 21 and 26 kDa) with an apparent t1/2 of about 15 min. The early cleavage may result from removal of a portion of the C-terminal CTP. In contrast to YhCG3, YhCG1 is converted to a major {alpha}-band at 19 kDa (Fig. 7Go) and ß-epitope-containing bands at 16 and 19 kDa (Fig. 8Go), perhaps attributable to cleavage at the internal CTP. To obtain these results with YhCG1, it was necessary to increase the exposure time during development of the Westerns, indicating the presence of only a small fraction of undigested and partially digested protein. The silver-stained gels suggest that YhCG1 may be degraded to small peptides and amino acids more extensively than YhCG3 (Fig. 6Go), and the various gels suggest that the N-terminal subunit is more extensively degraded than the C-terminal subunit.

Of note, samples incubated for the entirety of the experiment with no proteinase K displayed no endogenous proteinase activity (0 lanes), and reducing gels showed that there were no chain cleavages in hCG and the fusion proteins (data not shown). We also performed Western analysis under reducing conditions, but epitopes were evidently destroyed (data not shown). Since all of the proteins are glycosylated at potentially the same positions, it is not expected that minor differences in glycosyl complexity will result in the marked differences in proteinase K susceptibility observed here.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The data herein provide the first direct structural measurements comparing conformations of hCG and the single-chain proteins, YhCG1 and YhCG3. The CD spectra reveal conformational differences in the proteins studied. Limited proteolysis experiments confirmed the results obtained by CD spectroscopy and indicate that the N-terminal subunit in the single-chain hormones (e.g. the {alpha}-subunit in YhCG3 and the ß-subunit in YhCG1) may exhibit an increased sensitivity to proteinase K, thus suggesting a detrimental effect on folding of the subunit that is translated first in the single chain. The results obtained add significantly to our understanding of the structural consequences of translating the {alpha}- and ß-subunits in a single polypeptide chain and their ultimate folding into bioactive hormones.

The glycoprotein hormones, LH, CG, FSH, and TSH, comprise a family of proteins the biological potency of which is governed by their ability to form heterodimeric structures intracellularly before secretion and release into circulation. The biologically active proteins consist of a common {alpha}-subunit associated noncovalently with a hormone-specific ß-subunit. The crystal structures of hCG (4, 5) and hFSH (7) have provided considerable insight into the structure-function relationships of this family of cystine knot-containing hormones. Indeed, the growth factor-like cystine knot is fundamental to the structural integrity of the hormones by acting as a molecular scaffold on which the rest of the molecule is defined and thus was suggested to be integral to the proper presentation of hormone to receptor (1).

With the advent of single-chain glycoproteins (17, 18), the importance of the cystine knot to the biological activity of the hormones could be addressed, since the obligatory dimerization step in hormone secretion was bypassed. Indeed, several studies with single-chain hCG (22, 23) and hFSH (35) have now ascribed the primary function of the cystine knot to intracellular behavior and not to productive in vitro bioactivity. First generation yoked hormones were constructed as N-ß-{alpha}-C so as to utilize the CTP of ß as a linker and to retain the free C terminus of {alpha}, which is reported to be important in receptor binding and activation (10, 11, 12, 13). YhCG1 was able to bind to and activate LHR with properties similar to those of the native heterodimer. More recently, the reverse configuration (N-{alpha}-CTP-ß-C; YhCG3) was analyzed by our laboratory and others (19, 20, 21). These hormones bound receptor with lower affinity but retained the ability to transduce signal effectively. Moreover, an N-{alpha}-ß-C construct, lacking the CTP linker, was able to form heterodimeric-like immunoreactivity but was completely devoid of bioactivity (21). Thus, dimeric determinants alone do not satisfy the requirements for hormone activity. This point is supported by immunological evidence, where single-chain mutant hormones, which lack native-like heterodimeric structure, can still bind and activate LHR with wild-type-like activity (26). Therefore, it is becoming more apparent that distinct conformers of glycoprotein hormones can bind to and activate their receptor. Importantly, however, these provocative studies have not unveiled the extent or the nature of the purported conformational differences. To that end, we generated and purified significant quantities of YhCG1 and YhCG3 for direct structural comparisons with each other and with native hCG. Using the complementary and sensitive techniques, CD and limited proteolysis, several important characteristics of these hormones were gleaned.

First, both CD and limited proteolysis experiments confirmed that hCG, YhCG1, and YhCG3 represent distinct conformational entities. Although it is expected that the structure of YhCG3 may differ from that of YhCG1 and hCG, the marked contrasting properties of hCG and YhCG1 are notable. Second, the yoked CGs were more sensitive to proteinase K than dimeric hCG, perhaps suggesting a more compact structure in the native heterodimer. Third, the N-terminal subunit in the yoked CGs may display a decreased resistance to proteinase K, indicating a more open and flexible structure. Thus, the order of subunit connectivity is important to the folding of the subunits. The relevance of these general observations is discussed below with the individual constructs.

The CD spectrum for YhCG1 suggests that it may contain more helix than native hormone, whereas the limited proteolysis experiments for YhCG1 implicate a much less stable overall structure than hCG with a shortened proteolytic half-life and the lack of distinguishable (silver stain) products. These apparent contradictory properties are supported by evidence that linear (reduced, S-carboxymethylated) free ß-subunit and ß-derived peptides have significant helical propensities (36). Indeed, it is not known whether the appropriate disulfide bonding pattern is formed in YhCG1. However, it is conceivable that, given the inherent complexity of the disulfide bonding network in the ß-subunit alone, tethering the {alpha}-subunit to the C terminus may promote mispairing and alter the natural folding pathway for ß. Indeed, nonnative disulfide bonds have been reported to form during the folding of ß (6), and these may be kinetically trapped when the {alpha}-subunit is translated proximally. Increased aggregation has also been noted with the single-chain hCGs (21). Thus, with a configuration of N-ß-{alpha}-C, the ß-subunit may be destabilized by mispairing of disulfides promoting minor helical formation in the localized regions. Consequently, the {alpha}-subunit can fold effectively; hence, its stability as determined by Western analysis. The stable {alpha}-subunit can partially dimerize with the ß-subunit and form an effective, native-like agonist. This hypothesis is supported by the loss of some, but not all, of the heterodimeric epitopes in the N-ß-{alpha}-C configuration (26).

The YhCG3 CD spectrum displays a notable reduction in ellipticity, which extracts into a slight reduction in estimated secondary structure (helicity), although it is not significantly different from that of hCG. Also, the limited proteolysis noted in Western analyses shows that the resistance of the {alpha}-subunit to proteinase observed in native hCG and YhCG1 is diminished in YhCG3. Together, these data support the idea that the {alpha}-subunit, when tethered in N-{alpha}-CTP-ß-C context, may be unable to achieve its native fold. This change in structure would presumably affect association with ß. Thus, the {alpha}-subunit may exist in a quasi-free state. Certainly, this would explain the reduction in the intensity of negative ellipticity and the loss of immunoreactivity to {alpha}-subunit-specific antibodies (21), as the long loop of {alpha} would lack a defined structure (37), thus increasing this region’s susceptibility to proteolysis. Furthermore, residues {alpha}33–51, which are important to hCG binding to receptor (38, 39, 40, 41), are also located in this region; therefore, the arrangement of these residues would likely be altered, which, in turn, is manifested in the observed reduction in binding affinity.

A unique feature of the quaternary structure of hCG and hFSH is the seatbelt of the ß-subunit that wraps around the long loop of the {alpha}-subunit. In heterodimeric hCG, it has been proposed that the two subunits associate during biosynthesis with the closure of the seatbelt, i.e. formation of the ß-Cys-26-Cys-110 disulfide, occurring toward the latter stages of folding and assembly (6). Even so, functional holoprotein formation of hCG can also occur between subunits in which the ß-Cys-26-Cys-110 disulfide bond is intact (42). These and other results led Moyle and co-workers (42) to suggest that the seatbelt in free hCGß exists in a conformation that differs from that in the heterodimer. In their model, subunit association occurs with the long loop of {alpha} and the seatbelt of ß moving relative to each other to give the stable arrangement in the heterodimer. We propose that the major conformational differences between heterodimeric hCG, {alpha}-ß, and ß-{alpha} involve the {alpha}-long loop and the ß-seatbelt.

Single-chain glycoprotein hormones have become invaluable tools for the study of structure-function relationships. The experiments presented herein add significantly to our understanding of the effects of single-chain translation on the solution conformations of the heterodimeric glycoprotein hormones. Through direct structural data, it is evident that single-chain hCGs possess altered conformations that are not related to their in vitro bioactivities. The order in which the subunits are tethered affects the CD spectra of the hormones and the overall stabilities of the subunits. Furthermore, the marked differences observed in using both CD and limited proteolysis suggest that single-chain translation produces global conformational variation, in so much as the differences between native hCG and yoked hCGs are not localized to a single region. The ability of LHR to recognize multiple conformations suggests that the major evolutionary driving force for the heterodimeric structure of glycoprotein hormones may be secretory control and not hormone selectivity at the receptor interface.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning and Expression of YhCG1 and YhCG3
YhCG1 and YhCG3 were cloned as previously described (17, 19). A Flag tag (DYKDDDDK) was added to the 3'-end of both hormones using PCR and the 3'-primers,YhCG1:5'-TTACTTATCGTCATCGTCCTTGTAGTCAGATTTGTGATAATAAAC-3' and YhCG3:5'-GGCCTCGAGTTACTTATCGTCATCGTCCTTGTAGTCTTCTGGGAGGATCG-3'. Both hormones were cloned into the baculovirus transfer vector pVL1392/3 (PharMingen, San Diego, CA), which utilizes the polH promoter for expression very late in the baculovirus infection cycle. Of note, both proteins retained the signal sequences from the original 5'-proximal subunit. After cotransfection of the transfer vector and linearized baculovirus DNA (Autographa californica Nuclear Polyhedrosis Virus, AcNPV) Baculogold (PharMingen) into SF9 insect cells, medium from the cells was used for two more serial infections for amplification of the recombinant virus. High-titer (107–108 plaque forming units/ml) recombinant virus was used for expression in suspension-cultured Sf9 cells. A multiplicity of infection of 0.1 was used, and the cell medium was assayed 4 d post infection. An immunoradiometric assay (IRMA; Diagnostic Products, Los Angeles, CA) was used to quantify expression levels.

Protein Purification
Media were harvested from suspension cultures and clarified with two sequential centrifugation steps of 1,400 x g and 17,700 x g, respectively. Clarified expression medium was stirred vigorously at 4 C while ammonium sulfate was gradually added to 80% saturation followed by stirring overnight at 4 C. The protein pellet was harvested from the medium with two high-speed spins (17,700 x g), and the pellet was resuspended in TBS (50 mM Tris-HCl, pH 7.5; 150 mM NaCl). The resuspended pellet was then dialyzed extensively against TBS and again clarified with high-speed centrifugation (17,700 x g). The dialyzed and clarified protein sample was applied to an immunoaffinity resin containing the M2 Anti-Flag monoclonal antibody conjugated to agarose beads. Efficient binding of the proteins was achieved by cycling the sample over the column overnight at 4 C, after which the column was washed extensively with TBS (500–1000 ml, for a 1-ml column). The proteins were eluted with a 10–15 column volume wash with 0.1 M glycine, pH 3.5. The eluant was immediately neutralized via collection of fractions in microcentrifuge tubes preloaded with 1 M Tris-HCl, pH 8.0. The fractions were assayed using IRMA, and those containing hormone were pooled and concentrated as needed. Highly purified heterodimeric hCG, purified from pregnancy urine, was obtained from Dr. A. F. Parlow and the National Institute of Diabetes and Digestive and Kidney Diseases.

SDS-PAGE and Western Analysis
Protein samples were resolved with nonreducing 15% polyacrylamide gels and visualized by silver staining (50–100 ng protein sample loaded) and Western analysis (5–10 ng protein sample loaded) as previously described (43). Briefly, gels were fixed and transferred to polyvinylidine difluoride (Millipore Corp., Bedford, MA) using a tank transfer apparatus. The membrane was blocked (3% BSA, 0.2% Tween 20, TBS) and incubated with either a 1:5000 dilution of the {alpha}3 antiserum (rabbit polyclonal, kindly provided by Dr. Irving Boime, Washington University, St. Louis, MO), which is specific for the {alpha}-subunit of hCG and a 1:10,000 dilution of rabbit antiserum specific for the ß-subunit of hCG (also obtained from Dr. Irving Boime). The membrane was then washed and incubated with a donkey antirabbit IgG conjugated with horseradish peroxidase. After extensive washing of membrane, the protein was treated with enzyme-linked chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ) with visualization achieved by exposure of the membranes to film. The silver-stained gels and the gels used for Western analysis were densitometrically scanned.

In Vitro Bioassays
Characterization by the binding and cAMP induction patterns of the different hormones was achieved using methods previously described (19). All purified hormones were quantified using IRMA (Diagnostic Products) for the bioassays. Competitive binding assays were performed in 12-well tissue culture plates with HEK 293 cells stably expressing LHR. Cells were incubated with a fixed amount of 125I-hCG (100 pM) and increasing amounts of unlabeled hCG overnight at room temperature in Waymouth’s MB medium containing 0.1% BSA. Cells were washed twice with 1 N NaOH. The washes were collected and radioactivity measured using a {gamma}-counter. For cAMP induction, stably transfected LHR cells were stimulated in 12-well tissue culture plates for 30 min with increasing amounts of hormone in Waymouth’s MB medium containing 0.1% BSA and 0.8 mM of the phosphodiesterase inhibitor, isobutylmethylxanthine. The medium was removed, and the cells in each well were lysed with 1 ml of ethanol, followed by incubation overnight at -20 C. The ethanol was collected, centrifuged, dried, and resuspended in cAMP buffer that is compatible with the RIA used subsequently to quantify cAMP amounts (Perkin-Elmer Corp., Norwalk, CT).

Circular Dichroism
Protein concentrations were determined by UV absorption with the extinction coefficients for the proteins being estimated at 280 nm from the primary sequences assuming completely oxidized cystines (44). Purified protein samples were diluted to 10 µM and dialyzed against 5 mM phosphate buffer, pH 6.8. The dialyzed samples were filtered with a 0.1-µm filter and loaded into a 1-mm path length cell. The CD spectra were measured in the far UV at wavelengths between 250–190 nm using a Jasco 710 CD spectrometer (Jasco, Inc., Easton, MD). The measurements were obtained with the following spectrometer settings: band width, 1 nM; sensitivity, 50 millidegrees; response, 2 sec; scan speed, 20 nm/min; step resolution, 0.2 nm; starting wavelength, 250 nm; lowest wavelength, 190 nm; and five scans per sample. The data are presented as the mean ± SEM of three repeated measurements of five scans each with different protein preparations.

Limited Proteolysis
Proteinase K was added to purified hormone that was diluted to 10 µM in TBS (pH 7.5), 5 mM CaCl2 for a final enzyme-substrate ratio of 1:100 (wt/wt). All reactions were stopped simultaneously by the addition of 40% sodium dodecyl sulfate sample buffer, and the samples were boiled for 5 min and immediately resolved by 15% SDS-PAGE as described above. Nonspecific proteolysis in the hormone preparations and the proteinase K samples was analyzed by the incubation of samples containing hormone only and proteinase only for the longest digestion period. All of the samples were analyzed by Western analysis and silver staining as described above.


    ACKNOWLEDGMENTS
 
We thank Dr. Irving Boime for his generous gift of the {alpha}- and ß-subunit specific antisera and Ms. Judy Gray for excellent technical assistance.


    FOOTNOTES
 
This work was supported by NIH Research Grant DK-33973.

Abbreviations: CD, Circular dichroism; CG, chorionic gonadotropin; CTP, C-terminal peptide; hCG, human CG; IRMA, immunoradiometric assay; LHR, LH receptor; Y, yoked.

Received for publication September 10, 2002. Accepted for publication January 3, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Hearn MT, Gomme PT 2000 Molecular architecture and biorecognition processes of the cystine knot protein superfamily. I. The glycoprotein hormones. J Mol Recognit 13:223–278[CrossRef][Medline]
  2. Matzuk MM, Hsueh AJ, Lapolt P, Tsafriri A, Keene JL, Boime I 1990 The biological role of the carboxyl-terminal extension of human chorionic gonadotropin ß-subunit [published erratum appears in Endocrinology 1990, 126:2204]. Endocrinology 126:376–383
  3. Xie YB, Wang H, Segaloff DL 1990 Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity. J Biol Chem 265:21411–21414[Abstract/Free Full Text]
  4. Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW 1994 Crystal structure of human chorionic gonadotropin. Nature 369:455–461[CrossRef][Medline]
  5. Wu H, Lustbader JW, Liu Y, Canfield RE, Hendrickson WA 1994 Structure of human chorionic gonadotropin at 2.6 A resolution from MAD analysis of the selenomethionyl protein. Structure 2:545–558[Medline]
  6. Ruddon RW, Sherman SA, Bedows E 1996 Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin ß subunit. Protein Sci 5:1443–1452[Abstract/Free Full Text]
  7. Fox KM, Dias JA, Van Roey P 2001 Three-dimensional structure of human follicle-stimulating hormone. Mol Endocrinol 15:378–389[Abstract/Free Full Text]
  8. Narayan P, Gray J, Puett D 2002 Yoked complexes of human choriogonadotropin and the lutropin receptor: evidence that monomeric individual subunits are inactive. Mol Endocrinol 16:2733–2745[Abstract/Free Full Text]
  9. Grossmann M, Szkudlinski MW, Wong R, Dias JA, Ji TH, Weintraub BD 1997 Substitution of the seat-belt region of the thyroid-stimulating hormone (TSH) ß-subunit with the corresponding regions of choriogonadotropin or follitropin confers luteotropic but not follitropic activity to chimeric TSH. J Biol Chem 272:15532–15540[Abstract/Free Full Text]
  10. Kundu GC, Ji I, McCormick DJ, Ji TH 1996 Photoaffinity labeling of the lutropin receptor with synthetic peptide for carboxyl terminus of the human choriogonadotropin {alpha} subunit. J Biol Chem 271:11063–11066[Abstract/Free Full Text]
  11. Chen F, Wang Y, Puett D 1992 The carboxy-terminal region of the glycoprotein hormone {alpha}-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin. Mol Endocrinol 6:914–919[Abstract]
  12. Yoo J, Zeng H, Ji I, Murdoch WJ, Ji TH 1993 COOH-terminal amino acids of the {alpha} subunit play common and different roles in human choriogonadotropin and follitropin. J Biol Chem 268:13034–13042[Abstract/Free Full Text]
  13. Furuhashi M, Shikone T, Fares FA, Sugahara T, Hsueh AJW, Boime I 1995 Fusing the carboxy-terminal peptide of the chorionic gonadotropin (CG) ß-subunit to the common {alpha}-subunit: retention of O-linked glycosylation and enhanced in vivo bioactivity of chimeric human CG. Mol Endocrinol 9:54–63[Abstract]
  14. Jiang X, Dreano M, Buckler DR, Cheng S, Ythier A, Wu H, Hendrickson WA, El Tayar N 1995 Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone-receptor interactions. Structure 3:1341–1353[Medline]
  15. Bhowmick N, Huang J, Puett D, Isaacs NW, Lapthorn AJ 1996 Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor by site-directed mutagenesis and modeling. Mol Endocrinol 10:1147–1159[Abstract]
  16. Cosowsky L, Rao SN, Macdonald GJ, Papkoff H, Campbell RK, Moyle WR 1995 The groove between the {alpha}- and ß-subunits of hormones with lutropin (LH) activity appears to contact the LH receptor, and its conformation is changed during hormone binding. J Biol Chem 270:20011–20019[Abstract/Free Full Text]
  17. Narayan P, Wu C, Puett D 1995 Functional expression of yoked human chorionic gonadotropin in baculovirus-infected insect cells. Mol Endocrinol 9:1720–1726[Abstract]
  18. Sugahara T, Pixley MR, Minami S, Perlas E, Ben-Menahem D, Hsueh AJ, Boime I 1995 Biosynthesis of a biologically active single peptide chain containing the human common {alpha} and chorionic gonadotropin ß subunits in tandem. Proc Natl Acad Sci USA 92:2041–2045[Abstract]
  19. Narayan P, Gray J, Puett D 2000 A biologically active single chain human chorionic gonadotropin analog with altered receptor binding properties. Endocrinology 141:67–71[Abstract/Free Full Text]
  20. Sen Gupta C, Dighe RR 2000 Biological activity of single chain chorionic gonadotropin, hCG{alpha}ß, is decreased upon deletion of five carboxyl terminal amino acids of the {alpha} subunit without affecting its receptor binding. J Mol Endocrinol 24:157–164[Abstract/Free Full Text]
  21. Ben-Menahem D, Jablonka-Shariff A, Hyde RK, Pixley MR, Srivastava S, Berger P, Boime I 2001 The position of the {alpha} and ß subunits in a single chain variant of human chorionic gonadotropin affects the heterodimeric interaction of the subunits and receptor-binding epitopes. J Biol Chem 276:29871–29879[Abstract/Free Full Text]
  22. Ben-Menahem D, Kudo M, Pixley MR, Sato A, Suganuma N, Perlas E, Hsueh AJ, Boime I 1997 The biologic action of single-chain choriogonadotropin is not dependent on the individual disulfide bonds of the ß subunit. J Biol Chem 272:6827–6830[Abstract/Free Full Text]
  23. Sato A, Perlas E, Ben-Menahem D, Kudo M, Pixley MR, Furuhashi M, Hsueh AJ, Boime I 1997 Cystine knot of the gonadotropin {alpha} subunit is critical for intracellular behavior but not for in vitro biological activity. J Biol Chem 272:18098–18103[Abstract/Free Full Text]
  24. Ben-Menahem D, Hyde R, Pixley M, Berger P, Boime I 1999 Synthesis of multi-subunit domain gonadotropin complexes: a model for {alpha}/ß heterodimer formation. Biochemistry 38:15070–15077[CrossRef][Medline]
  25. Kanda M, Jablonka-Shariff A, Sato A, Pixley MR, Bos E, Hiro’oka T, Ben-Menahem D, Boime I 1999 Genetic fusion of an {alpha}-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-ß subunit genes: production of a bifunctional protein. Mol Endocrinol 13:1873–1881[Abstract/Free Full Text]
  26. Jackson AM, Berger P, Pixley M, Klein C, Hsueh AJ, Boime I 1999 The biological action of choriogonadotropin is not dependent on the complete native quaternary interactions between the subunits. Mol Endocrinol 13:2175–2188[Abstract/Free Full Text]
  27. Birken S, Maydelman Y, Gawinowicz MA 2000 Preparation and analysis of the common urinary forms of human chorionic gonadotropin. Methods 21:3–14[CrossRef][Medline]
  28. Woody RW 1995 Circular dichroism. Methods Enzymol 246:34–71[Medline]
  29. Holladay LA, Puett D 1975 Gonadotropin and subunit conformation. Arch Biochem Biophys 171:708–720[Medline]
  30. Erbel PJ, Haseley SR, Kamerling JP, Vliegenthart JF 2002 Studies on the relevance of the glycan at Asn-52 of the {alpha}-subunit of human chorionic gonadotropin in the {alpha}ß dimer. Biochem J 364:485–495[CrossRef][Medline]
  31. Greenfield NJ 1996 Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Anal Biochem 235:1–10[CrossRef][Medline]
  32. Provencher SW, Glockner J 1981 Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20:33–37[Medline]
  33. Chen YH, Yang JT, Chau KH 1974 Determination of the helix and ß form of proteins in aqueous solution by circular dichroism. Biochemistry 13:3350–3359[Medline]
  34. Johnson WC 1999 Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins 35:307–312[CrossRef][Medline]
  35. Hiro’oka T, Maassen D, Berger P, Boime I 2000 Disulfide bond mutations in follicle-stimulating hormone result in uncoupling of biological activity from intracellular behavior. Endocrinology 141:4751–4756[Abstract/Free Full Text]
  36. Puett D, Birken S 1989 Helix formation in reduced, S-carboxymethylated human choriogonadotropin ß subunit and tryptic peptides. J Protein Chem 8:779–794[Medline]
  37. Erbel PJ, Karimi-Nejad Y, De Beer T, Boelens R, Kamerling JP, Vliegenthart JF 1999 Solution structure of the {alpha}-subunit of human chorionic gonadotropin. Eur J Biochem 260:490–498[Abstract/Free Full Text]
  38. Reed DK, Ryan RJ, McCormick DJ 1991 Residues in the {alpha} subunit of human choriotropin that are important for interaction with the lutropin receptor. J Biol Chem 266:14251–14255[Abstract/Free Full Text]
  39. Bielinska M, Boime I 1992 Site-directed mutagenesis defines a domain in the gonadotropin {alpha}-subunit required for assembly with the chorionic gonadotropin ß-subunit. Mol Endocrinol 6:261–271[Abstract]
  40. Liu C, Roth KE, Shepard BA, Shaffer JB, Dias JA 1993 Site-directed alanine mutagenesis of Phe33, Arg35, and Arg42-Ser43-Lys44 in the human gonadotropin {alpha}-subunit. J Biol Chem 268:21613–21617[Abstract/Free Full Text]
  41. Xia H, Chen F, Puett D 1994 A region in the human glycoprotein hormone {alpha}-subunit important in holoprotein formation and receptor binding. Endocrinology 134:1768–1770[Abstract]
  42. Xing Y, Williams C, Campbell RK, Cook S, Knoppers M, Addona T, Altarocca V, Moyle WR 2001 Threading of a glycosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin subunits. Protein Sci 10:226–235[Abstract/Free Full Text]
  43. Fralish GB, Narayan P, Puett D 2001 High-level expression of a functional single-chain human chorionic gonadotropin-luteinizing hormone receptor ectodomain complex in insect cells. Endocrinology 142:1517–1524[Abstract/Free Full Text]
  44. Gill SC, von Hippel PH 1989 Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326[Medline]