The Biological Action of Choriogonadotropin Is Not Dependent on the Complete Native Quaternary Interactions between the Subunits

Alison M. Jackson, Peter Berger, Mary Pixley, Cynthia Klein, Aaron J. W. Hsueh and Irving Boime

Department of Molecular Biology and Pharmacology (A.M.J., M.P., I.B.) Washington University School of Medicine St Louis, Missouri 63110
Institute for Biomedical Aging Research (P.B.) Austrian Academy of Sciences A-6020 Innsbruck, Austria
Division of Reproductive Biology (C.K., A.W.J.H.) Department of Gynecology/Obstetrics Stanford University Medical Center Stanford, California 94305


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human CG (hCG) is a member of the glycoprotein hormone family characterized by a heterodimeric structure consisting of a common {alpha}-subunit noncovalently bound to a hormone-specific ß-subunit. The two subunits are highly intertwined and only the heterodimer is functional, implying that the quaternary structure is critical for biological activity. To assess the dependence of the bioactivity of hCG on the heterodimeric interactions, {alpha}- and ß-subunits bearing mutations that prevent assembly were covalently linked to form a single chain hCG. Receptor binding and signal transduction of these analogs were tested and their structural integrity analyzed using a panel of monoclonal antibodies (mAbs). These included dimer-specific mAbs, which react with at least four different epitope sites on the hormone, and some that react only with the free ß-subunit. We showed that there was significant loss of quaternary and tertiary structure in several regions of the molecule. This was most pronounced in single chains that had one of the disulfide bonds of the cystine knot disrupted in either the {alpha}- or ß-subunit. Despite these structural changes, the in vitro receptor binding and signal transduction of the single chain analogs were comparable to those of the nonmutated single chain, demonstrating that not all of the quaternary configuration of the hormone is necessary for biological activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glycoprotein hormone family is representative of a class of heterodimeric proteins that are dependent on the formation of the heterodimer for full biological activity. They include the placentally derived human CG (hCG) and also pituitary-derived FSH, TSH, and LH (1). These heterodimers have a common {alpha}-subunit and a hormone-specific ß-subunit. The crystal structure of hCG (2, 3) showed that the two subunits are intimately associated with each other along much of their surfaces, each subunit having remarkably similar folds with two hairpin loops at one end and a single loop at the other. The two loops of one subunit are adjacent to the single loop of the other. At the core of the hCG {alpha}- and ß-subunits is a cystine knot motif previously observed in several growth factors (4). The carboxy-terminal region in the ß-subunit forms a ‘seat belt’ around the {alpha}-subunit that stabilizes the native heterodimer. This structural configuration, together with the heterodimeric requirement for receptor binding, implies that the complex quaternary structure of the gonadotropins is crucial for biological activity. Recently a number of observations using single-chain glycoprotein hormone variants, where the {alpha}- and ß-subunits are covalently linked, have suggested that the quaternary configuration is not a prerequisite for receptor binding. Single-chain mutants of hFSH and hCG displayed normal receptor binding and signal transduction, despite modeling that suggested that some of the {alpha}-ß subunit alignments seen in the crystal structure could not be maintained (5). For example, molecular modeling of tethers lacking a linker sequence reveal that the alignment of the {alpha}/ß domains in the single chains differ substantially from that seen in the heterodimer. Moreover, CG single-chain mutants bearing deleted disulfide bonds in the cystine knot of either the {alpha}- or ß-subunits were biologically active (6, 7). These data suggested that the extensive interactions holding the subunits together in the wild type was not essential for receptor recognition. However, the extent of the structural loss in these single-chain variants was not known, and thus a clear relationship between the heterodimeric configuration and biological activity could not be assessed from the earlier work.

Here we sought to directly examine the importance of the integrity of the quaternary structure for the biological function of hCG. We introduced mutations into subunit regions that are known to be critical for assembly and secretion of the heterodimers (8, 9, 10). Those mutations that prevented subunit association were then generated by site-directed mutagenesis in the {alpha}- or ß- domains of single-chain hCG. This approach permits analysis of receptor binding properties of residues critical for assembly that were previously not possible due to the inability of mutated subunits to form heterodimer. These analogs were examined for structural changes and activity on the assumption that if they are biologically active and the {alpha}/ß domains not associated in a native configuration, it would indicate that the precise wild-type conformation is not essential for maximal hormone function. The structure of these single-chain analogs, which include mutations in the {alpha}-helix of the {alpha}- subunit and disulfide bond mutations in the {alpha}- and ß-subunits, was assessed using a panel of conformationally sensitive monoclonal antibodies (mAbs). This panel included eight mAbs with specificity for only the intact heterodimer and two that react only with free ß-subunit (11, 12, 13). We show that the single-chain analogs were disrupted in their heterodimeric configuration and yet biologically active. These results are consistent with the hypothesis that the quaternary configuration of the {alpha}/ß heterodimer is critical for secretion and intracellular stability but not for receptor binding and signal transduction.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the {alpha}-Helix of the {alpha}-Subunit
Assembly
The region near the {alpha}-helix on the {alpha}-subunit (amino acids 38–42; Fig. 1Go) has been implicated in the assembly (reaction) (8, 10, 14, 15, 16, 17, 18). Previously we showed that substitution of Thr 39 with Phe ({alpha}T39F) or Ala ({alpha}T39A) eliminated or reduced {alpha} heterodimer formation (8). To examine the role of the adjacent residues, an additional triple mutant was constructed, {alpha}39TPL41 changed to {alpha}39AAA41. This mutant, together with {alpha}T39F or {alpha}T39A variants, was cotransfected with the hCGß gene in Chinese hamster ovary (CHO) cells, and several clones expressing either the same amount of subunits or excess ß were isolated (Fig. 2Go). Assembly was determined by immunoprecipitating labeled subunits in the media with either {alpha}- and ß-antisera that recognize noncombined subunits and the corresponding heterodimers. Because the level of ß-subunit synthesized is the same or in excess, the amount of {alpha}-subunit precipitated by both antisera should be the same if complete heterodimer formation has occurred (8). In the case of cells expressing the wild-type heterodimer, both the {alpha}- and ß-antisera coprecipitated a comparable level of {alpha}-subunit (lanes 1 and 2). This implies that combination was quantitative. The two bands on the gels corresponding to the ß-subunit contain one or two asn-linked oligosaccharides (19). The assembly of all mutants was reduced although the extent varied, e.g. for the {alpha}T39A mutant, the efficiency of dimer formation was 10–30% (lanes 3 and 4), whereas for the {alpha}T39F (lanes 5 and 6) and the {alpha}39AAA41 (lanes 7 and 8) mutants, less than 5% of total {alpha}-subunit formed heterodimer. This is reflected by a parallel increase in the proportion of the free form of the {alpha}-subunit, which is more heterogeneous and migrates slower on SDS-PAGE than the dimer form (20) (compare lane 1 with lanes 3, 5, 7, and 9). This heterogeneity is due to hyperglycosylation of the asn-linked carbohydrates in the noncombined {alpha}-subunit (20 21B ). The effect of these mutations is apparently reversible since assembly of {alpha}39AAA41 is significantly increased in clones expressing more ß- than {alpha}-subunit (compare lanes 7 and 8 with lanes 9 and 10; see Discussion). That the triple alanine substitution dramatically reduces assembly is consistent with previous studies (10, 15), which found that determinants for assembly are not restricted to the Thr 39 but also include neighboring residues.



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Figure 1. Topological Map Describing the Relative Binding Sites of the mAbs Used in This Study

The epitope regions with known structural localization (ß1, ß6, ß7, {alpha}4, and c1-c3) are integrated onto the crystal structure (2 3 ) of the hCG heterodimer (A) and the CGß subunit (B). The epitope assignments are based on site-directed mutagenesis, peptide competition, and sandwich ELISAs (see Materials and Methods; Refs. 11, 12, 23, and 33). Antibodies whose epitopes are depicted within the same circle compete with one another for binding to hCG as well as mAbs in the overlapping circles (for clarity we have used m instead of mAb to designate the mAbs). The {alpha}- and ß-subunits are represented by green and blue, respectively. NH2 and COO correspond to the amino and carboxyl ends of the subunits, respectively. The asterisks along the {alpha}-subunit indicate amino acids 39–41. The hatched green segment denotes the carboxy-terminal region of the CGß subunit (amino acid residues 118–145). The structure of this sequence has not yet been elucidated. In panel B, the ß-subunit-specific epitopes of mAbs 64 and 68 are shown. Although the ß-subunit undergoes conformational changes during assembly, the dimer and noncombined ß-subunits are presented structurally identical to simplify the epitope assignments. (This ribbon diagram was created by Neil Isaacs).

 


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Figure 2. Assembly of CGß Wild-Type Subunit (WT) and {alpha}-Mutants

Threonine 39 was mutated to alanine ({alpha}T39A, lanes 3 and 4) or phenylalanine ({alpha}T39F, lanes 5 and 6); a triple alanine substitution was introduced in positions 39–41 (lanes 7–10). One clone is presented for each mutant except ß{alpha}39AAA41 where an additional clone with excess ß-subunit is shown (lanes 9 and 10). The WT heterodimer synthesized in transfected cells is seen in lanes 1 and 2. Lanes 1, 3, 5, 7, and 9 correspond to media precipitated by anti-{alpha}-serum, and lanes 2, 4, 6, 8, and 10 contain protein precipitated by anti-ß- serum. The immunoprecipitated proteins were resolved on SDS polyacrylamide gels containing ß-mercaptethanol. The migration of the dimer and free {alpha}-forms are shown. The extent of assembly was determined as a percent of the dimer form of the {alpha}-subunit precipitated by ß-antiserum to the total {alpha}-subunit pool (noncombined + dimer) collected with {alpha}-antiserum.

 
Biological Activity
Studies using hCG-derived peptides implicated the {alpha}39–41 sequence in heterodimer recognition for the receptor (14, 18). Assessing the role of the {alpha} 39–41 sequence on ligand binding by site-directed mutagenesis is difficult due to the inhibition of assembly when this region is altered. To by-pass the assembly step the above mutations were constructed in the {alpha}-domain of the single chain. These mutants were secreted efficiently, and their receptor binding affinity and signaling were tested on CHO cells expressing the human LH/CG receptor (Table 1Go). Both the ß{alpha}T39A and triple-alanine mutant bound the receptor with high affinity. However, ß{alpha}T39F showed a 42-fold reduction compared with the unmutated single chain (ß{alpha}) (Table 1Go). Similar results were obtained when the mutants were tested with the human kidney 293 line expressing the LH/CG receptor (data not shown). To assess whether there were changes in signal transduction, cAMP production was determined. While signal activation generally paralleled binding affinity (Table 1Go), ß{alpha}T39F exhibited a 9-fold reduction in signaling compared with the 42-fold decrease in binding affinity. This implies some uncoupling of the two events. The data show that threonine 39 in the {alpha}-subunit is not only associated with the assembly step but also involved in receptor binding.


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Table 1. Receptor Binding and Changes in cAMP Accumulation by Single-Chain hCG Analogs

 
Structural Analysis with mAbs
Although the single-chain ß{alpha}T39A and ß{alpha}39AAA41 mutants are biologically active, it is not known whether the mutated {alpha}- and unmodified ß-domains form a heterodimeric-like interaction. To examine this point, the single-chain analogs were analyzed with a panel of mAbs that recognize different epitopes sensitive to conformational changes. The mAbs used include eight dimer-specific mAbs (epitopes c1–c4; see Materials and Methods for definition of epitope regions) and two that bind only free ß-subunit (epitopes ß6 and ß7). If the {alpha}/ß heterodimeric interaction is disrupted, the dimer-specific mAbs will not recognize the single-chain mutants because the dimer-specific epitope is lost. However, if the domains are in a noncombined conformation, the free subunit- specific mAbs should detect them. Nonreducing Western blotting in the absence of heat denaturation showed that six of the eight dimer-specific mAbs, 10, 40, 53, 55, A407, and B109, reacted with all of the single-chain mutants. Figure 3Go shows representative blots for mAbs 40 (panel A), A407 (panel B), and 53 (panel C). In each blot, lanes 1 and 5 correspond to unmutated single-chain and hCG heterodimer, respectively. These dimer-specific mAbs gave a positive reaction with all the {alpha}-single-chain mutants (molecular mass = 47–50 kDa). These results show that while these {alpha}-mutants cannot assemble efficiently to form heterodimer, when they are incorporated into the single chain, some heterodimer-like epitopes are generated. It is curious that when the mutants are probed with mAbs 407 and 53, heterogeneity between 47 and 60 kDa is observed. These forms may be due to altered conformation and/or changes in oligosaccharide processing caused by the mutations. The mAbs also detected high molecular mass proteins, the identities of which are not clear, but aggregates of misfolded hCG variants and complexes formed during purification of urinary hCG have been observed (21A, 22). In previous studies of a series of tethered gonadotropin variants, there was no correlation between the proportion of aggregates formed by a particular mutant and the in vitro biological activity (6, 7). Thus, it is unlikely that the aggregates are contributing significantly to the overall potency of the tethered hormones. Further resolution will require purification and direct examination of their biological activity.



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Figure 3. Blot Analysis of hCG Single Chains Bearing Mutations at Residues 39–41 in the {alpha}-Domain

Panels A, B, and C show the blots probed with dimer-specific mAbs 40, A407, and 53, respectively. Equal amounts of the variants were loaded. The nonmutated hCG single chain (ß{alpha}) (lane 1) and hCG heterodimer (NIH CR127) (lane 5) are shown for each mAb.

 
In contrast to the above, dimer-specific mAb 26 (Fig. 4AGo) does not react with the single chain bearing either the ß{alpha}T39F (lane 2) or ß{alpha}39AAA41 (lane 3) mutation and only partially with ß{alpha}T39A variant (lane 4). This suggests that the mutations in the region {alpha}39–41 disrupted the mAb 26 epitope, paralleling the extent of the mutated subunit to form heterodimer (see Fig. 2Go). Another dimer-specific mAb, designated 45, displayed weak reactivity with all variants including the unmutated single chain (Fig. 4BGo). As described in Materials and Methods, this mAb binds to residues in the cystine knot region. The result implies that the single chains have lost some heterodimeric contacts at the mAb 45 epitope (see below). That the low immunoreactivity of the mutants to mAbs 26 and 45 was not due to denaturation during blotting was demonstrated by enzyme-linked immunosorbent assays (ELISAs) (Fig. 4CGo and Table 2AGo). The ß{alpha}T39A mutant had 14% cross-reactivity with mAb 26 compared with the unmutated single chain, and the ß{alpha}T39F and ß{alpha}39AAA41 variants were not detected (Table 2AGo). No ELISA signal was detected with dimer-specific mAb 45 (Fig. 4DGo and Table 2AGo). As a further control we also tested the reactivity of mAb 26 in an ELISA containing no Tween 20 to assess whether the detergent caused denaturation (Fig. 4CGo, inset). The absence of Tween did not significantly alter the ratio of immunoreactivity of the mutants compared with reaction mixtures containing detergent. Further ELISA experiments performed in the absence of Tween with mAbs 55, 64, and 68 did not affect the ratio of analog immunoreactivity (data not shown). As discussed in Materials and Methods, the amount of each {alpha}-mutein added to the ELISA reaction mixture was determined by a ß-subunit-specific mAb. Although the ß-domain is not mutated, such quantitation may be subject to overall conformational changes induced by the {alpha}-mutations. To address this point, the mutein concentrations were also determined for the ELISA by an RIA protocol containing polyclonal antiserum against the ß-subunit. We reasoned that such a quantitation method would be less susceptible to major conformational changes caused by the mutations. Using this assay the immunoreactivity of the mutants was comparable to the mAb-based quantitation (Table 2BGo). Taken together, these data support the blotting experiments that the heterodimeric interaction at epitopes corresponding to mAb 26 and 45 were disrupted in all of the single-chain variants.



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Figure 4. Reactivity of Dimer-Specific mAbs 26 and 45 with the Single-Chain Variants Containing Mutations in the 39–41 Sequence

Equal amount of variants (based on RIA) was probed with mAb 26 (panel A) or mAb 45 (panel B). Panels C and D represent the ELISAs with mAbs 26 and 45, respectively. The inset graph shows an ELISA mAb 26 in the absence of Tween.

 

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Table 2. ELISA Analysis of Threonine 39 Mutants

 
If, as discussed above, the {alpha}/ß heterodimeric interaction is disrupted in the mutants, they should be more accessible to mAbs that are specific for the free ß-subunit. To test this prediction, two mAbs, designated 64 and 68, which recognize epitopes in different regions of only the free ß-subunit (11, 23) were tested with the single-chain variants (Fig. 5Go). As expected, both mAbs did not detect hCG heterodimer (lane 5), whereas the noncombined ß-subunit in the CR127 preparation was detected (lane 5, Mr = 30 kDa). When these mAbs were tested against the unmutated single chain (lane 1), weak activity was observed, suggesting that some of the {alpha}- and ß-contact sites in the single chain do not form a tight heterodimeric complex. This is consistent with data obtained with dimer-specific mAb 45 above, which did not detect the unmutated single chain. The mAbs 64 (panel A) and 68 (panel B) reacted strongly to all three of the mutated single chains. Comparable data were obtained by ELISA (Table 2AGo); the mAbs reacted with the single-chain mutants in the order ß{alpha}T39F, ß{alpha}39AAA41 > ß{alpha}T39A > unmutated single chain, which is the inverse of reactivity seen with the heterodimer-specific mAb 26 (Fig. 4Go). When the mutants were assayed according to the polyclonal-based quantitation described above, their reactivity to mAbs 64 and 68 was 2- to 6-fold greater compared with CGß{alpha} (Table 2BGo). The data show that loss of heterodimeric specificity is associated with gain of reactivity to free subunit determinants and demonstrate that the mutations {alpha}T39F, {alpha}T39A, and {alpha}39AAA41 disrupted the {alpha} domain interactions in the single chain.



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Figure 5. Blots of Single-Chain Mutants Residues 39–41 in {alpha}-Domain Probed with ß-Subunit-Specific mAbs 64 (A) and 68 (B)

The 30 kDa corresponds to the migration of free CGß subunit.

 
Disulfide Bond Mutations in the Single-Chain hCG
Based on the hCG crystal structure, the five disulfide bonds in the {alpha}- subunit are at residues 10–60, 28–82, 32–84, 7–31, and 59–87 (2, 3); the six bonds of the ß-subunit are 9–57, 34–88, 38–90, 23–72, 93–100, and 26–110 (2, 3). In each subunit the first three bonds comprise the cystine knot (2, 3). Monomeric {alpha}- or CGß-subunits containing certain disulfide mutations are unable to fold appropriately and form heterodimers (24, 25, 26). However, when either a mutated {alpha}- or ß-domain was incorporated in the single chain, they were biologically active (6, 7). Earlier studies showed that the extent of heterodimer formation of the {alpha}-subunit bearing either the cystine mutations 10–60 or 32–84 was less than 5% (7). To assess the heterodimeric-like behavior of these {alpha}-disulfide bond mutants in the single chain, a Western blot analysis was performed using the mAbs described above; two representative dimer-specific mAbs 10 and 40 are shown (Fig. 6Go; see also Table 3Go). Each reacted with the unmutated single chain and the hCG heterodimer (lanes 1 and 4). In the case of mAb 10 (panel A), CGß{alpha} 10–60 was not detected (lane 2) but CGß{alpha} 32–84 was recognized (lane 3). No significant reactivity of mAb 40 with these mutants was seen (panel B, lanes 2 and 3). Similar results were obtained when blots were probed with the other dimer-specific mAbs described in Table 3Go (data not shown). The ELISA profiles also show a comparable decrease in immunoreactivity of the mutants to these dimer-specific mAbs (Table 3Go).



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Figure 6. Analysis of the Secreted Single Chains Containing the Disulfide Bond Mutations 10–60 and 32–84 in the {alpha}-Subunit Domain (A and B), and the hCGß Disulfide Bond Mutations 9–57, 34–88, and 38–90 (C, D, and E)

Concentrated media were quantitated by RIA and equal amounts of single chain or NIH hCG dimer were loaded on the gel. The mAbs 10 and 40 are dimer specific. A parallel blot was probed with anti-{alpha}-polyclonal serum (panel E).

 

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Table 3. ELISA Summary of Disulfide Bond hCG Mutants

 
Single chains containing the disulfide bond mutations in the ß-subunit domain were probed with the dimer-specific mAbs, and little or no reactivity was seen, e.g. mAbs 10 and 40 (Fig. 6Go, C and D, and Table 3Go). The absence of signal was not due to a preferential loss of the mutants since they were detected with polyclonal antiserum against the {alpha}-subunit (panel E). Clearly, the disulfide bond mutations in either the {alpha}- or ß-subunit disrupted the quaternary relationship between the subunit domains in the single chain.

As shown above for the {alpha}-helix mutants, these disulfide bond variants, compared with the nonmutated single chain, might be expected to manifest a greater signal with the ß-subunit-specific mAbs. Consistent with this prediction (Fig. 7Go), the {alpha}-mutants have an increased immunoreactivity to mAbs 64 (panel A) and 68 (panel B). These results were also supported by ELISA assays (panels C and D and Table 3Go). However, in contrast to these data, there was no detectable immunoreactivity of the ß-disulfide bond mutants to mAbs 64 or 68 except for the reactivity of ß38–90 with mAb 64 (Fig. 8Go). Moreover, reactivity of the ß-mutants was significantly reduced with five additional mAbs that recognize the dimer or noncombined ß-subunit (data not shown). The ß-mutants were not detected by ELISAs containing the ß-subunit-specific mAbs 64 and 68 (Fig. 8Go, C and D, and Table 3Go). Thus, in contrast to the {alpha}-disulfide bond single-chain mutants that are detected by mAbs 64 and 68, these mAbs fail to recognize epitopes on the ß-subunit (23). As performed above for the {alpha} 39–41 mutants, we tested the ELISA immunoreactivity in the absence of Tween with representative dimer-specific mAb, e.g. 55, and both the ß-subunit-specific mAbs 64 and 68; in all cases there were no significant differences in the ratio of immunoreactivities compared with assays containing detergent (data not shown).



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Figure 7. Immunoreactivity of ß-Subunit-Specific mAbs 64 and 68 with the {alpha}- Disulfide Single-Chain Mutants

Panels A/B and C/D are the Western blots and corresponding ELISAs, respectively. Note, the ordinate ELISA scales are not identical.

 


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Figure 8. Immunoreactivity of ß-Subunit-Specific mAbs 64 and 68 with the ß- Disulfide Single-Chain Mutants

Panels A/B are Western blots and C/D are the corresponding ELISAs. Note the different scales on the ordinates in the ELISA.

 
To assess whether the mutation-induced changes affected the ELISA quantitation using mAb 2 or mAb 132 (see Materials and Methods), we quantitated the disulfide bond mutants with the polyclonal-based RIA discussed above (see Materials and Methods). There are no changes in the ELISA when the mutants were screened with ß-specific mAbs 64 and 68 (Table 4Go). In the case of the immunoreactivities to the dimer-specific mAbs, those mutants that scored weak or not detectable by the mAb quantitation gave similar signals whether the muteins were quantitated by mAb or polyclonal antisera. There are variations with some of the mAbs and disulfide mutants when the two quantitation methods are compared (Table 4Go). The largest difference (5-fold increase) is seen when the {alpha}10–60 and ß9–57 mutants are probed with mAb 10. (When {alpha}10–60 is analyzed by Western blot with mAb 10, a significant reduction in signal is observed compared with the ELISA. The reason for this variation is not clear.) Although the signal is increased for some of the other muteins, the overall immunoreactivity is still 6–10 times less than the unmutated single chain. Despite these differences, the data obtained from two widely different quantitation protocols show that loss of heterodimeric immunoreactivity is markedly reduced with a corresponding increase in reactivity of the noncombined ß-subunit-specific epitopes. Taken together the results further support the hypothesis that these mutations disrupted the heterodimeric configuration of the CGß{alpha} single chain, but despite these modifications, the mutants nevertheless display high-affinity receptor binding.


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Table 4. ELISA Results of Disulfide Bond hCG Mutants Quantitated with Polyclonal Antiserum

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Many multisubunit proteins exert their biological function through different domains on each peptide chain, brought together by the quaternary interaction of the subunits (e.g. immunoglobulin, the GroEL chaperone, lactate dehydrogenase). A mutation that disrupts the assembly of the subunits leads to inactivity (27, 28), and this has been observed for the glycoprotein hormone family (1). However, whether or not the quaternary structure per se generates biological determinants has not been fully explored. Here we show that when the {alpha}- and ß-subunits of hCG are genetically linked as a single polypeptide chain, but prevented from forming the intimately associated dimer-like configuration, the biological activity is nevertheless retained. We approached this issue with two distinct sets of mutations. One set involved the amino acids in the {alpha}-subunit that have direct contact with the ß-subunit, and the other dealt with altering the tertiary structure of the subunits by mutating the disulfide bonds. The single-chain model was critical for these studies since it overcame the limitation of assembly defects that would hinder analysis of these regions as seen in earlier work because of low heterodimer production.

The sequence 39–41 of the {alpha}-subunit is contained in the long loop 2 which interfaces with loops 1 and 3 of the CGß subunit. Previous studies have demonstrated that mutations in this region inhibit assembly with the ß-subunit of CG (8, 10, 15) or FSH (16). Threonine 39 apparently occupies a pivotal role in this sequence; based on the crystal structure, it resides on the external face of the dimer, and there is a hydrogen bond between this residue and asparagine 52, which bears an oligosaccharide that is involved in assembly and biological activity (42). In addition, it has been shown that Thr 39 can be O-glycosylated in vivo, which prevents formation of the heterodimer (29, 30). Residues 40–50 in the {alpha}-subunit contain the only helical structure in the dimer, and Thr 39 occupies the NH2-terminal cap (31) of a helix. The cap apparently initiates helix formation (31). The hydrogen bond between Thr 39 and Asn 52 contributes to the secondary structure of the helix and thus mutation of this threonine residue presumably disrupts helical stability. The introduction of the bulky phenylalanine would lead to a greater perturbation of the helix. The single-chain data suggest that Thr 39 has a role in receptor binding because the binding of ß{alpha}T39F was inhibited 42-fold compared with the nonmutated control. Because there is a correlation between the inhibition of receptor binding of ß{alpha}T39F and the loss of immunoactivity to dimer mAb 26 with a corresponding gain of activity to free subunit-specific mAbs 64 and 68, it could be concluded that the quaternary structure is involved in the biological activity (Fig. 3Go). However, although the {alpha}39AAA41 mutant exhibits a similar structural change, as reflected by its decreased reactivity to mAb 26 and an increase in signal to mAb 64/68, it still binds to receptor with an affinity comparable to wild type. We suggest that the determinant associated with the {alpha}39–41 sequence in receptor binding can be uncoupled from its requirement for {alpha}/ß assembly and implies that Thr 39 interacts with the receptor.

Although {alpha}T39 is important for {alpha}/ß-subunit recognition, it is curious that mutations that reduce heterodimer formation did not have such an inhibitory effect on the association of {alpha}/ß-domains in the single chain. For example, the monomeric {alpha}-subunit bearing the T39F mutation does not assemble with the ß- subunit, but it forms a heterodimeric-like association when the mutant is incorporated into the single chain (Fig. 3Go). Because the {alpha}/ß-domains are covalently linked in the single chain, the local concentration of the domains at the {alpha}/ß-interface may be sufficient to promote the productive interaction of the two subunits. This is consistent with our data that indicate when the ratio of ß-subunit exceeds the {alpha}-subunit more heterodimer is observed. We suggest that the assembly reaction proceeds in at least two phases: the first involves the recognition by the subunits to initiate pairing, which occurs in the ER lumen through the possible aid of one or more chaperones and the second involves the final functional dimeric configuration of the subunits. This is supported by studies that show that synthesis of an assembly-competent hCGß subunit is associated with multiple intermediate forms (32).

Given the dramatic effects cysteine mutations have on the secretion and assembly of the individual subunits (7, 24, 25, 26), it was unexpected that disrupting the cystine knot in either the {alpha}- or ß-subunit domain would not significantly affect the receptor binding or signal transduction. Many of the mAbs used here are known to react with different regions of the hormone (11, 12, 23, 33, 39), and thus lack of mutant immunoreactivity is presumably due to altered conformation of the subunit(s) rather than abolishing recognition at a specific cysteine residue. These observations further support the hypothesis that all of the wild-type heterodimeric interactions are not essential for receptor binding and signal transduction. Clearly, domains from both subunits are necessary since free subunits are not active. It appears that if the crucial domains for receptor binding/signal transduction are present, hormone function is preserved; even some loss of tertiary structure is tolerated as shown by the reduced immunoreactivity of the ß-disulfide bond mutants to all mAbs that recognize the ß-subunit. Binding of one domain to the receptor may restore altered conformation of the other domain(s). However, although the native quaternary structure may not be essential for receptor activation, we cannot exclude the possibility that the function of the heterodimeric configuration is required to maintain circulatory half-life of the hormone.

The unmutated single-chain hCG itself has lost some quaternary structure since it is not recognized by dimer-specific mAb 45. Epitope mapping studies using mutagenesis (33) indirectly imply that this mAb binds in the vicinity of the cystine knots at the contact sites in the heterodimer (see Materials and Methods). Additionally, a free subunit-specific mAb, which also binds near the ß-subunit cystine knot (residue 89), reacts positively with the unmutated single chain. Therefore, there is some aberration in this area created by linking the C terminus of the ß-chain to the N terminus of the {alpha}-chain. That the unmutated single-chain hCG is as potent as the heterodimer, despite having such an open structure at the core of the molecule, is further evidence that neither all of the native heterodimeric configuration nor an intact cystine knot is a prerequisite for receptor binding. While we cannot exclude some native quaternary interactions in the mutants, e.g. the sequences responsible for receptor binding, our functional data are consistent with the conclusions from crystal structure analysis that the hCG dimer structure is open with relatively modest affinity between the subunits (3). These data imply that gonadotropins with different conformations can combine with the receptor and activate adenylate cyclase.

The data also support the hypothesis that the cystine knots are scaffolds in the subunits that play a role in heterodimer formation (6). It is interesting that the biologically active form of cystine knot-containing proteins is either a homo- or a heterodimer (34). This capacity to dimerize is critical for the glycoprotein hormones in which the common {alpha}-subunit is shared by four hormones. Arakawa et al. (35) have shown that the subunits of brain-derived neurotrophic factor and neurotrophin-3, members of the cystine knot family which exist as homodimers, formed biologically active heterodimers. It is intriguing to consider that the common {alpha}-subunit or ß-subunit may form dimers with subunits of other proteins that have a cystine knot motif. This may be a mechanism by which the free ß-subunit exhibits a growth factor effect observed on certain cancer cells (36); it could form a dimer with monomeric growth factors and then bind to a receptor on the tumor cells.

These studies have important biological implications for producing recombinant multisubunit proteins, since it may not be necessary to ensure that the product has the exact native conformation. It is known that, even in large protein-protein interfaces, only a few residues are actively involved in binding to receptor (37). Thus, the approach of covalently linking subunits from multimeric proteins, combined with mutagenesis, is a promising strategy for creating less complex molecules. The removal of nonessential residues could lead to determining critical structural requirements for signal transduction and thus facilitate the design of small peptide agonists or antagonists.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Enzymes used for DNA manipulation were purchased from Promega Corp. (Madison, WI) except for thermostable polymerase for PCR (Klentaq), which was obtained from Dr. W. Barnes (Washington University, St Louis, MO). Oligonucleotides used for mutagenesis and sequencing were prepared by Nucleic Acid Chemistry Laboratory, Washington University (St Louis, MO). [S35]Cysteine and Pro-mix were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Media and reagents for cell culture were prepared by Washington University Center for Basic Research (St Louis, MO). Purified hCG (CR127) was obtained from the NIH Pituitary Hormone Program (Baltimore, MD).

Antibodies
mAb B110 was obtained from Dr. W. Moyle (Robert Wood Johnson Medical School, Piscataway, NJ); B109 and A407 were obtained from Drs. S. Birken and R. Canfield (Columbia University, New York, NY). The Innsbruck (INN-) series of mAbs were described previously (11, 12). All mAbs are purified ascites except INN-hCG-10 and INN-hCG-40, which are unfractionated ascites. All INN-hCG-x mAbs will be referred to as mAb x. Rabbit polyclonal antiserum against hCGß- and {alpha}-subunits were prepared in this laboratory. The epitopes for these mAbs on hCG have been extensively characterized by two-site antibody compatibility assays, inter- and intraspecies cross-reactivities, peptide mapping, and site-directed mutagenesis (11, 12, 23, 33, 38, 39). The majority of epitopes are discontinuous, and the reactivity of the mAbs is conformationally dependent. Nine distinct epitope regions have been defined on the ß-subunit (ß1–ß9), six on the {alpha}-subunit ({alpha}1–{alpha}6), and four regions only present on the holohormone, termed c1 through c4 (Fig. 1Go). The ß6- and ß7-epitope regions are specific for the free ß-subunit. The epitope region ß1 involves CGß-subunit amino acid residues 10 and 60 (33). Residue 89 in CGß was also shown by mutagenesis to be involved in both ß1- and ß7-epitope regions (A. M. Jackson, P. Berger, A. J. Lapthorn, N. W. Isaacs, P. J. Delves, T. Lund, and I. Roitt, unpublished data)and ß1 overlaps with region ß7 and c3. Therefore it is likely that mAbs specific for epitope region c3 (mAb 45) and ß7 (mAb 68) bind to a similar area of the hormone (core of the molecule in Fig. 1Go).

Construction of {alpha}-Subunit Mutants and Single-Chain Analogs
The construction of pM2 plasmids containing {alpha}-subunit T39F and T39A was described earlier (8). The following primers were used for PCR overlap mutagenesis to generate the triple mutant {alpha}TPL39–41AAA:

1. 5'-TAC TTT GTC GAC AAA TGA TAA TTC AGT GAT TGA-3' SalI

2. 5'-AGA TCC GGA TCC ACA GTC AAC CGC CCT-3' BamHI

3. 5'-AGA GCA TAT CCC GCG GCC GCT AGG TCC AAG AAG-3'

4. 5'-CTT CTT GGA CCT AGC GGC CGC GGG ATA TGC TCT-3'

Sense and antisense primers 3 and 4 correspond to amino acid residues 35–45 of the {alpha}-subunit (bp 277–309), and primers 1 and 2 correspond to bp 42–65 and 709–729 in the 5'- and 3'-nontranslated sequence, respectively (43). The hCG {alpha} wild-type gene, subcloned in pM2 (18), was used with primers 1 and 3 to create the first fragment. Primers 2 and 4 generated the corresponding overlapping fragment. These fragments were then used as the templates with primers 1 and 2 to synthesize the complete {alpha}-subunit mutant, which resulted in a BamHI site at the 5'-end and a SalI site at the 3'-end. These sites were used to subclone the fragment into pM2. The single-chain analogs were engineered using the naturally occurring XbaI site at amino acid 35 of the {alpha}-subunit gene (7). An XbaI digest of the {alpha}-mutants excised a 1900-bp fragment, which was subcloned into an XbaI digest of pM2HA-CGß{alpha}(5) to generate single-chain variants. All constructs were sequenced to ensure no errors were introduced during the PCR.

Transfection, Clone Selection, Labeling, and Immunoprecipitation
Plasmids containing the mutant genes were transfected into CHO cells and screened as previously described (19, 40). Clones were immunoprecipitated as previously described (8, 24).

Receptor Binding and cAMP Assays
The single-chain variants were quantitated by an hCG RIA (Diagnostic Products Corp, Los Angeles, CA) using hCGß polyclonal antiserum. Conditioned media were incubated with CHO or human fetal kidney 293 cells stably transfected with human LH/CG receptor, and the binding affinity was determined as previously reported (41). The cAMP accumulation (intra- and extracellular) was determined using the NEN Life Science Products (Boston, MA) flashplate assay as per manufacturer’s instructions. Briefly, 5 x 104 CHO cells stably transfected with the LH/CG receptor were incubated for 2 h at room temperature with hCG dimer or mutants, [125I]cAMP was added, and the cells were incubated for 17 h at room temperature. The radioactivity in the flashplate was determined with a Packard Top Counter (Packard Instruments, Meriden, CT).

Western Blot and ELISA
Concentrated conditioned medium of single chains was quantitated by polyclonal RIA (Diagnostics Products, Los Angeles, CA) using either the hCGß antiserum in the kit or our {alpha}-antiserum. We elected to use this polyclonal- rather than a monoclonal-based assay to minimize the potential error in quantitation due to structural changes caused by a particular mutation. For mutants in the {alpha}-subunit domain of the single chain, antiserum against the ß-subunit was used whereas anti-{alpha}-serum was employed for mutations in the ß-subunit. In several studies using site-directed mutagenesis, we observed minimal changes in recovery when such muteins were immunoprecipitated with polyclonal antisera. Equivalent amounts of each protein were loaded onto 12.5% SDS-polyacrylamide gels in the absence of heat and ß-mercaptoethanol. Proteins were transferred onto nitrocellulose and probed with mAbs and visualized on film by chemiluminescence (Tropix, Bedford, MA). All experiments contained a blot probed with polyclonal antisera to ensure that comparable amounts of material was loaded on the gels.

To determine the immunoreactivity of the variants in solution assays, conditioned media were examined by ELISA. All test mAbs were titrated against purified hCG, except for the ß-subunit-specific mAbs, which were titrated with recombinant CGß subunit. The mAb used to determine the amount of each variant in the condition media was based on the mutated subunit. In the case of changes in the {alpha}-domain, mAb 2 was the capture mAb. This mAb, which recognizes the free and dimer forms of the CGß subunit, exhibited comparable binding to the {alpha}-mutants and nonmutated single chain. Likewise, for the mutations in the ß-subunit domain, mAb 132 was used since its epitope includes amino acids 17–22 of the {alpha}-subunit, which is minimally influenced by mutations on the ß-subunit. The detection of the complex was achieved with polyclonal ß-antiserum for mAb 2 and polyclonal {alpha}-antiserum for mAb 132.

The muteins were also quantitated for the ELISA using a polyclonal-based RIA; for mutations in the {alpha}-subunit, antiserum against the ß-subunit was used, whereas anti-{alpha}-serum was used for muteins containing changes in the ß-subunit domain. This quantitation protocol should be less sensitive to conformational changes induced by the mutations. When the two quantitation methods are compared, there was a 0- to 5-fold variation in the determinations for some of the muteins. Despite these differences the trends are unchanged.

Equal amounts of each analog were used in all analyses. The ELISA plates were prepared according to the following: 1) The wells (Corning, Inc., Corning, NY) were coated with 100 µl/well of the test (capture) mAb in carbonate-bicarbonate buffer [0.29% (wt/vol) NaHCO3; 0.17% (wt/vol) Na2CO, pH 9.6) at 4 C overnight at a concentration that yields a linear response to the appropriate standard; 2) After coating, blocking was performed with 200 µl 0.05% Tween-PBS containing 0.5% BSA; 3) Condition media containing variant were added; 4) Rabbit anti-hCGß or {alpha} sera (at a dilution titrated for each coated mAb) were added; 5) Alkaline phosphatase-conjugated goat antirabbit IgG (Sigma, St. Louis, MO) was added; and 6) The reactions were developed with p-nitrophenyl phosphate (1 µg/ml). At each step the plates were incubated for 1 h at 37 C, and the wells were washed three times with Tween-PBS between each step. After the addition of p-nitrophenyl phosphate, the plates were incubated at room temperature for 1 h, and absorbance at 405 nm was determined in an ELISA plate reader.


    ACKNOWLEDGMENTS
 
We thank Drs. Raymond Ruddon, Gabriel Waksman, David Ornitz, Vicenta Garcia-Campayo, David Ben-Menahem, and Carl Frieden for their comments regarding the manuscript. We are also grateful to Mary Wingate for the preparation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Irving Boime, Department of Pharmacology, Washington University Medical School, 660 South Euclid, St. Louis, Missouri 63110.

A.M.J. is a Lalor Foundation Research Fellow, and P.B. is funded by the Austrian Science Fund (P13652-Gen). This work has been supported by a grant from Organon.

Received for publication March 30, 1999. Revision received August 27, 1999. Accepted for publication September 9, 1999.


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