Advances in recombinant DNA technology: corifollitropin alfa, a hybrid molecule with sustained follicle-stimulating activity and reduced injection frequency.

BACKGROUND: Recombinant DNA technologies have been used to develop longer-acting therapeutic proteins. One approach is to introduce sequences containing additional glycosylation sites. Using this technique, a new chimeric gene has been developed containing the coding sequences of the FSH beta-subunit and the C-terminal peptide of the hCG beta-subunit, which bears four O-linked oligosaccharide binding sites. Co-expression of the alpha-subunit and the chimeric FSH beta-subunit produces a new recombinant molecule, named corifollitropin alfa, with a prolonged elimination half-life and enhanced in vivo bioactivity compared with wild-type FSH. METHODS: Medline searches by subject and additional searching by hand. RESULTS: Initial studies in pituitary suppressed female volunteers confirmed the extended half-life of the compound. Phase II studies have shown that corifollitropin alfa is able to induce and sustain multi-follicular growth for an entire week in women undergoing ovarian stimulation using GnRH antagonist co-treatment for IVF. Corifollitropin alfa regimens have been developed with dosages of 100 and 150 microg, for patients with body weight 60 kg, respectively. CONCLUSIONS: Corifollitropin alfa is the first long-acting hybrid molecule with sustained follicle-stimulating activity developed for the induction of multi-follicular growth along with GnRH antagonist co-treatment for IVF. This new treatment option may be simpler and more convenient for patients compared with conventional long protocols of daily FSH injections in combination with GnRH agonist co-treatment. The safety and efficacy of such regimens is currently being evaluated in large comparative phase III clinical trials. The development of corifollitropin alfa is the first step towards a new generation of recombinant gonadotrophins.

The efficacy of recombinant equine follicle stimulating hormone (reFSH) to promote follicular growth in mares using a follicular suppression model.

The efficacy of a recently engineered single chain recombinant equine follicle stimulating hormone (reFSH) was investigated in estrous cycling mares whose gonadotropins and follicular activity had been suppressed by concurrent treatment with progesterone and estradiol (P&E). Time of estrus was synchronized in 15 estrous cycling mares during the breeding season with prostaglandins F(2alpha) (PGF(2alpha)). The day after ovulation, mares were treated once daily with P&E for 14 days. Mares received a second injection of PGF(2alpha) on day 6 of the synchronized estrous cycle to induce luteolysis. On day 8 post-ovulation mares were randomly assigned to three groups: small dose reFSH-treatment group (0.5mg reFSH IV, twice daily); large dose reFSH-treatment group (0.85mg reFSH IV twice daily); control group (saline IV, twice daily). reFSH treatment occurred concurrently with the last week of P&E treatment. After a follicle or cohort of follicles reached 35mm in diameter, mares were injected with 0.75mg of recombinant equine luteinizing hormone (reLH) to induce ovulation. Post-treatment ovulation was assessed. Daily blood samples were collected for analysis of FSH, LH, estradiol, progesterone, and inhibin by radioimmunoassay (RIA). On the first day of reFSH/saline treatment, blood samples were collected periodically from 1h prior to treatment to 6h post-injection via an indwelling jugular catheter to determine acute changes in FSH concentrations. Monitoring of follicular activity, estrus, and ovulation was performed daily by utilizing a stallion and transrectal ultrasonography. A difference (por=35mm follicles (days 16-21) than controls. Mares treated with reFSH, at either dose, took less time (average: 2.95+/-0.42 days) to develop 2-3 times more pre-ovulatory follicles than control mares (7.8+/-0.51 days) (p

The expanding role of recombinant gonadotropins in assisted reproduction.

Using recombinant gonadotropins for assisted reproduction of domestic species is still in its infancy. Yet, the purity, potency and pathogen-free nature of recombinant gonadotropins make them attractive alternatives to tissue-derived gonadotropic agents. In this study, the authors summarize the work to date using recombinant gonadotropins to enhance the - fertility of domestic animals and they discussed their recent studies examining the biopotency of single chain analogues of human gonadotropins. In these studies, single chain analogues of follicle stimulating hormone (Fc alpha), chorionic gonadotropin (CG beta alpha) or a gonadotropin construct with dual activity (FcCG beta alpha) were administered to sheep pre-treated with antisera directed against GnRH. Ovulation was induced 3 days after analogue administration using hCG (1000 IU, iv). Although Fc alpha or CG beta alpha alone induced only modest oestradiol production during the pre-hCG period, serum concentrations of oestradiol were markedly increased (p < 0.05) 3 days after administration of FcCG beta alpha or the Fc alpha + CG beta alpha combination. Final ovarian weight was significantly increased (p < 0.05) in animals receiving Fc alpha, Fc alpha + CG beta alpha or FcCG beta alpha. Collectively, these observations demonstrate that the single chain analogues of the human gonadotropins are active in sheep.

The efficacy of a single chain recombinant equine luteinizing hormone (reLH) in mares: induction of ovulation, hormone profiles, and inter-ovulatory intervals.

The objectives of this study were to determine the efficacy of recombinant equine luteinizing hormone (reLH) in shortening the time to ovulation in cycling mares and to determine the effects of treatment on endogenous hormones and inter-ovulatory intervals. In study 1, mares of light horse breeds (3-20 years) were treated with either a vehicle, various doses of reLH, or human chorionic gonadotropin (hCG). Cycling mares were examined by palpation and ultrasound per rectum daily or every 12h from the time of treatment to ovulation. In studies 2 and 3, jugular blood samples were collected daily or every 12h from the time of treatment to ovulation for analysis of LH, follicle stimulating hormone (FSH), estradiol-17beta (E(2)), and progesterone (P(4)) by radioimmunoassays (RIA). Increasing doses of reLH (0.3, 0.6, 0.75, and 0.9 mg) showed increasing effectiveness at inducing ovulation within 48 h of treatment. Treatments with the 0.75 and 0.9 mg doses of reLH resulted in 90% and 80% ovulation rates, which were similar to hCG treatment (85.7%). Except for the early rise in LH after treatment with 0.5, 0.65, and 1.0mg of reLH, hormone profiles appeared to be similar between control and treated cycles. Inter-ovulatory intervals were similar between control and treatment cycles. In conclusion, reLH is a reliable and effective ovulatory agent that does not significantly alter endogenous hormone profiles or affect inter-ovulatory intervals.

Nude mice as a model for gonadotropin-induced adrenocortical neoplasia.

Certain inbred mice (e.g., DBA/2J, CE) develop sex steroid producing adrenocortical tumors following gonadectomy. This adrenal response is thought to result from an unopposed increase in circulating gonadotropins and/or a decrease in factor(s) of gonadal origin. To differentiate between these two possibilities, we utilized the NU/J strain of nude mice, which are immunologically compromised and therefore permissive to xenografts. One group of female nude mice was gonadectomized, while another group of females received xenografts of CHO cells stably transfected with human chorionic gonadotropin (hCG). After 1-2 months, subcapsular adrenocortical neoplasms containing sex steroid-producing cells were observed in both groups. We conclude that high levels of circulating gonadotropins are sufficient to induce adrenocortical tumorigenesis, even in the presence of intact gonads.

Independent activities of FSH and LH structurally confined in a single polypeptide: selective modification of the relative potencies of the hormones.

The human glycoprotein hormones CG, LH, FSH, and TSH are heterodimers composed of a common alpha subunit noncovalently associated with a hormone-specific beta subunit. Recently, it was reported that a covalently fused triple-domain gonadotropin analog containing FSH beta, CG beta, and alpha subunits was dually active because it bound to both FSH and human CG (hCG)/LH receptors. However, it is not known whether both activities can be uncoupled from each other or whether they change in tandem when modifications are made in the molecule. To address this point, we constructed a triple-domain analog containing FSH beta, LH beta, and alpha subunits, and variants of this analog differing in the carboxyl-terminal region of LH beta. All of the analogs exhibited bifunctional action, i.e. they bound to both LH/hCG and human FSH receptors. FSH binding and signal transduction were similar for all variants and differed less than 2-fold from that of the heterodimer. In contrast, the triple-domain variants manifested distinct individual differences in LH activity. Binding affinity of the longest variant was 30-fold lower than that of the heterodimer. Shortening the length of the LH beta carboxyl-terminal region resulted in decreasing affinities between 210- and more than 480-fold. The potency of adenylate cyclase activation for LH/hCG also decreased as the carboxyl length of LH beta subunit decreased. Thus, while minimally affecting the FSH activity, truncating the carboxyl end of the LH beta subunit in the triple-domain analogs alters the alignment of the LH beta-alpha domains, presumably at the junction between the subunits, and perturbs epitopes required for receptor binding. These data imply that the relative potencies of the two gonadotropin components of a triple-domain structure are independent from each other and can be selectively modified. Because there is a strong rationale for FSH/LH combinations for clinical protocols and patients exhibit variations in metabolic responses in the ratio of FSH/LH, the ability to vary the individual activities represents a potential addition to the therapeutic repertoire for treating infertility.

The position of the alpha and beta subunits in a single chain variant of human chorionic gonadotropin affects the heterodimeric interaction of the subunits and receptor-binding epitopes.

The glycoprotein hormone family represents a class of heterodimers, which include the placental hormone human chorionic gonadotropin (CG) and the anterior pituitary hormones follitropin, lutropin, and thyrotropin. They are composed of common alpha subunit and a hormone-specific beta subunit. Based on the CG crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, recent observations using single chain glycoprotein hormone analogs, where the beta and alpha subunits are linked (NH(2)-CGbeta-alpha; CGbetaalpha orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for receptor binding/signal transduction. To study the heterodimeric alignment of the two subunit domains in a single chain and its role in the intracellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of alpha was fused to the CGbeta amino terminus (NH(2)-alpha-CGbeta; alphaCGbeta orientation). The secretion rate of alphaCGbeta from transfected Chinese hamster ovary cells was less than that seen for CGbetaalpha. The alphaCGbeta tether was not recognized by dimer-specific monoclonal antibodies and did not bind to lutropin/CG receptor. To define if one or both subunit domains were modified in alphaCGbeta, it was co-transfected with a monomeric alpha or CGbeta gene. In each case, alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes were formed indicating that CG dimer-specific epitopes were established. The alphaCGbeta/alpha complex bound to receptor indicating that the beta domain in the alphaCGbeta tether was still functional. In contrast, no significant receptor binding of alphaCGbeta/CGbeta was observed indicating a major perturbation in the alpha domain. These results suggest that although dimeric-like determinants are present in both alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes, the receptor binding determinants in the alpha domain of the tether are absent. These results show that generating heterodimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are distinct from those associated with intracellular behavior.

Novel recombinant gonadotropins.

The gonadotropin hormones chorionic gonadotropin, luteinizing hormone and follicle-stimulating hormone are heterodimers that consist of a common alpha subunit noncovalently associated with a hormone-specific beta subunit. Site-directed mutagenesis and gene transfer techniques have been invaluable tools for elucidating structure-function determinants of these hormones. Here, we review how questions about the structural biology of these glycoprotein hormones have provided crucial information for creating analogs (agonists and antagonists) that can be used to treat infertility in the clinic. The ability to manipulate the protein structure of these hormones will enable the engineering of both long- and short-acting therapeutic agents.

Disulfide bond mutations in follicle-stimulating hormone result in uncoupling of biological activity from intracellular behavior.

The crystal structure of human CG reveals that each subunit is a member of the superfamily of cystine-knot growth factors. Although the distribution of the cysteine residues in all the beta-subunits is conserved, the conformation of the human FSH dimer differs from that of the CG/LH dimers. This suggests that the function of the cystine bonded loops in the human FSHbeta-subunit may differ from that in the CGbeta-subunit. To address this issue, we deleted two disulfide bonds in the FSHbeta domain: cys 20-104 and cys 28-82, which correspond to the disulfide bonds 26-110 and 34-88, respectively, in the CGbeta-subunit. The cys 26-110 bond is associated with the "seat-belt" region and cys 34-88 is a bond in the cystine knot. Coexpression of the wild-type alpha-subunit with the FSHbeta cysteine mutants in CHO cells revealed no detectable heterodimer. The FSHbeta mutants were then incorporated into a single chain where the beta-subunit is genetically fused to the alpha-subunit. In such a model, the rate-limiting subunit assembly step is by-passed and mutations that otherwise block heterodimer formation can be evaluated in terms of biological activity. Compared with the nonmutated single chain, the single-chain 28-82 mutant is secreted more slowly and its recovery is substantially reduced, whereas secretion and recovery of the 20-104 mutant was not significantly affected. The receptor binding affinity of the cys 28-82 mutant did not differ from wild-type and binding of the cys 20-104 mutant was decreased only 2-fold. The signal transduction data parallel the binding affinities, although the maximal accumulation of cAMP is less for the cys 20-104 mutant than that seen for cys 28-82 and nonmutated single-chains variants. These data support the hypothesis that the determinants for intracellular behavior and bioactivity of the gonadotropins are not the same, and that the cystine knot is a critical determinant for the formation of a stable, assembly-competent subunit. In addition, the data imply that the "seat-belt" conformation does not play a prominent role in the bioactivity of FSH.

Cellular localization of the human chorionic gonadotropin beta-subunit in transgenic mouse placenta.

CG is a human placental glycoprotein expressed in first-trimester trophoblasts. To examine the regulation of the CGbeta-subunit in an in vivo model, we previously constructed transgenic mice containing the CGbeta gene cluster and demonstrated its expression in the placenta. Here, we determine the cell type responsible for CGbeta synthesis in the mouse by immunohistochemical and in situ hybridization analyses. Unexpectedly, the protein and messenger RNA were not detected in trophoblast or elsewhere in the chorioallantoic placenta but in the parietal endoderm, a separate extraembryonic component of the placenta. The identity of this CGbeta-producing layer was confirmed by the presence of laminin A, a known protein of the parietal endoderm extracellular matrix. However, we observed heterogeneity, with respect to synthesis of laminin A and CGbeta; parietal endoderm cells expressing CGbeta at high levels synthesized less laminin A, and vice versa. The absence of CGbeta production in trophoblasts of the transgenic mouse demonstrates a lack of transcriptional equivalence between rodent and human trophoblasts. The data are consistent with the hypothesis that, in human placenta, one or more transcriptional factors coevolved as members of the CGbeta gene cluster underwent duplication.

The biological action of choriogonadotropin is not dependent on the complete native quaternary interactions between the subunits.

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 beta-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 beta-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 beta-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 beta-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.

Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein.

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.

Glycoprotein hormone structure-function and analog design.

Human chorionic gonadotropin (hCG), luteinizing hormone, follicle-stimulating hormone (FSH), and thyrotropin (TSH) are hormones that share a common alpha subunit but differ in their beta subunits. Recombinant DNA techniques, valuable tools for structure-function analyses, provide an approach for designing therapeutic analogs. FSH is used clinically to stimulate the ovarian follicles for in vitro fertilization and to initiate follicular maturation in women with infertility problems. The CG beta subunit contains a carboxy-terminal extension (CTP) with four serine O-linked oligosaccharides, which is important for the long half-life of hCG. A clinical problem of FSH is its relatively short half-life in circulation. Fusing CTP to the FSH beta coding sequence increased the in vivo potency of the resulting FSH dimer over three-fold. Analogs of the other hormones containing CTP also increase their biologic half-life. Subunit assembly is vital to the function of these hormones. To address whether alpha and beta subunits can be synthesized as one chain and also maintain biological activity, a chimera comprised of the hCG beta subunit genetically fused to the alpha subunit was constructed. The resulting polypeptide was efficiently secreted and displayed an increased biologic activity in vitro and in vivo. Similarly, the single-chain form of FSH also retained in vivo activity. Since subunit dissociation inactivates the activity of the heterodimer, single-chain analogs should have longer biological half-lives. These analogs represent suitable substrates for engineering potent and stable agonists and antagonists.

Synthesis of multi-subunit domain gonadotropin complexes: a model for alpha/beta heterodimer formation.

The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH) are heterodimers, composed of a common alpha subunit assembled to a hormone-specific beta subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers, but not as multimers. Little information is available regarding the steps associated with the assembly reaction. It is unclear if the initial alpha beta engagement results either in the formation of only mature heterodimer or if the nascent complex is reversible and can undergo an exchange of subunits or combine transiently with an additional subunit. This is relevant for the case of LH and FSH, because both are synthesized in the same cell (i.e., pituitary gonadotrophs) and several of the alpha subunit sequences required for association with either the LH beta or FSH beta subunits are different. Such features could favor the generation of short-lived, multi-subunit forms prior to completion of assembly. Previously, we showed that the CG beta or FSH beta subunit genes can be genetically fused to the alpha gene to produce biologically active single chains, CG beta alpha and F beta alpha, respectively. Studies using monoclonal antibodies sensitive to the conformation of the hCG subunits suggested that in contrast to the highly compact heterodimer, the interactions between the beta and alpha domains in the single chain are in a more relaxed configuration. That the tethered domains do not interact tightly predicts that they could combine with an additional subunit to form triple domain complexes. We tested this point by cotransfecting CHO cells with the genes encoding F beta alpha and the CG beta subunit or the CG beta alpha and FSH beta monomer. The CG beta subunit combined noncovalently with F beta alpha to form a F beta alpha/CG beta complex. Ternary complex formation was not restricted to a specific set of single chain/monomeric subunit, because a CG beta alpha/FSH beta complex was also detected implying that triple domain intermediates could be transiently generated along the secretory pathway. Monoclonal antibodies specific for the CG heterodimer recognized the F beta alpha/CG beta complex, which suggests that the epitopes unique for dimeric CG were established. In addition, media containing F beta alpha/CG beta displayed high-affinity binding to both CG and FSH receptors. The presence of CG activity is presumptive for the existence of a functional F beta alpha/CG beta complex, because neither F beta alpha nor the uncombined CG beta subunit binds to CG receptor. These data show that the alpha subunit of the tether, although covalently linked to the FSH beta domain, can functionally interact with a different beta subunit implying that the contacts in the nascent alpha beta dimer are reversible. The formation of a functional single chain/subunit complex was not restricted to the FSH single chain/CG beta subunit since CG single chain interacts with the monomeric FSH beta subunit and exhibits FSH activity. The presence of the triple domain configuration does not abolish bioactivity, suggesting that although the gonadotropins are heterodimers, the cognate receptor is capable of recognizing a larger ligand composed of three subunit domains.

Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis.

The role of FSH in gonadal tumorigenesis and, in particular, in human ovarian cancer has been debated. It is also unclear what role the elevated FSH levels in the inhibin-deficient mouse play in the gonadal tumorigenesis. To directly assess the role of FSH in gonadal growth, differentiation, and gonadal tumorigenesis, we have generated both gain-of-function and loss-of-function transgenic mutant mice. In the gain-of-function model, we have generated transgenic mice that ectopically overexpress human FSH from multiple tissues using a mouse metallothionein-1 promoter, achieving levels far exceeding those seen in postmenopausal women. Male transgenic mice are infertile despite normal testicular development and demonstrate enlarged seminal vesicles secondary to elevated serum testosterone levels. Female transgenic mice develop highly hemorrhagic and cystic ovaries, have elevated serum estradiol and progesterone levels, and are infertile, mimicking the features of human ovarian hyperstimulation and polycystic ovarian syndromes. Furthermore, the female transgenic mice develop enlarged and cystic kidneys and die between 6-13 weeks as a result of urinary bladder obstruction. In a complementary loss-of-function approach, we have generated double-homozygous mutant mice that lack both inhibin and FSH by a genetic intercross. In contrast to male mice lacking inhibin alone, 95% of which die of a cancer cachexia-like syndrome by 12 weeks of age, only 30% of the double-mutant male mice lacking both FSH and inhibin die by 1 yr of age. The remaining double-mutant male mice develop slow-growing and less hemorrhagic testicular tumors, which are noted after 12 weeks of age, and have minimal cachexia. Similarly, the double-mutant female mice develop slow-growing, less hemorrhagic ovarian tumors, and 70% of these mice live beyond 17 weeks. The double-mutant mice demonstrate minimal cachexia in contrast to female mice lacking only inhibin, which develop highly hemorrhagic ovarian tumors, leading to cachexia and death by 17 weeks of age in 95% of the cases. The milder cachexia-like symptoms of the inhibin and FSH double-mutant mice are correlated with low levels of serum estradiol and activin A and reduced levels of aromatase mRNA in the gonadal tumors. Based on these and our previous genetic analyses, we conclude that elevated FSH levels do not directly cause gonadal tumors. However, these results suggest FSH is an important trophic modifier factor for gonadal tumorigenesis in inhibin-deficient mice.

Maternal-fetal tolerance is maintained despite transgene-driven trophoblast expression of MHC class I, and defects in Fas and its ligand.

During mammalian pregnancy, one or more semiallogeneic fetuses gestate in direct contact with the maternal circulation and uterine tissue. However, a damaging maternal immune response is not normally provoked. We studied two possible mechanisms for this maternal-fetal tolerance, alone and in combination. First, we directly tested the hypothesis that the striking absence of MHC class I molecules on most placenta trophoblasts protects the fetus from maternal immune attack, by creating transgenic mice which express Ld in giant cell trophoblasts. Second, because Fas ligand (FasL) may contribute to immune privilege, we tested whether functional FasL expression by the fetus, or Fas expression by the mother, contributes to successful reproduction in a fully allogeneic breeding. Our data indicate that neither abnormal expression of MHC class I in giant cells, nor disruption of the Fas-FasL system, nor a combination of these two defects, has an adverse effect on pregnancy outcome. These results suggest that during healthy allogeneic pregnancy, down-regulation of MHC class I and expression of FasL on placenta are not critical events, and other factors must prevent a harmful maternal immune response.

Dissociation of early folding events from assembly of the human lutropin beta-subunit.

The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common alpha-subunit and beta-subunits that confer biological specificity. LHbeta and CGbeta share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHbeta assembles with the alpha-subunit more slowly than does hCGbeta, and only a fraction of the LHbeta synthesized is secreted, whereas CGbeta is secreted efficiently. To understand why the assembly and secretion of these related beta-subunits differ, we studied the folding of LHbeta in CHO cells transfected with either the LHbeta gene alone, or in cells cotransfected with the gene expressing the common alpha-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pbeta1, to the second major folding form, pbeta2, did not differ significantly from the pbeta1-to-pbeta2 conversion of CGbeta, suggesting that variations between the intracellular fates of the two beta-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGbeta, where the folding to pbeta2 results in an assembly-competent product, apparently greater than 90% of the LH pbeta2 recovered from LHbeta-transfected CHO cells was assembly incompetent, accounting for inefficient LHbeta assembly with the alpha-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGbeta, all 12 LHbeta cysteine residues formed S-S linkages as soon as pbeta2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pbeta2 and excess urinary alpha-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGbeta, LHbeta homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respective beta-subunits.

The carboxyl-terminal region is a determinant for the intracellular behavior of the chorionic gonadotropin beta subunit: effects on the processing of the Asn-linked oligosaccharides.

The placental hormone human CG (hCG) consists of two noncovalently linked alpha- and beta-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common alpha subunit but differ in their structurally distinct beta subunits. The CGbeta subunit is distinguished among the beta subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGbeta subunit (CGbeta114) with the alpha-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGbeta and CGbeta114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.

Conversion of thyrotropin heterodimer to a biologically active single-chain.

TSH and the gonadotropins, FSH, LH, and CG are a family of heterodimeric glycoprotein hormones composed of a common alpha-subunit noncovalently linked to a hormone specific beta-subunit. Assembly of alpha- and beta-subunits is essential for hormone-specific posttranslational modifications, receptor binding, and bioactivity. Structure-function studies of TSH and gonadotropins using site-directed mutagenesis can often affect folding, assembly, and secretion of the hormone. To circumvent these difficulties, recently, the gonadotropin heterodimers were converted to single chains. Here we converted the hTSH heterodimer to a biologically active single chain by genetically fusing the amino terminal end of the common alpha-subunit to the carboxyl terminal end of hTSHbeta in the presence or absence of hCGbeta carboxyl terminal peptide (CTP), which was used as a linker. Wild-type hTSH and the single chains were expressed in Chinese hamster ovary (CHO) cells, and they were efficiently secreted. Although the secretion rate of the single chain was 3-fold higher than that of hTSH wild-type. Moreover, the secretion of the single chain in the presence of the CTP linker was dramatically increased. On the other hand, receptor binding and in vitro bioactivity of the single chains were similar to that of hTSH wild-type. These data indicate the potential of the single chain approach to further investigate structure-function relationships of TSH.

Cystine knot of the gonadotropin alpha subunit is critical for intracellular behavior but not for in vitro biological activity.

The common alpha subunit of glycoprotein hormones contains five disulfide bonds. Based on the published crystal structure, the assignments are 7-31, 59-87, 10-60, 28-82, and 32-84; the last three comprise the cystine knot, a structure also seen in a variety of growth factors. Previously, we demonstrated that the efficiency of secretion and the ability to form heterodimers by alpha subunits bearing single cysteine residue mutants in the cystine knot were significantly reduced. These results suggested that the cystine knot is critical for the intracellular integrity of the subunit. To assess if the presence of the free thiol affected the secretion kinetics, we constructed paired cysteine mutants of each disulfide bond of the alpha subunit. The secretion rate for these monomers was comparable with wild type except for the alpha-10-60 mutant, which was 40% lower. The recovery of the alpha7-31 and alpha59-87 mutants was greater than 95%, whereas for the cystine knot mutants, it was 20-40%. Co-expression of the wild-type chorionic gonadotropin beta subunit with double cysteine mutants did not enhance the recovery of alpha mutants in the media. Moreover, compared with wild-type, the efficiency of heterodimer formation of the alpha10-60 or alpha32-84 mutants was less than 5%. Because subunit assembly is required for biological activity, studies on the role of these disulfide bonds in signal transduction were not possible. To bypass the assembly step, we exploited the single chain model, where the alpha and beta subunits are genetically fused. The recovery of secreted tethered gonadotropins bearing mutations in the cystine knot was increased significantly. Although dimer-specific monoclonal antibodies discriminated the conformation of single chain alpha10-60 and alpha32-84 mutants from the native heterodimer, these mutants were nevertheless biologically active. Thus, individual bonds of cystine knot are important for secretion and heterodimer formation but not for in vitro bioactivity. Moreover, the data suggest that the native heterodimer configuration is not a prerequisite for receptor binding or signal transduction.