Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops)

Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for ¹²⁵I-hCG, (0.9–2.5 × 10⁹ M^?1, and 0.7–1.64 × 10⁹ M^?1 respectively, for green and rhesus monkeys) and low binding capacity (0.343–0.682 fmol/mg and 0.198–0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 10⁹ M^?1) and low binding capacity (1.04 fmol/mg tissue homogenate) for ¹²⁵I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.     

Selective radioimmunoassays for human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG).

Highly specific radioimmunoassay systems were developed for measurement of hLH and hCG using antisera purified by affinity chromarography or simple adsorption to select the antibodies reacting specifically with either gonadotropin. Such systems permit specific measurement of lLH and hCG in samples containing both. These assays are suitable for various clinical and physiological studies, particularly, study of pituitary functions in the presence of chorionic or trophoblastic secretion.     

Opposite contribution of two ligand-selective determinants in the N-terminal hormone-binding exodomain of human gonadotropin receptors

The nine leucine-rich repeat-containing exodomains of the human FSH receptor (hFSH-R) and the human LH/chorionic gonadotropin receptor (hLH-R) harbor molecular determinants that allow the mutually exclusive binding of human FSH (hFSH) and human LH (hLH)/human chorionic gonadotropin (hCG) when these hormones are present in physiological concentrations. Previously, we have shown that the beta-strands of hLH-R leucine-rich repeats 3 and 6 can confer full hCG/hLH responsiveness and binding when simultaneously introduced into a hFSH-R background without affecting the receptor's responsiveness to hFSH. In the present study, we have determined the nature of contribution of each of these two beta-strands in conferring hCG/hLH responsiveness to this mutant hFSH-R. Human LH-R beta-strand 3 appeared to function as a positive hCG/hLH determinant by increasing the hCG/hLH responsiveness of the hFSH-R. In contrast, mutagenesis of hFSH-R beta-strand 6, rather than the introduction of its corresponding hLH-R beta-strand, appeared to allow the interaction of hCG/hLH with the hFSH-R. Hence, hFSH-R beta-strand 6 functions as a negative determinant and, as such, restrains binding of hCG/hLH to the hFSH-R. Detailed mutagenic analysis revealed that the ability of the hFSH-R to interact with hCG/hLH depends primarily on the identity of two amino acids (Asn104, a positive LH-R determinant, and Lys179 a negative FSH-R determinant) that are situated on the C-terminal ends of beta-strands 3 and 6, respectively.     

Characterization of estrogen and antiestrogen binding to the cytosol and microsomes of breast tumors

The binding of [3H]estradiol and [3H]hydroxytamoxifen to the cytosol and microsomal fractions of several human breast tumors was investigated. By washing microsomal membranes with a KCl-free or a KCl-containing medium we could distinguish between intrinsic, extrinsic and contaminant estradiol binding sites in these membranes. We observed that treatment of the microsomes with low salt medium removes about 80% of the total estradiol binding sites, whereas 20% are not extractable. The concentration of unextractable [3H]estradiol binding sites in the microsomes varies in proportion to the level of cytosolic estrogen receptors (ER). About 10% of the total extranuclear specific estrogen binding sites was consistently found tightly associated to the microsomal fraction, which displays an affinity for estradiol (Kd = 0.1-0.6 nM) similar to that of the cytosolic ER. The displacement of [3H]estradiol with unlabeled hormone or with the antiestrogens, nafoxidine, enclomiphene and tamoxifen (TAM) exhibits identical IC50 values either in the cytosol or in the microsomal membranes. On the other hand, the microsomal fraction of breast tumors also binds [3H]hydroxyTAM, but with higher capacity and lower affinity than those of the cytosolic fraction. Furthermore, we did not observe correlation between the concentrations of ER and of antiestrogen binding sites (AEBS) in the tumors. These results indicate that microsomal membranes of human breast tumors contain estrogen binding sites which may be related to the cytosol ER recycling and that specific AEBS are predominantly localized in this membrane system. Furthermore, it is shown that the magnitude of estradiol binding to microsomes depends on the ER positive degree of the tumors, whereas the magnitude of the antiestrogen binding to the microsomes is independent of the ER status of the tumors.     

Specific binding of androgen and androgen-receptor complex by microsomes from rat ventral prostate

Microsomes from rat ventral prostate show the presence of a high affinity-low capacity population of androgen-binding sites with affinity for ionic exchange resin similar to that of cytosol androgen receptor (AR), as manifested by similar results obtained with hydroxylapatite. The affinity for mibolerone was similar for both forms (Ka = 0.5-2.9 x 10(10) M-1). The membrane-bound form can be extracted in hypotonic buffer, with retention of binding properties. Isotonic sucrose allowed higher degree of extractability of the microsomal AR than 10% (v/v) glycerol. The presence of hormone lends stability to the microsomal AR, while high salt or nonionic detergents have a deleterious effect on their longevity. The microsomal receptor form is not sensitive to serine-proteases as opposed to the cytosol AR. After exhaustive extraction of binding sites, microsomes are capable of accepting cytosol mibolerone-receptor complexes to a level corresponding to the concentration of depleted binding sites; microsomes from non-target tissue do not manifest such capability. Microsomal AR complexes do not bind DNA and they are not activated after heat treatment. Mixed preparations of extracted microsomal complexes with cytosol complexes showed heat-induced increased ability to bind DNA to the same level of diluted cytosol complex alone, indicating the absence of a microsomal inhibitor of DNA binding. The results indicate the co-existence of a non-DNA binding form of the AR in the microsomal membranes with the classical DNA binding form of the AR present in the cytosol of ventral prostate homogenates.     

Comparison of the ability of seven gonadotropin preparations from different mammalian sources to interact with the adenylyl cyclase system in corpora lutea from rabbits and rats

The effects of guanine nucleotides and magnesium (Mg2+) on the interaction of seven different gonadotropin preparations with their rabbit and rat luteal receptors were studied and compared to the ability of these gonadotropins to stimulate luteal adenylyl cyclase activity. In both the rabbit and rat, human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) were less efficacious than the other gonadotropin preparations in stimulating luteal adenylyl cyclase activity and thus behaved as partial agonists. Addition of 2 mM MgCl2 increased the affinity of the rat luteal receptors for all seven gonadotropins tested, while in the rabbit Mg2+ increased the affinities for porcine, bovine, ovine, rat and rabbit LH but did not significantly alter the affinities for hCG or hLH. In no instance did the addition of 100 microM GTP alter the affinity of the receptor from that observed in the absence or presence of Mg2+. A positive correlation existed for both species between the Kd values calculated from binding experiments and the Kact values obtained in adenylyl cyclase assays suggesting that the specific gonadotropin-binding sites present in rabbit and rat luteal membranes represent receptors which mediate the stimulatory effect of LH. The magnitude of the Mg2+-induced increase in affinity of a given gonadotropin preparation for its receptor was correlated with the efficacy with which that gonadotropin stimulated luteal adenylyl cyclase activity in both the rabbit and rat.     

The differential binding affinities of the luteinizing hormone (LH)/choriogonadotropin receptor for LH and choriogonadotropin are dictated by different extracellular domain residues

The high degree of amino acid sequence homology and the divergent ligand binding affinities of the rat (r) and human (h) LH receptors (LHRs) allowed us to identify amino acid residues of their extracellular domain that are responsible for the different binding affinities of bovine (b) and hLH, and human choriogonadotropin (hCG) to the hLHR and rLHR. Because of the proposed importance of the beta-sheets of the leucine-rich repeats (LRRs) of the extracellular domain of the LHR on hormone binding, we examined 10 divergent residues present in these regions by analyzing two complementary sets of mutants in which hLHR residues were substituted with the corresponding rLHR residues and vice versa. These experiments resulted in the identification of a single residue (a Ile or Ser in the C-terminal end of LRR2 of the hLHR or rLHR, respectively) that is important for hLH binding affinity. Surprisingly, however, this residue does not affect hCG or for bLH binding affinity. In fact, the results obtained with bLH and hCG show that several of the divergent residues in the beta-sheets of LRR1-9 affect bLH binding affinity, but none of them affect hCG binding affinity. Importantly, our results also emphasize the involvement of residues outside of the beta-sheets of the LRRs of the LHR in ligand binding affinity. This finding has to be considered in future models of the interaction of LH/CG with the LHR.     

Ligand selectivity of gonadotropin receptors. Role of the beta-strands of extracellular leucine-rich repeats 3 and 6 of the human luteinizing hormone receptor

The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hFSH-R) and human luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is determined by their approximately 350 amino acid-long N-terminal receptor exodomains that allow the mutually exclusive binding of human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) when these hormones are present in physiological concentrations. The exodomains of each of these receptors consist of a nine-leucine-rich repeat-containing subdomain (LRR subdomain) flanked by N- and C-terminal cysteine-rich subdomains. Chimeric receptors, in which the structural subdomains of the hFSH-R exodomain were substituted with those of the hLH-R, showed a similar high responsiveness to human chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR subdomain of the hLH-R. In addition, these chimeric receptors showed no responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor exodomains are predicted to adopt a horseshoe-like conformation, of which the hormone-binding concave surface is composed of nine parallel beta-strands. Receptors in which individual beta-strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG and hLH selectivity is predominantly determined by hLH-R beta-strands 3 and 6. A mutant receptor in which the hFSH-R beta-strands 3 and 6 were substituted simultaneously with their hLH-R counterparts displayed a responsiveness to hCG and hLH similar to that of the wild type hLH-R. Responsiveness to hFSH was not affected by most beta-strand substitutions, suggesting the involvement of multiple low-impact determinants for this hormone.     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.     

Binding characteristics of progesterone and antiprogestin ZK 98.299 in human endometrial and myometrial cytosol

The binding of ZK 98.299, a synthetic progesterone antagonist, with human endometrium and myometrium cytosol was studied and compared with that of progesterone. Progesterone showed specific saturable binding to its receptors in both endometrium and myometrium. ZK 98.299 and progesterone were mutually competitive for binding to progesterone receptors; however, the relative binding affinity of ZK 98.299 was 16% that of progesterone. ZK 98.299 exchanged the progesterone-labelled receptor sites. [3H]ZK 98.299 showed specific binding which was linearly related to the cytosol protein concentration. The binding was not saturable at 15 nM of ligand. The binding capacity and binding affinity of ZK 98.299 receptor was less than that of progesterone. Progesterone also partially displaced the binding of [3H]ZK 98.299. This study suggest that ZK 98.299 and progesterone both bind to the same protein. However, whether ZK 98.299 binds to progesterone receptors alone or even to other functionally related sites is not known. It appears that ZK 98.299 when present in higher concentration than progesterone would be an effective receptor ligand.     

Ontogeny of the gonadal receptor for luteinizing hormone in the pig

Homogenates of porcine ovaries and testes collected between 70 d post coitum and 42 d post partum were incubated with radiolabelled human chorionic gonadotropin (hCG) to determine the presence and relative amounts of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors. Specific binding of (125)I-hCG to ovaries and testes occurred at all stages of fetal and postnatal development. Ovarian tissue possessed relatively low affinity, high capacity LH/hCG binding sites that were most numerous at Day 80 of gestation and decreased thereafter. In contrast, high affinity, low capacity LH/hCG binding sites were found in the testes. In males, the total number of LH/hCG binding sites remained stable until near term and then increased with age, but the number of sites per gram of testicular tissue did not change (P>0.05). In summary, differential binding of LH/hCG in gonadal tissue occurred in male and female piglets during pre- and post-natal periods, and this binding reflected the known differential pattern of development of the male and female gonad.     

Characterization studies of a rat hepatic cytosolic androgen-binding protein

A rat hepatic cytosolic [3H]methyltrienolone (R1881) binding protein was studied under various conditions. This protein was also compared with the male-specific high capacity--low affinity estrogen-binding protein derived from the same cytosolic fraction. Analysis of the R1881 binding protein in adult (60-85 days old) male rat liver cytosol indicated the presence of a high affinity--low capacity binding site (Kd = 0.3 nM; Bmax = 5.9 fmol/mg) and a lower affinity--higher capacity component (Kd = 10.4 nM; Bmax = 131 fmol/mg). The latter component was eliminated by addition of triamcinolone or cortisol to the assay mixture. Steroid binding to the high affinity R1881 site was specific for testosterone, dihydrotestosterone, androstenedione, and mibolerone, with a moderate specificity to cyproterone acetate, flutamide hydroxide, and estradiol. Saturation studies indicated that these steroids were binding to the same or a similar high affinity component except for flutamide hydroxide which produced nonsaturable displacement. The high affinity site had no specificity for progesterone, diethylstilbestrol, or cortisol. Like the high capacity--low affinity protein, this protein was not present in the immature, adult, or 10-day ovariectomized adult female. However, unlike the high capacity--low affinity protein, it was present in low quantities in the immature male. In addition, castration of the adult for 18 h, 4 days, or 10 days or hypophysectomy for 10-17 days did not have a significant effect on the high affinity component compared with the controls. Testosterone administration to these animals did not alter this protein binding. These studies indicate that a specific, high affinity--low capacity androgen-binding protein exists in rat hepatic cytosol. Furthermore, this protein shows age and sex dependency, but its presence is not affected by altering gonadal or hypophyseal factors in the adult male.     

Gonadotropin binding factor(s). Extraction of high affinity gonadotropin binding sites from rat testis and partial characterization of their interaction with human follitropin, lutropin, and choriogonadotropin.

Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal. These studies reveal that: 1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH. The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4 degrees, 27 degrees, and 37 degrees. 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors. 3. The equilibrium dissociation constant (Kd) of 125I-hFSH binding to the soluble factor was 6.0 +/- 0.58 X 10(-10) M, consistent with the values obtained from the membrane binding studies. Similarly, the Kd value for 125I-hLH to the soluble factor(s) was 3.33 +/- 0.3 X 10(-9) M, comparable to the values obtained from the membrane binding studies. Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH. Furthermore, two classes of binding sites for 125I-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 +/- 0.5 X 10(-11) M and 1.35 +/- 1.2 X 10(-9) M. The apparent Hill coefficient of interaction of 125I-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites. 4. The binding of 125I-hLH and 125I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher Kd for hLH (294 +/- 13.8 mug/ml) compared to hFSH (66.6 +/- 4 mug/ml). These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 X 10(-9) M to 6.0 X 10(-10) M versus the ratio of 294 mug/ml to 66.6 mug/ml. 5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin. The binding of FSH at low factor concentrations was found to be 5- to 10-fold greater than prolactin, growth hormone, and albumin. 6. The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood. 7. The effect of ethanol upon solubilization of the factor(s) has been investigated. The factor(s) can be extracted with buffer or water alone. However, 10 to 25% of ethanol (v/v) facilitates the process of solubilization. The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes. The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent. 8. Finally the effect of soluble factors on classical membrane binding was investigated...     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

A method for determination of the specific activity of receptor-bound human chorionic gonadotropin (hCG).

Precise knowledge of the specific activity (S.A., muc/mug) of the receptor bound radiolabelled hormone is required for study of the stoichiometry of peptide hormone-receptor interactios. A radioligand receptor assay using 131-I-hCG as tracer and transplantable mouse luteoma homogenates (Biol. Reprod. 8:550, 1973) as a source of receptor was used as a model to determine the specific activity of receptor bound 125-I-hCG. Progressive saturation of the gonadotropin receptor by 125-I-hCG suggests the presence of a high affinity-low capacity binding event (saturating between 14 and 37 ng/100 mg homogenate) that does not distinguish between non-radioactive hCG and 125-I-hCG, and a low affinity-high capacity binding event (saturating between 240 and 270 ng/100 mg homogenate) that shows a preference for non-radioactive hCG over 125-I-hCG. Parallelism between bound 125-I-hCG and non-radioactive hCG in terms of competition with tracer 131-I-hCG could only be demonstrated for the high affinity event.     

Biochemical evidence that human placental lactogen and human chorionic gonadotropin are not stored in cytoplasmic secretion granules

The intracellular storage sites for the human placental hormones placental lactogen (hPL) and chorionic gonadotropin (hCG) are unknown. To determine whether hPL and hCG are stored in cytoplasmic secretion granules, we have compared the localization of hPL and hCG in placental homogenates following differential and density-gradient centrifugations to those of prolactin (PRL) and luteinizing hormone (LH) in human and rat pituitary homogenates. In the differential centrifugation studies, 93.1 +/- 4.1% (mean +/- SE) of the hPL and 79.4 +/- 6.0% of the hCG were detected in the postmicrosomal supernatant of placental homogenates. In contrast, 95-98% of the hPRL and hLH in the pituitary homogenates were detected in particulate fractions. Following centrifugation on sucrose-density gradients, particulate hPL and hCG were distributed diffusely throughout the gradients, while greater than 90% of the pituitary hormones sedimented as single peaks with densities of 1.22 g/cm3. When human placental and rat pituitary tissues were homogenized together prior to differential and density-gradient centrifugations, similar marked differences were observed between the distribution of the placental and pituitary hormones. These results strongly suggest that the placental hormones hPL and hCG, unlike pituitary PRL and LH, are not stored in large secretory granules. Differences in the intracellular storage sites of the hormones may explain, in part, differences in the regulation of peptide hormone secretion by placental and pituitary tissues.     

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.     

Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction. These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit. The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins. The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants. In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system. A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity. Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active. The preliminary data also suggested that P. pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated. This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.     

Chemical synthesis of peptide fragments of the hormone-specific beta-subunit of human follicle-stimulating hormone

In order to determine the specific antigenic determinants of human follicle-stimulating hormone (hFSH), hFSH-beta peptides with amino acid residues 33-49 (V2), 95-118 (V3), 76-118 (V3 + 1/2 C2), 1-33 (V1 + C1), 22-33 (1/2C1), and 95-107 (V3 + 1/4C2) according to the nomenclature of Stewart and Stewart [Stewart, M., & Stewart, F. (1977) J. Mol. Biol. 116, 175] as well as additional peptides with the residues 93-107, 91-107, 89-107, 87-107, and 85-107 were chemically synthesized. The peptides were examined in radioimmunoassay systems of FSH, luteinizing hormone (LH), or human chorionic gonadotropin (hCG). V3 + 1/2C2 and V1 + C1 showed immunological activity, whereas the other peptides did not. Antibodies were raised in rabbits against these peptides and examined for specific binding with hFSH, LH, thyroid-stimulating hormone (TSH), and hCG. V3 + 1/2C2 as well as V1 + C1 produced antisera, which specifically bound hFSH, hLH, and hTSH, indicating that the amino acid sequences contained in hFSH-beta peptides V3 + 1/2C2 and V1 + C1 share common antigenic sites with hLH and hTSH. Antisera were produced in rabbits against hFSH-beta, against reduced and S-aminoethylated hFSH-beta (AE-FSH-beta), and against AE-FSH-beta coupled to hemocyanin. Reduced and S-aminoethylated beta-subunit of FSH-beta coupled with hemocyanin produced antisera in rabbits that specifically bound only hFSH and not hLH, hTSH, or hCG.