INTRODUCTION

Human chorionic gonadotropin (CG),¹ lutropin (LH), follitropin, and thyrotropin are members of the glycoprotein hormone family that share a common  subunit but differ in their hormone-specific  subunits. The amino acid sequence of the  subunit is identical within a species (1), suggesting that the tertiary structure of the  subunit requires a degree of flexibility to adapt to a unique conformation relative to the  domain(s). The human subunit has 10 highly conserved cysteine residues, which form five disulfide bonds. Based on the crystal structure of the subunit in hCG, the proposed pairs are 10-60, 28-82, 32-84, 7-31, and 59-87 (Fig. 1) (2, 3). Bonds 28-32 and 32-84 comprise a ring structure penetrated by a bond bridging cysteine residues 10 and 60 resulting in a core that forms three hairpin loops. This structure is a common feature to the large growth factor family including tumor growth factor-, activins, nerve growth factor, and platelet-derived growth factor (4-8). Chemical reduction (1) or site-specific mutations of individual cysteine residues in either the common  or CG subunit, alters dimer assembly and secretion rate (9, 10). Mutants lacking either the 7-31 or 59-87 bond were secreted (10), and heterodimers containing the 7-31 mutation bound to the LH/hCG receptor with an affinity comparable with the wild-type hCG, whereas the 59-87 mutant interacted with a lower binding affinity. However, mutants comprising the cystine knot (10-60, 28-82, and 32-84) were not secreted in sufficient quantities to allow examination of the biological activity. To address this issue, we used a single chain gonadotropin model where the CG subunit was genetically fused to the  subunit (11, 12) to promote more efficient secretion of proteins bearing the  and  subunit domains in the same complex. Using this approach, the rate-limiting step of subunit assembly could be circumvented, and mutations that otherwise block dimer formation could be evaluated. Here we show that single chains containing mutations in the cystine knot (10-60, 28-82, and 32-84) are secreted and biologically active, implying that quaternary relationships between the subunits in the heterodimer primarily influence the intracellular behavior rather than receptor binding/signal transduction.

Disulfide bond assignments of the common human  subunit.

MATERIALS AND METHODS

Enzymes for preparing vectors and for polymerase chain reaction were purchased from Promega (Madison, WI), Boehringer Mannheim, or Stratagene (La Jolla, CA). Oligonucleotides were prepared by the Washington University Sequencing Facility. Cell culture media and reagents were prepared by the Washington University Center for Basic Cancer Research (St. Louis, MO). Fetal bovine serum, dialyzed fetal bovine serum, and the neomycin analogue, G418, were purchased from Life Technologies, Inc. For metabolic labeling, [³⁵S]cysteine-methionine (Promix) (Amersham Corp.) or [³⁵S]cysteine (ICN, Irvine, CA) were used. Monoclonal antibodies A407 and B109 were gifts from Dr. Robert Canfield and Dr. Steve Birken (Columbia University, New York).

Construction of  Cysteine Mutants

Previously, the intracellular behavior of single cysteine mutants was assessed (10, 13). Double mutants were only constructed that deleted the 7-31 and 59-87 disulfide bridges. Here, we designed all of the analogs to contain double substitutions for each disulfide bond that were based on the assignments from the recently published crystal structure (2, 3). Thus, both monomeric  subunit and the single chain analogs containing such modifications were constructed.

The  cysteine mutants previously constructed in vector pM² were used (10). Exon III of the  subunit contains a unique XbaI restriction site within the sequence encoding amino acid residues 34 and 35 (Fig. 2A). Because the vector also contains a single XbaI site, fragments bearing 10, 28, or 32 mutations were exchanged with XbaI-digested pM² containing 60, 82, or 84 substitutions. This resulted in double cysteine mutants 10-60, 28-82, and 32-84, respectively. Construction of the pM² 7-31 and pM² 59-87 were described previously (10).

Single chain variants were constructed with the carboxyl end of the  subunit fused to the amino end of the  subunit using overlap polymerase chain reaction mutagenesis (11) (Fig. 2B). All constructs generated by polymerase chain reaction were sequenced to ensure that the final products contained no misincorporated residues.

Transfection, selection of stable CHO clones, metabolic labeling, and immunoprecipitation with subunit-specific antiserum were previously described (10, 14, 15). Analysis by Western blotting was performed using conditioned media that were concentrated with Centriprep-10 columns (Amicon, Beverly, MA). Samples were loaded on 12.5% SDS-polyacrylamide gels and electroblotted to nitrocellulose (Hybond ECL, Amersham International, UK), and the proteins were detected by the Western Light kit (Tropix, Bedford, MA).

To determine the biological activity, the variants were quantitated either by an hCG RIA (Diagnostic Products Corporation, Los Angeles, CA) containing CG polyclonal antiserum or an hCG dimer-specific enzyme-linked immunosorbent assay kit (Organon, Oss, The Netherlands). Both assays gave comparable results. Conditioned media were incubated with human fetal kidney 293 cells stably transfected with human LH/CG receptor, and the binding affinities (10) and cAMP accumulation were determined (16). Iodination of hCG (CR-127; 14,900 IU/mg) was performed as described (17); the specific activity and maximum binding of ¹²⁵I-labeled hCG, as determined by radioligand receptor assay (18), were 53,000 cpm/ng and 40%, respectively. Nonspecific binding was determined by adding a 1000-fold excess of unlabeled ligand (Pregnyl, Organon); specific binding routinely was 10-12% of total ¹²⁵I-labeled hCG added. The cAMP levels were also determined in 293 cells expressing human LH receptors by radioimmune assay (16).

RESULTS

Intracellular Behavior of Cys Mutations in the Monomeric  Subunit

The proposed disulfide bonds in the  subunit are at positions 7-31, 10-60, 28-82, 32-84, and 59-87 (Fig. 1). Previously, we demonstrated that  subunits containing single mutations at Cys-10, -28, -60, -82, and -84 were not secreted and in most cases were degraded intracellularly (10). These mutants also failed to assemble with hCG subunit, whereas mutants with alterations at Cys-7, -31, -32, -59, or -87 were secreted and assembly-competent. Because a free thiol group could alter intracellular behavior (9, 10, 19, 20), double mutants in the  subunit were constructed using the assignments based on the crystallographic analysis (2, 3). To assess secretion/stability of these mutants, transfected cells were labeled with [³⁵S]cysteine and subjected to pulse-chase analysis. The intracellular (lysate) and extracellular (media) forms were immunoprecipitated and resolved on SDS gels (Fig. 3). Except for the 10-60 variant, the secretion rate of the mutants and wild-type subunit were comparable. As described previously, the WT, 7-31, and 59-87 mutants were efficiently secreted because greater than 95% of the subunits were recovered from the media (10). In contrast, the recovery of the 10-60, 28-82, and 32-84 double mutants was 21, 41, and 20%, respectively (Fig. 3; Table I). The decreased recovery was not due to lower synthesis but rather to enhanced degradation, since at zero time of chase the intracellular accumulation of the mutants is comparable (Fig. 3). These data suggest the cystine knot structure is critical for maximum  subunit secretion. Although the secreted non-combined WT is heterogeneous, the media form of 10-60 is more homogeneous, suggesting that a post-translational modification is affected by this mutation.

Secretion kinetics of  subunit cysteine mutants.

Table I. Secretion of  subunit cysteine mutants Values represent the mean ± S.E. of at least two experiments. Variant t1/2a Recovery Monomerb Dimerc min %  WT 130  ± 12 92  ± 4 83  ± 1 7-31 135  ± 18d >95d 77  ± 6 10-60 195  ± 34 21  ± 6 <5 28-82 123  ± 30e 41  ± 6e 49  ± 5 32-84 114  ± 6e 20  ± 4e <5 59-87 142  ± 36d >95d 90  ± 5 a Time when half of the maximal signal is detected in the media with free  subunit only. b The amount of hormone retrieved from the media at steady state and expressed as a fraction of the total (lysate + medium). c Clones co-expressing hCG WT and  subunits were pulse-labeled and chased for up to 24 h. The samples from each clone were divided into two aliquots and immunoprecipitated with  or hCG antisera. Recovery is determined by comparing the quantity of  subunit in the secreted dimer (detected with  antiserum) with the total  subunit pool (lysate + media) using polyclonal  subunit antiserum. d Data from previous study (10). e Mean ± range of two experiments.

To evaluate the efficiency of dimer formation, the  constructs were co-transfected with the hCG gene (21), and clones synthesizing excess  subunit were selected to ensure that it would not limit assembly. The cells were labeled, and equal aliquots of lysate/medium were precipitated with subunit-specific polyclonal antisera (Fig. 4, Table I). Recovery of the 10-60 and 32-84 mutants in the secreted heterodimer was less than 5% although synthesis of all the mutants was at significant levels. In the case of the 28-82 mutant (50% recovery), the combination efficiency is relatively unaffected, since the amount of  subunit precipitated by either  or  subunit antiserum was comparable. Pulse-chase experiments demonstrated that the 7-31, 28-82, and 59-87 mutants combined efficiently with the  subunit, and their recovery paralleled the uncombined  subunit synthesized in the absence of co-transfected  subunit (Table I). The low amount of dimers containing the 10-60 or 32-84 mutant reflects a decrease in combination efficiency and/or an enhanced intracellular degradation of the mutated subunit. The data show that the disulfide bonds 10-60 and 32-84 (and to a lesser extent 28-82) are critical for assembly.

Clones expressing hCGWT and either WT, 10-60, 28-82, or 32-84 were labeled for 1 h (Lysates) or 7 h (Media).

Due to decreased dimer accumulation in the media, studies on the biologic action of the heterodimers containing the 10-60 or 32-84 mutation are difficult. However, if the  subunit is covalently linked to the  subunit, the rate-limiting assembly step is bypassed, and sufficient material could be obtained to examine signal transduction. The disulfide bond mutants described above were linked to the wild-type  subunit to form single chain analogs (see "Materials and Methods"), and the intracellular behavior was examined by pulse-chase experiments. The secretion kinetics of the single chain variants (Fig. 5, Table II) paralleled the corresponding monomers. The variants including the nonmutated tether (CG) were secreted at a comparable t1/2 (CG t1/2 = 106 min), while the single chain 10-60 mutant is secreted much slower (t1/2 = 244 min; see Table II). Thus, even in the single chain construct, the 10-60 bond is critical for secretion. However, compared with the corresponding heterodimers (compare Tables I and II), recovery of the mutants 10-60 and 32-84 dramatically increases when incorporated in a single chain.

Secretion kinetics of  cysteine mutants in CG single chain.

Table II. Secretion of  mutants in CG tether See the legend of Table I for description of quantitation. CG corresponds to the nonmutated CG single chain. Variant t1/2 Recovery min % CG 106  ± 4 74  ± 5 7-31 107  ± 5 81  ± 5 10-60 244  ± 36 36  ± 3 28-82 102  ± 15 55  ± 5 32-84 138  ± 32 45  ± 6 59-87 102  ± 13 87  ± 4

Because little heterodimer is formed with  mutants 10-60 and 32-84, we suspected that the conformation of the single chain bearing these mutated  domains would be changed. To address this issue, conditioned media were examined by Western blot and probed by two dimer-specific monoclonal antibodies: A407 and B109 (22-26) (Fig. 6). An A407 epitope resides in amino acid residues 5 and 6, 11-13, and 81 of the  subunit (23). Under nonreducing conditions, A407 recognizes 7-31, 28-82, 59-87, nonmutated CG single chain, and CG heterodimer (Fig. 6A). However, 10-60 and 32-84 are barely detected (Fig. 6A). These low signals are not due to a difference in the recovery of the blotted protein, since reprobing with polyclonal  antiserum shows comparable signals for all mutants, including 10-60 and 32-84 (Fig. 6B). Moreover, it is apparent that the migration of the mutants under nonreduced conditions is altered by the mutations, implying that the conformation of the single chain mutants and CG are not the same.

Conformational difference of  Cys-mutated single chains.

Similar data were obtained with B109, which is specific for the dimer form of the  subunit (24-26) (Fig. 6C). Tethers containing mutations 7-31, 28-82, and 59-87 and the nonmutated are recognized as well as the heterodimer, but single chains containing either the 10-60 or 32-84 mutation show very weak signal compared with the same blotted membrane using CG antiserum (Fig. 6D). Both A407 and B109 recognized higher molecular weight proteins in CG 28-82 and 59-87. They are likely noncovalently linked because when the samples are boiled under nonreducing conditions, the "aggregates" disappeared (data not shown). (It is not clear why the aggregates are detected by monoclonal antibodies but not by polyclonal antiserum; we are currently purifying these proteins for extensive characterization.) These results suggest that the conformation of single chain mutants 10-60 and 32-84 differ substantially from the nonmutated single chain and the heterodimeric forms.

Because we could now obtain sufficient quantities of single chains bearing the cystine knot mutants, the influence of the disulfide bonds on receptor binding/signal transduction was examined using human kidney 293 cells expressing the LH/hCG receptor. For these bioassays, conditioned media were quantitated with a -specific polyclonal based radioimmune assay (see "Materials and Methods"). It is apparent that except for the 59-87 mutant, the binding affinity of all of the mutants was comparable with the nonmutated tether (Fig. 7A, Table III). The binding affinity of single chain 59-87 was reduced 10-fold, similar to that for the heterodimer containing this mutation in the monomeric  subunit as previously reported (10).

Table III. Biological activity of  cysteine mutants in CG single chain Binding IC50a cAMP ED50b Coupling factor (IC50/ED50)c ng/ml ng/ml CGd 23.3  ± 7.9 4.3  ± 0.8 3.4  ± 1.4 7-31 6.7  ± 1.7 7.4  ± 1.8 1.0  ± 0.3 10-60 26.9  ± 8.4 11.9  ± 3.5e 2.9  ± 1.2e 28-82 6.5  ± 2.1 6.9  ± 0.5 1.0  ± 0.2 32-84 14.1  ± 3.1 12.9  ± 3.6 1.6  ± 0.6 59-87 254.3  ± 55.4 43.2  ± 15.5 8.1  ± 2.7 a Concentrations required to displace 50% of the maximal 125I-hCG bound. Values are presented as mean ± S.E. of at least three independent experiments. b Concentration inducing half-maximal cAMP levels. Data are the mean ± S.E. of at least two independent experiments. c The ratio of IC50 versus the ED50. Numbers are shown as mean ± S.E. of at least two independent experiment sets using same samples. d CG corresponds to the nonmutated CG single chain. e Mean ± range of two experiments.

Adenylate cyclase activation by the single chain was also determined (Fig. 7B). The data demonstrate that signal transduction parallels receptor binding affinity, suggesting that the determinant(s) necessary for bioactivity is conserved between CG heterodimer and single chain (Fig. 7B, Table III). Thus, despite differences in the intracellular behavior and immunoreactivity to conformationally sensitive monoclonal antibodies, the mutants are nevertheless biologically active in vitro, with coupling of receptor binding to signal transduction unaffected by these structural modifications.

DISCUSSION

Within a species, the amino acid sequence of the  subunit is identical (1), and it is the unique structure of the  subunit that ensures binding of each dimer to its cognate receptor. The  subunit, therefore, must be very flexible, since it combines with each of the four glycoprotein  subunits and  subunits from different species (27). Further evidence for this adaptability is the conformational changes occurring during dimer formation (for review, see Refs. 1 and 28-34). Presumably, the configuration of the disulfide bonds of the  subunit is critical for its interaction with the  subunit. Previous studies on these bonds in the monomeric  subunit demonstrated that single mutations in the cystine knot blocked secretion and/or inhibited heterodimer formation (10). Although the results implicated a critical role for these bonds in the intracellular behavior of the subunit, we could not exclude the possibility that the free thiol, rather than the disrupted bond per se, led to the observed changes (9, 10, 19, 20). To address this issue, double cysteine mutations of all the proposed pairs were constructed in the  subunit. It was apparent that mutations in the knot altered the recovery of the subunit from the media. Recovery of variants containing mutations outside the cystine knot, 7-31 and 59-87, was comparable with the wild type  subunit. These changes in the secretion patterns presumably reflect alterations in the folding of the subunit. Consistent with this hypothesis are the experiments showing that, compared with the wild-type  subunit, the cystine knot variants 10-60 and 32-84 combined much less efficiently to the  subunit, with less than 5% of these mutants recovered as dimers. Thus, assessing the role of the 10-60 and 32-84 bonds in the biological action of hCG was precluded. Because tethers containing either mutation at 10-60 or 32-84 were recovered in the media, we could determine their biologic activity. Unexpectedly, it was observed that all of the cystine knot and the 7-31 mutants exhibited high affinity binding for the receptor. When corrected for binding, signal transduction was unchanged regardless of the mutation. These results imply that disulfide bonds of the cystine knot are required for maximal secretion/assembly but that for receptor recognition, not all of the bonds in the core are needed. The single chain bearing the 59-87 mutant exhibited a 10-fold decrease in receptor binding. Several studies have demonstrated the importance of the C-terminal residues 88-92 in the  subunit for maximum binding affinity (1, 35-38). It is likely that disrupting the 59-87 bond perturbed the configuration of the carboxyl-terminal region, leading to the observed decrease in receptor binding. That the altered intracellular behavior of these tethers is not accompanied by a significant change in biologic activity, especially the 10-60 and 32-84 mutants, suggests that the receptor could recognize different conformations of the hormone. While this conclusion is supported by the absence of recognition by the dimer-specific monoclonal antibodies, we cannot exclude a greater liability of the epitopes in the 10-60 and 32-84 mutants to the SDS-polyacrylamide gel electrophoresis conditions used.

Given that perturbing the cystine knot affects the intracellular behavior of both the monomeric  subunit and the single chain molecule, the conformation of this region is likely to be relatively conserved regardless of the associated hormone-specific  subunit. This fixed core determinant may be necessary for the "escort" (42, 43) function of the  subunit, which is critical for rescuing the pituitary  subunits from the endoplasmic reticulum because few, if any, uncombined LH, follitropin, and thyrotropin  subunits are secreted in the absence of co-expressed  subunit (34, 39, 40). Thus, we would propose that the major conformational changes of the  subunit resulting from assembly with the  subunit occurs in regions outside of the cystine knot, i.e. the three hairpin loops (2, 3, 23, 41). Consistent with this interpretation, the recovery of the variants with the 7-31 and 59-87 mutations, which lie outside the knot, was comparable with the wild type.

That the intracellular fate of single chains with mutations of paired cysteines and corresponding monomers parallel each other suggests that a misfolded epitope is not recognized by an intracellular transport component and/or that the mutants are trapped by a chaperone, which leads to enhanced degradation. Although the  domain increases the recovery of the mutated single chains, e.g. in CG10-60 and CG32-84, they are still less than that of the wild type. Thus, even the presence of the  subunit domain is not sufficient to completely override the  Cys mutations.

Other members of the cystine knot growth factor superfamily display similar characteristics (6-8). Mutations of the paired cysteine residues within the knot of tumor growth factor-1 or activin dramatically reduced secretion (6, 7). Thus, together with our data presented here, the cystine knot is apparently the basic frame of the glycoprotein hormone subunits and several growth factors and represents a critical determinant for secretion and assembly of functional ligand. Receptor recognition is apparently less dependent on the configuration of this structure. The data also show the importance of the single chain approach to study structure-function of multisubunit proteins where dependence on the assembly step is essential for biologic action.