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The glycoprotein hormone (GPH) system is fundamentally significant in regulating the physiology of chordates, such as thyroid activity and gonadal function. However, the knowledge of the GPH system in the primitive chordate ascidian species is largely lacking. Here, we reported an ancestral GPH system in the ascidian (Styela clava), which consists of GPH α subunit (Sc-GPA2), GPH β subunit (Sc-GPB5), and the cognate leucine-rich repeat-containing G protein-coupled receptor (Sc-GPHR). Comparative structure analysis revealed that distinct from vertebrate GPH β subunits, Sc-GPB5 was less conserved, showing an atypical N-terminal sequence with a type II transmembrane domain instead of a typical signal peptide. By investigating the presence of recombinant Sc-GPA2 and Sc-GPB5 in cell lysates and culture media of HEK293T cells, we confirmed that these two subunits could be secreted out of the cells via distinct secretory pathways. The deglycosylation experiments demonstrated that N-linked glycosylation only occurred on the conserved cysteine residue (N78) of Sc-GPA2, whereas Sc-GPB5 was non-glycosylated. Although Sc-GPB5 exhibited distinct topology and biochemical properties in contrast to its chordate counterparts, it could still interact with Sc-GPA2 to form a heterodimer. The Sc-GPHR was then confirmed to be activated by tethered Sc-GPA2/GPB5 heterodimer on the Gs-cAMP pathway, suggesting that Sc-GPA2/GPB5 heterodimer-initiated Gs-cAMP signaling pathway is evolutionarily conserved in chordates. Furthermore, in situ hybridization and RT-PCR results revealed the co-expression patterns of Sc-GPA2 and Sc-GPB5 with Sc-GPHR transcripts, respectively in ascidian larvae and adults, highlighting the potential functions of Sc-GPA2/GPB5 heterodimer as an autocrine/paracrine neurohormone in regulating metamorphosis of larvae and physiological functions of adults. Our study systematically investigated the GPA2/GPB5-GPHR system in ascidian for the first time, which offers insights into understanding the function and evolution of the GPH system within the chordate lineage., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

A novel heterodimeric glycoprotein hormone (GpH) comprised of alpha (GpA2) and beta (GpB5) subunits was discovered in 2002 and called thyrostimulin for its ability to activate the TSH receptor in mammals, but its central function in vertebrates has not been firmly established. We report here the cloning and expression of lamprey (l)GpB5, and its ability to heterodimerize with lGpA2 to form a functional l-thyrostimulin. The full-length cDNA of lGpB5 encodes 174 amino acids with ten conserved cysteine residues and one glycosylation site that is conserved with other vertebrate GpB5 sequences. Phylogenetic and synteny analyses support that lGpB5 belongs to the vertebrate GpB5 clade. Heterodimerization of lGpB5 and lGpA2 was shown by nickel pull-down of histidine-tagged recombinant subunits. RNA transcripts of lGpB5 were detected in the pituitary of lampreys during both parasitic and adult life stages. Intraperitoneal injection with lGnRH-III (100 μg/kg) increased pituitary lGpA2, lGpB5, and lGpHβ mRNA expression in sexually mature, adult female lampreys. A recombinant l-thyrostimulin produced by expression of a fusion gene in Pichia pastoris activated lamprey GpH receptors I and II as measured by cAMP enzymeimmunoassay. In contrast to jawed vertebrates that have pituitary LH, FSH, and TSH, our data support that lampreys only have two functional pituitary GpHs, lGpH and l-thyrostimulin, which consist of lGpA2 and unique beta subunits. It is hypothesized that lGpH and l-thyrostimulin differentially regulate reproductive and thyroid activities in some unknown way(s) in lampreys., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hypo-glycosylated hFSH(21/18) (possesses FSHβ(21) and FSHβ(18)bands) was isolated from hLH preparations by immunoaffinity chromatography followed by gel filtration. Fully-glycosylated hFSH(24) was prepared by combining the fully-glycosylated FSHβ(24) variant with hCGα and isolating the heterodimer. The hFSH(21/18) glycoform preparation was significantly smaller than the hFSH(24) preparation and possessed 60% oligomannose glycans, which is unusual for hFSH. Hypo-glycosylated hFSH(21/18) was 9- to 26-fold more active than fully-glycosylated hFSH(24) in FSH radioligand assays. Significantly greater binding of (125)I-hFSH(21/18) tracer than hFSH(24) tracer was observed in all competitive binding assays. In addition, higher binding of hFSH(21/18) was noted in association and saturation binding assays, in which twice as much hFSH(21/18) was bound as hFSH(24). This suggests that more ligand binding sites are available to hFSH(21/18) in FSHR than to hFSH(24). Hypo-glycosylated hFSH(21/18) also bound rat FSHRs more rapidly, exhibiting almost no lag in binding, whereas hFSH(24) specific binding proceeded very slowly for almost the first hour of incubation., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Background: Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) both have a role to play in follicular development during the natural menstrual cycle. LH supplementation during controlled ovarian stimulation (COS) for assisted reproductive technology (ART) is used for patients with hypogonadotropic hypogonadism. However, the use of exogenous LH in COS in normogonadotropic women undergoing ART is the subject of debate. The aim of this study was to investigate characteristics of infertile women who received the 2:1 formulation of follitropin alfa and lutropin alfa (indicated for stimulation of follicular development in women with severe LH and FSH deficiency) in German clinical practice., Methods: A 3-year, multicentre, open-label, observational/non-interventional, post-marketing surveillance study of women (21-45 years) undergoing ART. Primary endpoint: reason for prescribing the 2:1 formulation of follitropin alfa and lutropin alfa. Secondary variables included: COS duration/dose; oocytes retrieved; fertilization; clinical pregnancy; ovarian hyperstimulation syndrome (OHSS)., Results: In total, 2220 cycles were assessed; at least one reason for prescribing the 2:1 formulation was given in 1834/2220 (82.6%) cycles. Most common reasons were: poor ovarian response (POR) (39.4%), low baseline LH (17.8%), and age (13.8%). COS: mean dose of the 2:1 formulation on first day, 183.1/91.5 IU; mean duration, 10.8 days. In 2173/2220 (97.9%) cycles, human chorionic gonadotrophin was administered. Oocyte pick-up (OPU) was attempted in 2108/2220 (95.0%) cycles; mean (standard deviation) 8.0 (5.4) oocytes retrieved/OPU cycle. Fertilization (≥1 oocyte fertilized) rates: in vitro fertilization (IVF), 391/439 (89.1%) cycles; intracytoplasmic sperm injection (ICSI)/IVF + ICSI, 1524/1613 (94.5%) cycles. Clinical pregnancy rate: all cycles, 25.9%; embryo transfer cycles, 31.3%. OHSS: hospitalization for OHSS, 8 (0.36%) cycles, Grade 2, 60 (2.7%), and Grade 3, 1 (0.05%)., Conclusions: In German routine clinical practice, the most common reasons for using the 2:1 formulation of follitropin alfa and lutropin alfa for women undergoing ART were POR, low baseline LH, and age. Severe OHSS incidence was low and similar to that reported previously.

Described herein is the first total chemical synthesis of the unique α-subunit of the human glycoprotein hormone (α-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, α-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared. One contains the disaccharide chitobiose at the natural N-glycosylation sites. The other contains dodecamer oligosaccharides at these same sites. The dodecamer sugar is a consensus sequence incorporating the key features associated with human glycoproteins.

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and β genes with [TSHβ- carboxyl-terminal peptide (CTP)-α] or without (TSHβ-α) the CTP of human chorionic gonadotropin β-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHβ-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T(4) in circulation after 2 and 4 h of treatment. However, the level of T(4) after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHβ-α and TSHβ-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHβ-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

Enzymatic deglycosylation followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples. Specific glycosidases were used to remove sugars from glycoproteins in a controlled fashion leaving the protein core intact; the resulting change in molecular weight could be detected as shifts in gel mobility. Alternatively, glycan-sensitive reagents were used to visualize the intensity of glycoprotein bands before and after enzyme treatment. The ease of use of these techniques, which require only basic laboratory instrumentation and reagents, makes them the methodology of choice for initial glycobiology studies. These protocols are also well suited to screen for optimal expression conditions, since multiple glycoprotein samples can be processed at once.

Background: The pregnancy hormone human chorionic gonadotropin (hCG) and its free subunits (hCG alpha, hCG beta) are produced in the male reproductive tract and found in high concentrations in seminal fluid, in particular hCG alpha. This study aimed to elucidate changes in peptide hormone profiles in patients showing abnormal semen analyses and to determine the genuineness of the highly abundant hCG alpha., Methods: Seminal plasma was obtained from 45 male patients undergoing semen analysis during infertility workups. Comprehensive peptide hormone profiles were established by a panel of immunofluorometric assays for hCG, hCG alpha, hCG beta and its metabolite hCG beta core fragment, placental lactogen, growth hormone and prolactin in seminal plasma of patients with abnormal semen analysis results (n = 29) versus normozoospermic men (n = 16). The molecular identity of large hyperglycosylated hCG alpha was analyzed by mass-spectrometry and selective deglycosylation., Results: hCG alpha levels were found to be significantly lower in men with impaired semen quality (1346 +/- 191 vs. 2753 +/- 533 ng/ml, P = 0.022). Moreover, patients with reduced sperm count had reduced intact hCG levels compared with normozoospermic men (0.097 +/- 0.022 vs. 0.203 +/- 0.040 ng/ml, P = 0.028). Using mass-spectrometry, the biochemical identity of hCG alpha purified from seminal plasma was verified. Under non-reducing conditions in SDS-PAGE, hCG alpha isolated from seminal plasma migrated in a manner comparable with large free hCG alpha with an apparent molecular mass (Mr, app) of 24 kDa, while hCG alpha dissociated from pregnancy-derived holo-hCG migrated at approximately 22 kDa. After deglycosylation with PNGase F under denaturing conditions, all hCG alpha variants showed an Mr, app of 15 kDa, indicating identical amino acid backbones., Conclusions: The findings indicate a pathophysiological relevance of hCG, particularly its free alpha subunit, in spermatogenesis. The alternative glycosylation pattern on the free large hCG alpha in seminal plasma might reflect a modified function of this subunit in the male reproductive tract.

Background: The development of new fertility treatment options has facilitated individualized assisted reproductive technology (ART) protocols to improve outcomes. Manufacturing improvements to recombinant human follitropin alfa have allowed precise dosing based on mass (filled-by-mass; FbM) rather than bioactivity (filled-by-bioassay; FbIU). Continued monitoring and reporting of follitropin alfa treatment outcomes in routine clinical practice is essential., Objective: To provide an overview of the frequency of different controlled ovarian-stimulation protocols used in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles in new European Union member states, and to provide post-registration efficacy and safety data on follitropin alfa., Research Design and Methods: A 2-year, prospective, observational, multicentre, Phase IV study conducted at ART clinics in the Czech Republic, Estonia, Latvia, Lithuania, Poland, Slovakia and Slovenia. Women aged 18-47 years undergoing ovarian stimulation with follitropin alfa for conventional IVF or ICSI were eligible for inclusion. The main treatment outcome was cumulative clinical pregnancy rate. Data were analysed descriptively., Results: Clinical pregnancy outcomes were available for 4055 of 4085 (99.3%) patients. In total, 1897 (46.8%) patients used follitropin alfa FbIU; 2133 (52.6%) used follitropin alfa FbM. Clinical pregnancy was achieved by 39.5% (1603/4055) of patients. A greater proportion of patients with polycystic ovary syndrome achieved a clinical pregnancy than those with endometriosis (41.8% vs 37.8%, respectively). A higher cumulative pregnancy rate was observed with the use of follitropin alfa FbM than follitropin alfa FbIU (41.3% vs 37.8%, respectively; p = 0.02)., Conclusions: This study represents the most comprehensive audit of individualized ART in clinical practice in Central and Eastern Europe. Overall, clinical pregnancy was achieved by 39.5% of patients after stimulation with follitropin alfa. The use of follitropin alfa FbM resulted in a higher cumulative pregnancy rate than did the FbIU formulation. However, limitations of the study include the observational and non-comparative study design, and descriptive nature of statistical analyses; furthermore, the study was not designed to make direct comparisons between the success rates of different ovarian-stimulation protocols.

Bovine follicle-stimulating hormone (bFSH), a pituitary gonadotropin, is a heterodimer hormone that consists of a common alpha-subunit non-covalently associated with the hormone-specific beta-subunit. Unfortunately, expression levels of recombinant bFSH or its subunits are invariably low. We report here the secretory expression of biologically active bFSHalpha and bFSHbeta subunit in the methylotrophic yeast Hansenula polymorpha. A slightly higher level of expression of recombinant bFSH subunits was achieved by using the Saccharomyces cerevisiae-derived calnexin (ScCne1) as a chaperone in engineered H. polymorpha strains. The preliminary data also suggested that bFSH subunits expressed in H. polymorpha appeared to be less-glycosylated. This isoform had been shown to be 80% increase in in vivo bioactivity compared with the hyperglycosylated Pichia pastoris-derived recombinant bFSHalpha/beta. More sophisticated applications of bFSH would profit from the assembled less-glycosylated heterodimer.

Background: DNA aptamers are single-stranded nucleotide sequences that bind specifically to target molecules. By combining the advantages of PCR for amplifying specific DNA sequences and aptamer technology, we have developed a new strategy to detect target molecules such as proteins., Methods: Ovine follicle-stimulating hormone alpha subunit (oFSHalpha) was used as the model protein to generate a specific DNA aptamer via an in vitro evolutionary process. A targeted regional-mapping approach and a target-capturing assay were used to identify the binding region on the aptamer molecule. In the detection assay, referred to as "aptamer-based regionally protected PCR" (ARP-PCR), the aptamer was allowed to bind to the target protein in solution before digestion with DNase I. The region of the aptamer bound to the target was protected from DNase I cleavage. The target-binding region of the aptamer protected from the enzymatic treatment was then amplified by the PCR., Results: Aptamers against oFSHalpha were generated. Six sequences of 20 selected aptamer clones were identical. This aptamer sequence was divided into 4 regions according to the aptamer's secondary structure. From examination of the target-binding ability of each region, we determined the specific binding region, for which primers were designed. With the aptamer and primers to detect oFSHalpha by means of the ARP-PCR method, we were able to detect the target protein at concentrations as low as 10(-14) mol/L., Conclusions: Combining the use of a DNA aptamer with the PCR is a potentially useful analytic tool for detection of proteins at low concentrations. .

Background: Cystine-knot (cys-knot) structure is found in a rather large number of secreted proteins and glycoproteins belonging to the TGFbeta and glycoprotein hormone (GPH) superfamilies, many of which are involved in endocrine control of reproduction. In these molecules, the cys-knot is formed by a disulfide (SS) bridge penetrating a ring formed by 8, 9 or 10 amino-acid residues among which four are cysteine residues forming two SS bridges. The glycoprotein hormones Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), Thyroid-Stimulating Hormone (TSH) and Chorionic Gonadotropin (CG) are heterodimers consisting of non-covalently associated alpha and beta subunits that possess cys-knots with 8-amino-acyl (8aa) rings. In order to get better insight in the structural evolution of glycoprotein hormones, we examined the number and organization of SS bridges in the sequences of human 8-aa-ring cys-knot proteins having 7 (gremlins), 9 (cerberus, DAN), 10 (GPA2, GPB5, GPHalpha) and 12 (GPHbeta) cysteine residues in their sequence., Discussion: The comparison indicated that the common GPH-alpha subunit exhibits a SS bridge organization resembling that of DAN and GPA2 but possesses a unique bridge linking an additional cysteine inside the ring to the most N-terminal cysteine residue. The specific GPHbeta subunits also exhibit a SS bridge organization close to that of DAN but it has two additional C-terminal cysteine residues which are involved in the formation of the "seat belt" fastened by a SS "buckle" that ensures the stability of the heterodimeric structure of GPHs. GPA2 and GPB5 exhibit no cys residue potentially involved in interchain SS bridge and GPB5 does not possess a sequence homologous to that of the seatbelt in GPH beta-subunits. GPA2 and GPB5 are thus not expected to form a stable heterodimer at low concentration in circulation., Summary: The 8-aa cys-knot proteins GPA2 and GPB5 are expected to form a heterodimer only at concentrations above 0.1 microM: this would be consistent with a short-term paracrine role but not with an endocrine role after dilution in circulation. Consequently, GPA2 and GPB5 could exert separate endocrine roles either during development and/or during adult life of both vertebrates and invertebrates.

The objective of this study was to determine whether beta human chorionic gonadotropin (hCG) (CGB) subunits and alpha hCG (CGA) subunits are expressed and the hCG dimer is produced in normal human cyclic endometrium. Endometrial specimens were collected for histological dating from women undergoing treatment in our division of human reproduction. RNA from normal secretory endometrium was extracted, and CGB and CGA gene expression was assessed by semiquantitative PCR. Adequate secretory endometrial specimens were homogenized using protease inhibitors. Proteins present in the supernatant were separated electrophoretically, and molecular hCG isoforms were detected by Western blot. The supernatant hCG concentrations were measured by ELISA. We characterized hCG and leukocytes in endometrial specimens by immunohistochemistry. Uterine flushing was performed to confirm endometrial hCG secretion into the uterine fluid. A full-length CGB mRNA encompassing the exon 1 promoter region and the structure exons 2 and 3 (including the C-terminal peptide) was expressed in normal secretory endometrial specimens (similar to CGA) during the early secretory phase of the menstrual cycle, up to an optimum at the midsecretory to late secretory phases. In homogenate supernatants obtained from normal secretory endometrium, hormone concentrations of dimeric hCG were approximately 5 mU per 10 mg of tissue, compared with considerably smaller concentrations of corresponding single free CGB subunit. Single chains of CGB, CGA, and dimeric molecular hCG isoforms were found in endometrial specimens by Western blot. Glandular endometrial hCG production is demonstrated immunohistochemically, with an increase toward the late secretory phase vs. the early secretory phase of the normal secretory menstrual cycle. However, glandular hCG release is diminished or absent in the dyssynchronous or missing endometrial secretory transformation. Endogenous endometrial hCG may be important for implantation and maintenance of pregnancy.

The cDNAs of three gonadotropin (GTH) subunits (GTHalpha, FSHbeta, and LHbeta) and two GTH receptors (FSHR and LHR) from pituitary and gonads of black porgy were cloned. The nucleotide sequences of the GTHalpha, FSHbeta, and LHbeta cDNA were 354, 363, and 414 base pairs (bps) in length with open reading frames (ORF) encoding peptides of 117, 120, and 137 amino acids, respectively. The FSHR and LHR cDNA was 2118 and 2076 bps in length with ORFs encoding peptides of 705 and 691 amino acids, respectively. To study the mechanism of the estradiol-17beta (E(2)) action, we examined the expression pattern of GTH subunit mRNAs in pituitary and GTH-receptor mRNAs in gonads, and the changes of plasma E(2) level when E(2) treatment was applied to immature black porgy. E(2) treatment increased mRNA expression levels of the genes and plasma E(2) levels, indicating that E(2) stimulated the increases in GTH subunit and GTH-receptor mRNAs. These data indicate that E(2) plays an important regulatory role in the brain-pituitary-gonad axis of immature black porgy. We provide the molecular characterization and expression of the GTH subunits and GTH receptors during sex change in the protandrous black porgy.

Objective: To compare the efficacy, tolerability, and convenience of two formulations of the follitropin-alpha (Gonal-f) pen device versus the conventional form in Iranian women undergoing ovarian stimulation for intracytoplasmic sperm injection., Design: Randomized, single-center trial, parallel-group, single blind., Setting: Tertiary referral center, University Hospital., Patient(s): A total of 100 patients undergoing intracytoplasmic sperm injection., Intervention(s): After down-regulation with busereline acetate, patients were randomized to receive the pen device or the conventional syringe of follitropin-alpha. A computer-generated randomization list was used to allocate the patients to one of these two groups., Main Outcome Measure(s): The primary outcomes were patients' satisfaction, convenience, occurrence of local tolerance symptoms, and pain. Total dose of follitropin-alpha, duration of follitropin-alpha treatment, number of oocyte retrieved, number of viable embryos, and clinical pregnancies were secondary outcome measures. Data collection was performed by means of a questionnaire designed for the purpose of this study. The pain scored according to the Visual Analogue Scale., Result(s): Self-administration and patients' satisfaction were significantly higher in the pen device group than the conventional syringe group. Local reactions at injection sites and pain were significantly higher in the conventional syringe group than in the pen device group. There were no statistically differences in secondary outcome measures and convenience between two groups., Conclusion(s): Among the Iranian patients that we studied, the pen device of Gonal-f is safe, convenient, and less painful, with more patients' satisfaction than the conventional syringe form, but both forms have equal efficacy in intracytoplasmic sperm injection cycles.

Doping with (glyco)protein hormones represent an extremely challenging, analytical problem as nearly all are constitutively present at low concentrations that fluctuate according to circadian or alternative periodical, or external stimuli. Thus the mere concentration in a biological sample is only resolutive when this surpasses extreme values. As the vast majority of these molecules are produced by recombinant DNA technology it is believed that the exogenous molecules could bear the signature of the host cell. In particular, these could comprise structural differences originated from co or post-translational differences. In this study we have employed both proteomics and glycomics strategies to compare recombinant and urinary human chorionic gonadotrophin in order to evaluate this hypothesis. As anticipated the recombinant hormone could be shown to contain N-glycolyl neuraminic acid, a sialic acid that cannot be produced by humans. Furthermore, differences were observed in the overall glycosylation, in particular the presence of abundant hybrid-type glycans that were much less pronounced in the recombinant species. These differences were determined to occur predominantly in the alpha-subunit for which antidoping strategies focussed on these elements could be used for both chorionic gonadotrophin and lutrophin as they share the same alpha-subunit., (Copyright 2008 John Wiley & Sons, Ltd.)

The gonadotrophins LH, FSH and human (h) CG are non-covalent heterodimers composed of a common alpha and the hormone-unique beta subunit. LH regulates the production of androgens and progestins in the follicle, and the levels of these steroids are critical for the ovarian function. Structural features of the gonadotrophins involved in the steroidogenic response of the ovary are not completely understood. As an approach to address how the topology of the ligand affects steroidogenesis we exploited the single-chain (SC) gonadotrophin methodology because manipulating the relative position of the tethered subunit domains in SC hCG analogs enabled to change in the conformation, secretion, receptor binding and adenylyl cyclase activity. We genetically engineered a SC bovine LH analog with a linker derived from the CTP domain of the hCGbeta subunit, NH2-alpha-CTP-LHbeta-COOH (denoted as alphaCTPLHbeta; AB configuration) and evaluated the secretion form transfected CHO cells and steroidogenesis in follicular derived cells in comparison to the variant NH2-LHbeta-CTP-alpha-COOH (LHbetaCTPalpha; BA configuration). The secretion of the analogs from CHO cells was quantitative, and that of alphaCTPLHbeta was more efficient than that of LHbetaCTPalpha The experiments suggested that both variants were N- and O- glycosylated, though the posttranslational modifications are likely to be non-identical in the AB and BA analogs. The analogs stimulated progesterone secretion by immortalized rat granulosa cells that express the rat LH receptor but the EC50 of alphaCTPLHbeta (AB orientation) was higher by 20 fold, as compared to LHbetaCTPalpha (BA). In primary cultures of bovine theca cells, alphaCTPLHbeta stimulated progesterone release with a reduced sensitivity (by at least 50 folds) and smaller magnitude over the basal levels (about 3 folds) relative to LHbetaCTPalpha. In contrast, the accumulation of androstenedione in the media of the same primary cultures appeared to be nearly identical. As a result, the androstenedione/progesterone ratio for the alphaCTPLHbeta analog was significantly increased relative to LHbetaCTPalpha (2-3 folds). This unequal response suggests a distinct regulation of progesterone and androstenedione biosynthesis. Our data demonstrate major differences in steroid balance following stimulation of the receptor with structural LH analogs and provide further insight into gonadotrophin regulation of ovarian steroid production.

In multimeric glycoproteins, like glycoprotein hormones, mutual subunit interactions are required for correct folding, assembly, and transport in the secretory pathway. However, character and time course of these interactions need further elucidation. The influence of the glycoprotein hormone alpha-subunit (GPHalpha) on the folding of the human chorionic gonadotropin (hCG) beta-subunit (hCGbeta) in hCG alphabeta-heterodimers was investigated in [(35)S]Met/Cys-labeled JEG-3 cells. Completeness of disulfide bridge formation during the time course of folding was estimated by labeling with [(3)H]N-ethylmaleinimide of free thiol groups not yet consumed. Subunit association took place between immature hCGbeta (high (3)H/(35)S ratio) and almost completely folded GPHalpha. Analysis revealed a highly dynamic maturation process comprising of at least eight main hCGbeta folding intermediates (molecular masses from 107 to 28 kDa) that could be micro-preparatively isolated and characterized. These hCGbeta variants developed while being associated with GPHalpha. The 107-kDa variant was identified as a complex with calnexin. In contrast to hCG alphabeta-heterodimers, free nonassociated hCGbeta, free large GPHalpha, and GPHalphaalpha homodimers showed a fast-track-like processing in the secretory pathway. At 10 min before hCG secretion, sialylation of these variants had already been completed in the late Golgi, whereas hCG alphabeta-heterodimers had still not arrived medial Golgi. This shows that the GPHalpha in the hCG alphabeta-heterodimers decelerates the maturation of the hCGbeta portion in the heterodimer complex. This results in a postponed approval of hCG alphabeta-heterodimers by the endoplasmic reticulum quality control unlike GPHalphaalpha homodimers, free hCGbeta, and GPHalpha subunits.

The dynamics of glycoprotein hormone alpha-subunit (GPHalpha) maturation and GPHalpha alpha homodimer formation were studied in presence (JEG-3 choriocarcinoma cells) and absence (HeLa cells) of hCGbeta. In both cases, the major initially occurring GPHalpha variant in [35S]Met/Cys-labeled cells carried two N-glycans (M(r app) = 22 kDa). Moreover, a mono-N-glycosylated in vivo association-incompetent GPHalpha variant (M(r app) = 18 kDa) was observed. In JEG-3 cells the early 22-kDa GPHalpha either associated with hCGbeta, or showed self-association to yield GPHalpha alpha homodimers, or was later converted into heavily glycosylated large free GPHalpha (M(r app) = 24 kDa). Micro-preparative isolation of intracellular GPHalpha alpha homodimers of JEG-3 cells and their conversion by reduction revealed that they consisted of 22-kDa GPHalpha monomers and not of large free GPHalpha. In HeLa cells, the large free GPHalpha variant was not observed, whereas GPHalpha alpha homodimers were present. Intracellularly, early GPHalpha alpha homodimers (35 kDa) and late variants (JEG-3: 44 kDa, HeLa: 39 kDa) were found. Both cell types secreted 45 kDa GPHalpha alpha homodimers. Large free GPHalpha and GPHalpha alpha homodimers were more rapidly sialylated than hCG alphabeta-heterodimers indicating a sequestration mechanism in the secretory pathway. In GPHalpha alpha homo- as well as hCG alphabeta-heterodimers the subunit interaction site, located on loop 2 of GPHalpha (amino acids 33-42), became immunologically inaccessible indicating similar spatial orientation of GPHalpha in both types of dimers. The studies demonstrate the formation, in vivo dynamics of GPHalpha alpha homodimers, and the pathways of the cellular metabolism of variants of GPHalpha, monoglycosylated GPHalpha and large free GPHalpha.

We have explored the possibility to use 14 different monoclonal antibodies in order to follow the formation of the respective epitopes during the biosynthesis of hCG subunits and their association in JEG-3 choriocarcinoma cells using pulse (30s to 5 min)-chase (0-180 min) experiments. We found central cystine knot epitope structures (epitope beta1) to be formed immediately and simultaneously with epitopes on the protruding hCG-beta loops 1 and 3. We found also differences in the time-dependent folding of beta2 and beta4 epitopes, which are highly overlapping structures on the loops 1+3. These differences were reinforced by decreasing the temperature during the pulse-chase experiments to 25 degrees C. Moreover, we describe for the first time an intracellular intact hCG beta-subunit form that showed the transient expression of the hCG-beta-core fragment epitope beta11 in the course of the maturation of this subunit which casts new light on the presence of hCG-beta-core fragment in Down's syndrome, tumors and pregnancy.

Follicle stimulating hormone (FSH) is secreted from the pituitary gland to regulate reproduction in vertebrates. FSH signals through a G-protein coupled receptor (FSHR) on the target cell surface. We describe here the strategy to produce a soluble FSH-FSHR complex that involves the co-secretion of a truncated FSHR ectodomain (FSHR(HB)) and a covalently linked FSHalphabeta heterodimer from baculovirus-infected insect cells. FSH binds to FSHR(HB) with a high affinity comparable to that for the full-length receptor. The crystal structure of the FSH-FSHR(HB) complex provides explanations for the high affinity and specificity of FSH interaction with FSHR, and it shows an unexpected dimerization of these complexes. Here we also compare the crystal structure with theoretical models of the FSH-FSHR-binding mode. We conclude that the FSH-FSHR(HB) structure gives an authentic representation of FSH binding to intact FSHR.

All mammals exhibit pituitary-specific expression of LH and FSH, whereas placental expression of gonadotropins has been reported only in primates and equids. Some cetaceans, such as dolphins, have a long gestational period and a sexual cycle of about 27 days almost comparable with that of humans. Histologically, dolphins have an epitheliochorial placentae that resembles placentas of Perissodactyla including horses. In the present study, we cloned cDNAs encoding gonadotropins and observed their immunohistochemical localization in the placenta of bottlenose dolphin. The cDNAs obtained encoded 120 amino acids for the alpha-subunit (including 96 amino acids of mature proteins), and 141 amino acids for the beta-subunit (including 121 amino acids of mature proteins). The sequence of the alpha-subunit was similar to that in the pig (Artiodactyla) pituitary glycoprotein hormone [96.7% homology at amino acids (aa) level], and the sequence of the beta-subunit was similar to that of luteinizing hormone (LH) in the pig [94.3% homology at aa level] and white rhinoceros (Perissodactyla) [93.3% homology at aa level]. Of interest, dolphin LHbeta lacks carboxyl-terminal-peptides (CTP). This fact suggests that CTP are not essential for placental expression of gonadotropin in dolphins. Immunohistochemical observations employing anti-ovine LHbeta antibody revealed positive staining in the villositycal tissue. Our observations suggest placental expression of gonadotropin homologues in cetaceans and possible evolutionary conservation of placentae-derived hormonal control of ovarian functions during pregnancy.

Follicle stimulating hormone (FSH) is one of the important hormones that regulate gonadal functions. This hormone is glycosylated, and the glycans greatly influence the biological properties. In the present study the negatively charged glycopeptides of equine and human pituitary follicle stimulating hormone (eFSH and hFSH) have been characterized in a glycosylation site-specific manner using FT-ICR-MS and Edman sequencing. The characteristic pattern of glycan distribution at each glycosylation site has been deduced and compared between horse and human FSH preparations. The data suggest that site-specific differences exist between glycoforms of human and equine FSH. For instance, except for one site in the beta subunit (Asn7) of hFSH all other sites in both species have sulfated glycoforms. Also, glycoforms at Asn52 of hFSH are all complex type, whereas in eFSH, both complex and hybrid structures exist at this site. There is also a higher percentage of sulfated glycans in the latter site compared to the former. This is the first study that characterizes the glycans from this hormone in a glycosylation site-specific manner, and these data can be used to begin correlative studies between glycosylation structure and hormone function.

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.

The three dimensional structures of the C(1)X(2)G (3)(X(3))X(4)C(5) motif of hCG, which is considered to be important for noncovalent assembly of the alpha- and beta-subunits of glycoprotein hormones were analyzed to assess the importance of glycine (Gly) (G) at site X(3) in the motif by the conformational energy calculation using computational procedures. In the C(1)M(2)G (3)(X(3))C(4)C(5) motif of the alpha-subunit, Ramachandran plot analysis showing the allowed area of the dihedral angles demonstrated that only a Gly residue was allowed at site X(3). In calculating collision with surrounding atoms as a monomer the possible main chain models of the C(1)A(2)G(3)(X(3))Y(4)C(5) motif in the beta-subunit showed that only alanine (Ala) (A) or Gly at site X(3) is allowed to alleviate the collision with the cysteine (Cys) (C) residues which form a disulfide bridge. A mutant of the beta-subunit with the C(1)A(2)A(3)(X(3))Y(4)C(5) motif (Ala at site X(3)) may not compose a heterodimer with the alpha-subunit because of interference of intermolecular hydrogen bond formation. These findings indicate that the Gly residue at site X(3) (G(3)) in the motif is essential for heterodimer formation of glycoprotein hormones. The significance of similar motifs found in various human proteins other than glycoprotein hormones was suggested.

We report the nucleotide and deduced amino acid sequences of the pituitary glycoprotein hormone common alpha-subunit, and the FSH and LH beta-subunits of the elk (Cervus elaphus). These sequences were obtained by RT-PCR of pituitary gland polyadenylated RNA. A partial genomic fragment of the elk follicle stimulating hormone beta-subunit was also amplified from genomic DNA and sequenced. These sequences show high similarity to other related domestic species, including sika deer, ovine, bovine, porcine and equine. The presently reported cDNAs were used as probes for Northern analysis of pituitary gland and muscle samples from elk and sheep. Mature mRNA transcripts of approximately 700, 1700 and 550 bases were expressed in the pituitary gland but not in muscle tissue, for the glycoprotein hormone common alpha-subunit and FSH and LH beta-subunits, respectively. These observations are consistent with reports for other related species. The genomic fragment of elk FSH beta-subunit revealed an intronic microsatellite of 6 CT repeats, which differs from the 19 CT repeats documented for the sheep genomic sequence. We propose that differences in the CT repeats of the elk FSH beta-subunit gene may be used as a marker for elk parentage testing. The elk DNA sequences presently reported represent a unique addition to the growing collection of the pituitary hormone genes for various vertebrates, which will facilitate evolutionary and phylogenetic studies of the gonadotropin genes. In addition, this information and the cloned cDNAs will be useful for studying seasonal expression of the elk pituitary glycoprotein genes.

The unique structures of human choriogonadotropin (hCG) and related glycoprotein hormones make them well suited for studies of protein folding in the endoplasmic reticulum. hCG is stabilized by a strand of its beta-subunit that has been likened to a "seatbelt" because it surrounds alpha-subunit loop 2 and its end is "latched" by an intrasubunit disulfide bond to the beta-subunit core. As shown here, assembly begins when parts of the NH(2) terminus, cysteine knot, and loops 1 and 3 of the alpha-subunit dock reversibly with parts of the NH(2) terminus, cystine knot, and loop 2 of the hCG beta-subunit. Whereas the seatbelt can contribute to the stability of the docked subunit complex, it interferes with docking and/or destabilizes the docked complex when it is unlatched. This explains why most hCG is assembled by threading the glycosylated end of alpha-subunit loop 2 beneath the latched seatbelt rather than by wrapping the unlatched seatbelt around this loop. hCG assembly appears to be limited by the need to disrupt the disulfide that stabilizes the small seatbelt loop prior to threading. We postulate that assembly depends on a "zipper-like" sequential formation of intersubunit and intrasubunit hydrogen bonds between backbone atoms of several residues in the beta-subunit cystine knot, alpha-subunit loop 2, and the small seatbelt loop. The resulting intersubunit beta-sheet enhances the stability of the seatbelt loop disulfide, which shortens the seatbelt and secures the heterodimer. Formation of this disulfide also explains the ability of the seatbelt loop to facilitate latching during assembly by the wraparound pathway.

All three human glycoprotein hormone heterodimers are assembled in the endoplasmic reticulum by threading the glycosylated end of alpha-subunit loop two (alpha2) beneath a disulfide "latched" strand of the beta-subunit known as the "seatbelt." This remarkable event occurs efficiently even though the seatbelt effectively blocks the reverse process, thereby stabilizing each heterodimer. Studies described here show that assembly is facilitated by the formation, disruption, and reformation of a loop within the seatbelt that is stabilized by the most easily reduced disulfide in the free beta-subunit. We refer to this disulfide as the "tensor" because it shortens the seatbelt, thereby securing the heterodimer. Formation of the tensor disulfide appears to precede and facilitate seatbelt latching in most human choriogonadotropin beta-subunit molecules. Subsequent disruption of the tensor disulfide elongates the seatbelt, thereby increasing the space beneath the seatbelt and the beta-subunit core. This permits the formation of hydrogen bonds between backbone atoms of the beta-subunit cystine knot and the tensor loop with backbone atoms in loop alpha2, a process that causes the glycosylated end of loop alpha2 to be threaded between the seatbelt and the beta-subunit core. Contacts between the tensor loop and loop alpha2 promote reformation of the tensor disulfide, which explains why it is more stable in the heterodimer than in the uncombined beta-subunit. These findings unravel the puzzling nature of how a threading mechanism can be used in the endoplasmic reticulum to assemble glycoprotein hormones that have essential roles in vertebrate reproduction and thyroid function.

Vertebrate glycoprotein hormone heterodimers are stabilized by a strand of their beta-subunits known as the "seatbelt" that is wrapped around loop 2 of their alpha-subunits (alpha2). The cysteine that terminates the seatbelt is "latched" by a disulfide to a cysteine in beta-subunit loop 1 (beta1) of all vertebrate hormones except some teleost follitropins (teFSH), wherein it is latched to a cysteine in the beta-subunit NH(2) terminus. As reported here, teFSH analogs of human choriogonadotropin (hCG) are assembled by a pathway in which the subunits dock before the seatbelt is latched; assembly is completed by wrapping the seatbelt around loop alpha2 and latching it to the NH(2) terminus. This differs from hCG assembly, which occurs by threading the glycosylated end of loop alpha2 beneath the latched seatbelt through a hole in the beta-subunit. The seatbelt is the part of the beta-subunit that has the greatest influence on biological function. Changes in its sequence during the divergence of lutropins, follitropins, and thyrotropins and the speciation of teleost fish may have impeded heterodimer assembly by a threading mechanism, as observed when the hCG seatbelt was replaced with its salmon FSH counterpart. Whereas wrapping is less efficient than threading, it may have facilitated natural experimentation with the composition of the seatbelt during the co-evolution of glycoprotein hormones and their receptors. Migration of the seatbelt latch site to the NH(2)-terminal end of the beta-subunit would have facilitated teFSH assembly by a wraparound mechanism and may have contributed also to its ability to distinguish lutropin and follitropin receptors.

Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the alpha-subunit straddles a hole in the beta-subunit. This hole is formed when a cysteine at the end of a beta-subunit strand known as the "seatbelt" becomes "latched" by a disulfide to a cysteine in the beta-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the alpha-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the alpha-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the alpha-subunit after the subunits dock. Here we show that this "wraparound" process can be used to assemble disulfide cross-linked human choriogonadotropin analogs that contain an additional alpha-subunit cysteine, but only if the normal beta-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of alpha-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.

LH, FSH, and chorionic gonadotropin (CG) are comprised of a common alpha subunit and a hormone-specific beta subunit. Using Madin-Darby canine kidney (MDCK) epithelial cells to examine the polarized secretion of human CG/LH, we previously reported that CG and LH were detected in the apical and basolateral compartments, respectively, and the carboxyl terminal end of the CGbeta subunit contains a strong apical signal. Here we show that the carboxyl seven amino acids in the LHbeta subunit contribute to the basolateral secretion of LH, and an LH chimera bearing the CGbeta apical signal is redirected from the basolateral to the apical compartments. Because LH and FSH are synthesized in the same cell, we also compared the secretion polarity of LH with FSH. MDCK cells expressing the FSH dimer displayed an almost equal distribution of protein into the apical and basolateral compartments. Given that the LHbeta and CGbeta carboxy terminal sequences, which differ from that in the FSHbeta subunit, occupy a pivotal role in their polarized behavior, the results support the hypothesis that pituitary exit of LH and FSH occur via different secretion pathways, and are released spatially from the pituitary via different circulatory routes.

Camelids produce functional antibodies devoid of light chains. Autonomous heavy chain variable (V(H)H) domains in these molecules have adapted to the absence of the light chain in the following ways: bulky hydrophobic residues replace small aliphatic residues in the former light chain interface, and residues from the third complementarity-determining region (CDR3) pack against the framework and stabilize the global V(H)H domain fold. To determine the specific roles of CDR3 residues in framework stabilization, we used nai;ve phage-displayed libraries, combinatorial alanine-scanning mutagenesis and biophysical characterization of purified proteins. Our results indicate that in the most stable scaffolds, the structural residues in CDR3 reside near the boundaries of the loop and pack against the framework to form a small hydrophobic core. These results allow us to differentiate between structural CDR3 residues that should remain fixed, and CDR3 residues that are tolerant to substitution and can therefore be varied to generate functional diversity within phage-displayed libraries. These methods and insights can be applied to the rapid design of heavy chain scaffolds for the identification of novel ligands using synthetic, antibody-phage libraries. In addition, they shed light on the relationships between CDR3 sequence diversity and the structural stability of the V(H)H domain fold.

Reproduction is controlled by two pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This study reports the cloning, sequence analysis, and gene expression of gonadotropin (GTH) subunits from the European sea bass (Dicentrarchus labrax). The GTH subunits were cloned from a sea bass brain-pituitary cDNA library. The nucleotide sequences of the common alpha, the FSHbeta, and the LHbeta subunit cDNAs were 625, 521, and 591 base pair (bp) long, respectively, encoding for mature peptides of 94, 105, and 115 amino acids (aa), respectively. Sequence analysis showed that sea bass FSHbeta is more similar to higher vertebrate FSHbeta's (35-37%) than to LHbeta's (26-30%), whereas sea bass LHbeta is more similar to LHbeta's (40-53%) than to FSHbeta's (26-41%). Phylogenetic analysis of fish GTH sequences grouped the beta subunits into two groups, FSH and LH, distributed into four classes, corresponding to the accepted divisions of Elopomorphs, Ostariophysis, Salmonids, and Percomorphs. A dot-blot technique was developed to analyze GTH pituitary mRNA levels during the reproductive cycle of male sea bass. From October (initiation of gametogenesis) to February (spermiation), the expression of all three subunits in the pituitary increased in parallel, concomitantly with the gonadosomatic index (GSI) and the accumulation of LH protein in the pituitary, all values declining sharply at post-spermiation. This study demonstrates that the pituitary of sea bass contains two gonadotropin hormones and that both gonadotropins are probably involved in the control of gametogenesis, gamete maturation, and spermiation.

Human CG is a pregnancy marker secreted by the placenta, and it utilizes the same receptors as does LH. Human CG is a heterodimer, and its subunits are expressed in tissues other than placenta. Similarly, LH/hCG receptors are also expressed in multiple tissues; however, the physiological significance of this expression is unknown. Free hCGbeta is efficiently secreted in vitro in transfected cells and is highly expressed in many human cancers; however, the biological effects of free hCGbeta in vivo are unknown. To study in vivo consequences of elevated levels of free hCGbeta and hCG dimer in both male and female reproductive physiology, we used mouse metallothionein 1 promoter to generate multiple lines of transgenic mice that overexpressed either one or both subunits of hCG. Although mice expressing the glycoprotein hormone alpha subunit are normal and fertile, both male and female transgenic mice overexpressing only the hormone-specific hCGbeta subunit are infertile. The hCGbeta subunit-expressing transgenic female mice progressively develop cystic ovaries, whereas the male transgenic mice are infertile but otherwise are not phenotypically discernible. In contrast, both the male and female transgenic mice coexpressing high levels of the hCG subunits (i.e., the hCG dimer) demonstrate multiple reproductive defects. The male transgenic mice have Leydig cell hyperplasia, very high levels of serum testosterone, reduced testis size, and dramatically enlarged seminal vesicles and are infertile and display overly aggressive behavior when caged with females. The female transgenic mice are also infertile, have elevated levels of serum estradiol, and progressively develop hemorrhagic and cystic ovaries with thecal layer enlargement and stromal cell proliferation and degenerating kidneys. These results suggest that the in vivo biological effects of ectopically expressed free hCGbeta subunit are distinct from those of the hCG dimer and are gender specific. These transgenic mice are useful models for studying the biology of free hCGbeta subunit, for further analyzing the gain of function effects of hCG during early Leydig cell development, and for studying the roles of hCG in ovarian and kidney pathophysiology and function.

We have isolated a part of the gene for the pituitary glycoprotein hormone common alpha subunit (PGHalpha) and the whole gene for the follicle-stimulating hormone beta subunit (FSHbeta) in the Japanese crested ibis (Nipponia nippon), a critically endangered bird species in East Asia. The nucleotide sequence of a part of the PGHalpha gene (5026 bp) contained three exons holding the whole coding and 3' untranslated regions, but lacked a 5' untranslated region. Its exon-intron structure was similar to that in mammals, but different from that in teleosts in the location of the second intron. For the FSHbeta gene, the nucleotide sequence of 7633 bp was assembled from two phage clones. The exon-intron structure of three exons and two introns was similar to that observed in mammals and teleosts. In the putative promoter region of the ibis FSHbeta gene, a progesterone responsive element (PRE)-like sequence and two AP-1 responsive element-like sequences reported in the ovine FSHbeta gene were not conserved in complete form. The increased number of ATTTA motifs in the putative 3' untranslated region in comparison with those in Japanese quail and chicken FSHbeta cDNA suggested that more rapid degradation of FSHbeta mRNA occurs in this species. Deduced amino acid sequences of the ibis PGHalpha and FSHbeta showed high similarities with those of the corresponding subunits of other avian species. This is the first report on the genomic sequences of the PGHalpha and FSHbeta in an avian species.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds that seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This paper describes the results of our studies on the role of the disulfide bonds of hCG-beta in heterodimer formation with the alpha-subunit. Six disulfide peptides incorporating each of the six disulfide bonds of hCG-beta were screened, along with their linear counterparts, for their ability to competitively inhibit the recombination of alpha- and beta-subunits. The disulfide peptides Cys (9-57), Cys (34-88) and Cys (38-90) were found to inhibit the alpha/beta recombination whereas the remaining three disulfide peptides viz. Cys (23-72), Cys (26-110) and Cys (93-100) did not exhibit any inhibition activity. Interestingly, none of the linear peptides could inhibit the alpha/beta recombination. Results clearly demonstrate that the disulfide bonds Cys(9)-Cys(57), Cys(34)-Cys(88) and Cys(38)-Cys(90) of the beta-subunit of hCG are crucial for heterodimer formation with the alpha-subunit thus providing experimental confirmation of the conclusions from the crystal structure of the hormone.

Human choriogonadotropin (hCG) contains an alpha-subunit, common to other members of the glycoprotein hormone family, and a unique beta-subunit that determines hormone specificity. It is generally thought that heterodimer formation is obligatory for full hormonal activity, although other studies have indicated that individual subunits and homodimeric hCGbeta were capable of low affinity binding to the LH receptor (LHR) and subsequent activation. Previously, we constructed two yoked hormone (hCG)-LHR complexes, where the two hormone subunits and the heptahelical receptor were engineered to form single polypeptide chains, i.e. N-beta-alpha-LHR-C and N-alpha-beta-LHR-C. Expression of both complexes led to constitutive stimulation of cAMP production. In the present study, we investigated whether the human alpha-subunit and hCGbeta can act as functional agonists when covalently attached to or coexpressed with the LH receptor. Our initial results showed that hCGbeta, but not alpha, was able to activate LHR with an increase in intracellular cAMP in human embryonic kidney 293 cells but not in Chinese hamster ovary or COS-7 cells. Further examination of this apparent cell-specific agonist activity of hCGbeta revealed that low levels of endogenous alpha-subunit were expressed in human embryonic kidney 293 cells, thus enabling sufficient amounts of active heterodimer to form with the transfected hCGbeta to activate LHR. The studies in Chinese hamster ovary and COS-7 cells clearly demonstrate that, even under experimental conditions where hormone-receptor interactions are maximized, individual subunits of hCG can not act as functional agonists, at least in their monomeric form.

The X-ray structure of a ternary complex between human chorionic gonadotropin hormone (hCG) and two Fvs recognizing its alpha and beta subunits has been recently determined. The Fvs recognize the elongated hCG molecule by its two ends, one being the Leu-12-Cys-29 loop of the alpha subunit. We have designed and synthesized a 17-amino-acid peptide (named PepH14) derived from the sequence of this antigenic loop with the purpose of mimicking its three-dimensional structure and its affinity for antibodies. We have determined the solution structure of PepH14 by homonuclear NMR spectroscopy and derived distance restraints. Comparison of this structure with that of the corresponding antigenic loop of alpha-hCG reveals strong conformational similarities. In particular, the two pairs of residues that establish crucial contacts with the Fv fragment share the same conformation in PepH14 and in the authentic hormone loop. We propose a three-dimensional model of interaction of PepH14 with a llama V(HH) (V(HH)-H14) fragment cloned from a single-chain llama immunoglobulin raised against alpha-hCG. This model has been constrained by the chemical shift variations of the H14 1HN and 15N resonances monitored upon binding with PepH14. Mapping of the backbone chemical shift variations on the V(HH) structure determined by NMR indicates that PepH14 binds to V(HH)-H14 and forms a complex using the three complementary determining regions (CDRs). They define a shallow groove encompassing residues Thr-31, Ala-56, Tyr-59 and Trp-104 which have been shown to be in conformational exchange [Renisio, Pérez, Czisch, Guenneugues, Bornet, Frenken, Cambillau and Darbon (2002) Proteins 47, 546-555] and also Phe-37 and Ala-50. This groove is close to the hydrophobic interface area observed between VH and VL domains in Fvs from classical antibodies, which explains the rather lateral binding of PepH14 on the V(HH).

The canonical members of the human glycoprotein hormone subunit family of cystine knot-forming polypeptides include the common alpha-subunit, and four beta-subunit genes, FSHbeta, LHbeta, TSHbeta, and hCGbeta. Using pairwise sequence analysis of the complete human genome, we have identified two novel glycoprotein hormone subunit-related genes. Based on unique sequence similarity to the alpha- and beta-subunits of glycoprotein hormones, they were named glycoprotein-alpha2 (GPA2) and glycoprotein-beta5 (GPB5), respectively. PCR analysis using a panel of human cDNAs from 14 different tissues demonstrated that GPB5 is similar to other beta-subunits showing restricted tissue expression, mainly in pituitary and brain. In contrast, the GPA2 transcript is found in diverse tissues. Furthermore, immunoreactive GPA2 and GPB5 were detected in the anterior pituitary of mouse and frog, whereas the expression of GPA2 and GPB5 in transfected cells resulted in the secretion of recombinant polypeptides in conditioned medium. After GenBank searches in lower organisms, glycoprotein hormone beta-subunit-related genes were identified from the genome of nematode Caenorhabditis elegans, hookworm Ancylostoma caninum, and Drosophila melanogaster. The evolutionary conservation of these invertebrate homologs can be seen in several key sequence characteristics, and the data suggest that the glycoprotein hormone beta-subunit gene ancestor evolved before the emergence of bilateral metazoa, thus providing a better understanding of the evolution of this group of classic polypeptide hormones and their receptors. Studies of the complete inventory of genes homologous to glycoprotein hormone subunits in the human genome and lower organisms will allow future functional characterization and identification of their respective receptors.

hCG is a glycoprotein hormone composed of two dissimilar subunits. This hormone is not only heterogeneous in peptide structure but also in combination of subunits and carbohydrate structure. Common hCG-related molecules include hCG, hyperglycosylated hCG, nicked hCG, hCG missing the beta-subunit C-terminal peptide, free alpha-subunit, free beta-subunit, nicked free b-subunit and urine beta-core fragment. This article discusses the structures these hCG-related molecules and their occurrences in early pregnancy, 7-week to term pregnancy, hydatidiform mole (preevacuation and postevacuation), persistent gestational trophoblastic disease, choriocarcinoma and other malignancies. Multiple serum hCG tests are evaluated, and their abilities to detect the multiple hCG-related molecules are investigated. The accuracy of different serum hCG tests in detecting hCG and hCG-related molecules in patients with gestational trophoblastic diseases is evaluated. The findings of persistent low hCG values in the absence of pregnancy or an identifiable malignancy are examined. In addition, the false positive hCG assay problem is discussed. False positive hCG tests have led to many incidences in which gestational trophoblastic disease has been erroneously diagnosed and needlessly treated. hCG tests are identified that give a disproportionate number of false positive results. Finally, guidelines are presented for selecting an hCG test.

Hybrid hormone preparations were prepared by combining intact and Asn(56)-deglycosylated (N(56)dg) equine (e) LH or FSH alpha subunit preparations with truncated, des(121-149)eLH beta (eLH beta t), immunopurified, intact eLH beta or equine chorionic gonadotropin beta (eCG beta) preparations, and eFSH beta. The LH receptor-binding potencies of N(56)dg-eLH alpha:eLH beta t and N(56)dg-eFSH alpha:eLH beta t hybrids were equivalent to that of eLH; however, both N(56)dg-alpha preparations were only 3-4% as active as eLH in the rat testis Leydig cell bioassay. In the granulosa cell FSH bioassay, eLH alpha:eLH beta t stimulated progesterone synthesis and induced aromatase activity, while N(56)dg-eLH alpha:eLH beta t was completely inactive at doses up to 5 microg. N(56)dg-eLH alpha:eLH beta t inhibited progesterone production and aromatase induction elicited by 0.3 ng eFSH or 2 ng human (h) FSH. The inhibitory activities of N(56)dg-eLH alpha:eLH beta and N(56)dg-eCG alpha:eLH beta t were only 10% that of N(56)dg-eLH alpha:eLH beta t. N(56)dg-eLH alpha:eCG beta did not inhibit progesterone synthesis stimulated by eFSH at all and appeared to further stimulate aromatase induction at the highest dose tested. Preincubation of N(56)dg-eLH alpha:eLH beta and N(56)dg-eLH alpha:eLH beta t for 72 h at 37 C resulted in no loss of FSH receptor-binding activity. Preincubation resulted in 50% loss of receptor-binding activity by the eFSH preparation due to subunit dissociation, while 88% of N(56)dg-eLH alpha:eFSH beta activity was lost following 72 h, 37 C preincubation. While alpha Asn(56) oligosaccharide had no effect on eLH beta hybrid stability, it did contribute to the stability of the eFSH heterodimer.

Gonadal function is controlled by lutropins and follitropins, heterodimeric cystine knot proteins that have nearly identical alpha-subunits. These heterodimeric proteins are stabilized by a portion of the hormone-specific beta-subunit termed the "seatbelt" that is wrapped around alpha-subunit loop 2 (alpha 2). Here we show that replacing human chorionic gonadotropin (hCG) alpha 2 residue Lys51 with cysteine or alanine nearly abolished its lutropin activity, an observation that implies that alpha Lys51 has a key role in hormone activity. The activity of the heterodimer containing alpha K51C, but not that containing alpha K51A, was increased substantially when beta-subunit seatbelt residue beta Asp99 was converted to cysteine. As had been reported by others, heterodimers containing alpha K51C and beta D99C were crosslinked by a disulfide. The finding that an intersubunit disulfide restored some of the activity lost by replacing alpha Lys51 suggests that this residue is not crucial for receptor binding or signaling and also that hCG and related hormones may be particularly sensitive to mutations that alter interactions between their subunits. We propose the unique structures of hCG and related family members may permit some subunit movement in the heterodimer, making it difficult to deduce key residues involved in receptor contacts simply by correlating the activities of hormone analogs with their amino acid sequences.

Temperature-dependent dissociation of porcine luteinizing hormone (pLH) and of two of its glycoforms was studied by a combination of SDS-PAGE and micro-scale size-exclusion HPLC in parallel with the study of co-operative folding by high-sensitivity differential scanning calorimetry (HS-DSC). The transition temperature of dissociation of pLH at pH 7.0 as quantified by SDS-PAGE, HPLC and residual activity in radioreceptor assay was found to match exactly the transition temperature of its unfolding as measured by HS-DSC. Free alpha- and beta-subunits did not exhibit any unfolding transition in the same conditions. The microcalorimetric data for two pLH isoforms exhibiting different glycosylations were identical to those of a preparation of non-separated isoforms. It is concluded that: (a) free subunits exhibit no co-operative folding (i.e. no stable three-dimensional structure) and co-operative folding occurs only in alphabeta heterodimers; (b) the co-operative folding is responsible for the stability of the association of subunits; and (c) the heterogeneity of carbohydrate chains does not affect the stability of folding and association of subunits. The fastening of the "seat-belt" of the beta-subunit embracing the alpha-subunit by the Cysbeta26-beta110 disulfide bridge had been postulated to play a role in the preservation of the dimeric structure of gonadotropins. The present work shows that dissociation of subunits is directly related to their loss of common co-operative folding.

Monoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free alpha-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-alpha hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free alpha-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free alpha-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary hormones either as free subunits or intact molecules as follows: alpha hCG 100%; intact hCG 1.8%; beta hCG 0.14%; alpha FSH 24.5%; intact FSH 0.8%; beta FSH 0.09%; alpha LH 20.5%; intact LH 0.9%; beta LH 0.08%; alpha TSH 50.5%; intact TSH 3.7%; beta TSH 0.07%; The affinity constant (K) of this Mab with respect to free alpha-subunit of hCG was found to be 1.5 x 10(7) I/mol as determined by the simple antibody dilution analysis method.

Three of the five disulfide bonds in the glycoprotein hormone alpha-subunit (GPH-alpha) form a cystine knot motif that stabilizes a three-loop antiparallel structure. Previously, we described a mutant (alpha(k)) that contained only the three knot disulfide bonds and demonstrated that the cystine knot was necessary and sufficient for efficient GPH-alpha folding and secretion. In this study, we used alpha(k) as a model to study the intracellular GPH-alpha folding pathway. Cystine knot formation proceeded through a 1-disulfide intermediate that contained the 28-82 disulfide bond. Formation of disulfide bond 10-60, then disulfide bond 32-84, followed the formation of 28-82. Whether the two non-cystine knot bonds 7-31 and 59-87 could form independent of the knot was also tested. Disulfide bond 7-31 formed rapidly, whereas 59-87 did not form when all cysteine residues of the cystine knot were converted to alanine, suggesting that 7-31 forms early in the folding pathway and that 59-87 forms during or after cystine knot formation. Finally, loop 2 of GPH-alpha has been shown to be very flexible, suggesting that loop 2 does not actively drive GPH-alpha folding. To test this, we replaced residues 36-55 in the flexible loop 2 with an artificially flexible glycine chain. Consistent with our hypothesis, folding and secretion were unaffected when loop 2 was replaced with the glycine chain. Based on these findings, we describe a model for the intracellular folding pathway of GPH-alpha and discuss how these findings may provide insight into the folding mechanisms of other cystine knot-containing proteins.

The O-glycosylation sites for equine LHss (eLHss) and eCGss were identified by solid-phase Edman degradation of four glycopeptides derived from the C-terminal region. Both subunits were O-glycosylated at the same 12 positions, rather than the 4-6 sites anticipated. These sites were partially glycosylated, with carbohydrate attachment ranging from 20% to 100% for eCGss and from 10% to 100% for eLHss. When the C-terminal peptide containing all but one of the O-linked oligosaccharides was removed by mild acid hydrolysis of either eLHss or eCGss, hybrid hormones could be obtained by reassociating eLHalpha,eFSHalpha, or eCGalpha with the truncated ss subunit derivatives. These hybrid hormones were identical in LH receptor-binding activity when des(121-149)eLHss or des(121-149)eCGss were combined with the same alpha subunit preparation. Thus, O-glycosylation appears to be responsible for the ss subunit contribution to the substantial difference in LH receptor-binding activity between eLH and eCG. Comparison of the equid LH/CGss sequences with those available for the primate CGss subunits indicated a greater conservation of glycosylation patterns in the former.

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit but differ in their hormone-specific beta-subunits. Both subunits have five and six disulfide bonds, respectively, which consist of cystine knot structure. It is evident from numerous studies that the structure of beta-subunits is rigid, whereas that of alpha-subunit is flexible and can be molded by a beta-subunit. Previously, we reported that secreted forms of a mutants where either cysteine residue in the disulfide bond 7-31 or 59-87 was converted to alanine contained a disulfide-linked homodimer in addition to a monomer. To study whether the hCGbeta-subunit affects the conformations of alpha mutants, alpha-subunits lacking either the 7-31 or 59-87 disulfide bond were expressed with wild-type (WT) hCGbeta in Chinese hamster ovary cells, and homodimer formation and glycosylation of dimerized alpha-subunit were assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-alpha or -hCGbeta serum, digestion with endoglycosidase-H or -F, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. Our data showed that a homodimer was not observed in the half-Cys mutants except one, where cysteine at position 7 was converted to alanine, in the presence of beta-subunit. This finding indicated that hCGbeta-subunit rescued the a half-Cys mutants from the formation of intermolecular disulfide-linked homodimer by preferentially combining with the alpha mutants. In both free WT and all mutants treated with endoglycosidase-H, no or faint bands were recognized as the same migration as seen in endoglycosidase-F treatment. Even in the endoglycosidase-H sensitive cases, the amount of sensitive alpha-subunits was less than 5% of total alpha-subunits. In contrast to free alpha-subunits, distinct endoglycosidase-H sensitive bands were seen in both WT and mutants, although the ratio was various. We concluded that hCGbeta-subunit affects the folding and glycosylation of the alpha-subunit mutants.

In this review article, the reader is introduced to recent advances in our knowledge on a subset of the cystine knot superfamily of homo- and hetero-dimeric proteins, from the perspective of the endocrine glycoprotein hormone family of proteins: follitropin (FSH), Iutropin (LH), thyrotropin. (TSH) and chorionic gonadotropin (CG). Subsequent papers will address the structure-function behaviour of other members of this increasingly significant family of proteins, including various members of the transforming growth factor-beta (TGF-beta) family of proteins, the activins, inhibins, bone morphogenic growth factor, platelet derived growth factor-beta, nerve growth factor and more than 35 other proteins with similar topological features. In the present review article, specific emphasis has been placed on advances with the glycoprotein hormones (GPHs) that have facilitated greater insight into their physiological functions, molecular structures and most importantly the basis of the molecular recognition events that lead to the formation of hetero-dimeric structures as well as their specific and selective recognition by their corresponding receptors and antibodies. Thus, this review article focuses on the structural motifs involved in receptor recognition and the current techniques available to identify these regions, including the role of immunological methodology, peptide fragment design and synthesis and mutagenesis to delineate their structure-function relationships and molecular recognition behaviour., (Copyright 2000 John Wiley & Sons, Ltd.)

Previous work has indicated that, during the process of gametogenesis in salmon, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are differentially synthesized and released. Although substantial information is available on the regulation of LH in many fish species, relatively little is known about the regulation of FSH biosynthesis and secretion or the regulation of two types of alpha subunit in salmon. In this study, the effects of salmon gonadotropin-releasing hormone (sGnRH) on in vitro secretion of FSH, and alpha1, alpha2, LH beta, and FSH beta subunit gene expression were investigated in coho salmon (Oncorhynchus kisutch) using primary pituitary cell cultures. To quantify FSH beta, LH beta, alpha1, and alpha2 subunit transcript levels, a multiplex RNase protection assay (RPA) was developed. Probes for the beta subunits of coho salmon FSH and LH were available from previous studies. To generate probes for the alpha subunit RPAs, alpha1 and alpha2 subunit cDNAs were cloned using reverse transcriptase PCR. Release of FSH and LH into cell culture medium was quantified by radioimmunoassays. The effects of sGnRH on gonadotropin release and gene expression were tested at two points during the spring (April and May) prior to spawning in the autumn; a period when plasma and pituitary FSH levels are increasing and females are in early stages of secondary oocyte growth. In both experiments, sGnRH increased steady-state mRNA levels of FSH beta, alpha1, and alpha2, whereas LH beta mRNA levels were not detectable. Secretion of FSH was stimulated by sGnRH in a concentration-dependent manner. Medium LH was not detectable in the first experiment (April) and was measurable only after sGnRH treatment in the second experiment (May). Control levels of medium FSH and transcripts for FSH beta and alpha1 subunits increased approximately fourfold between April and May, whereas alpha2 transcript levels remained relatively constant, suggesting that the seasonal increase in FSH release may involve increased production of alpha1. Therefore, sGnRH has direct stimulatory effects on both secretion of FSH and FSH subunit biosynthesis, most likely due to increased transcription. However, alterations in rates of transcript degradation cannot be ruled out., (Copyright 2000 Academic Press.)

The common glycoprotein hormone alpha-subunit (GPH-alpha) contains five intramolecular disulfide bonds, three of which form a cystine knot motif (10-60, 28-82, and 32-84). By converting each pair of cysteine residues of a given disulfide bond to alanine, we have studied the role of individual disulfide bonds in GPH-alpha folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin beta-subunit (hCG-beta). Mutation of non-cystine knot disulfide bond 7-31, bond 59-87, or both (leaving only the cystine knot) resulted in an efficiently secreted folding form that was indistinguishable from wild type. Conversely, the cystine knot mutants were inefficiently secreted (<25%). Furthermore, mutation of the cystine knot disulfide bonds resulted in multiple folding intermediates containing 1, 2, or 4 disulfide bonds. High performance liquid chromatographic separation of intracellular and secreted forms of the folding intermediates demonstrated that the most folded forms were preferentially secreted and combined with hCG-beta. From these studies we conclude that: (i) the cystine knot of GPH-alpha is necessary and sufficient for folding and (ii) there is a direct correlation between the extent of GPH-alpha folding, its ability to be secreted, and its ability to heterodimerize with hCG-beta.