Glycoprotein Hormone Family Research Articles

Using Evolutionary Genomics to Discover Hormones and Receptors

All human genes have undergone hundreds of millions of years of evolution to have today’s sequences and associated functions. At the same time, ligands and receptors have also undergone co-evolution. Evolutionary methods can be used to find ligands and receptors that have not yet been discovered, leading to new clinical diagnoses and treatments. This talk will deal with: 1. From the perspective of homologous genes and paralogous genes, the co-evolution of the glycoprotein hormone family, including FSH, LH, TSH, and hCG together with their receptors. 2. Create clinically useful long-acting FSH based on the evolutionary approach. 3. Use the evolution method to find five orphan LGR receptors, followed by the identification of receptors for the ligands, relaxin and INSL3, as well as the ligand for LGR4. 4. Use evolutionary methods to discover a new human placental hormone (placensin) and a new gonadal hormone in fish (gonacin), both with important metabolic functions. 5. Does the chicken or the egg come first during evolution? Which came first, the ligand or the receptor?

Beta-HCG expression on IHC in a case of small cell carcinoma lung: A rare case report

Introduction: Beta-human chorionic gonadotropin (β-HCG) belongs to the glycoprotein hormone family and is usually used to exclude pregnancy in patients. It comprises of two subunits (α and β polypeptide chains) which are joined by noncovalent bonds. HCG is a specific marker for trophoblastic tumors of placenta and gestational tumors. Ectopic secretion of β-HCG by other tumors is rare. Lung cancer with beta-HCG production is rare, and only a few case reports have been published in the literature. Case history: We reported a case of 40 years old male who presented with a history of cough, chest pain, abdominal pain and superior vena cava syndrome. On CT chest mediastinal mass, right sided pleural effusion along with right lung collapse was noted. Biopsy was taken from the lesion. Biopsy was processed accordingly and IHC was performed and final diagnosis of small cell carcinoma lung with beta-HCG expression on IHC was made. Discussion and conclusion: Based on reports in the medical literature, beta-HCG expressing lung tumors are very rare. No case reports on beta HCG expressing small cell carcinoma of lung could be found by us in the reported literature. We reported an extremely rare case of beta-HCG expressing small cell carcinoma lung using IHC in a 40 years old male patient.

Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

BackgroundEquine chorionic gonadotropin (eCG), which comprises highly glycosylated α-subunit and β-subunit, is a unique member of the glycoprotein hormone family as it elicits both follicle-stimulating hormone (FSH)-like and luteinizing hormone (LH)-like responses in non-equid species. To examine the biological function of glycosylated sites in eCG, the following glycosylation site mutants were constructed: eCGβ/αΔ56, substitution of Asn56 of α-subunit with Gln; eCGβ-D/α, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the β-subunit; eCGβ-D/αΔ56, double mutant. The recombinant eCG (rec-eCG) mutants were expressed in Chinese hamster ovary suspension (CHO-S) cells. The FSH-like and LH-like activities of the mutants were examined using CHO-K1 cells expressing rat lutropin/CG receptor (rLH/CGR) and rat FSH receptor (rFSHR).ResultsBoth rec-eCGβ/α and rec-eCGβ/αΔ56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCGβ-D/α and eCGβ-D/αΔ56, which lack approximately 12 O-linked glycosylation sites, was slightly delayed. The expression levels of all mutants were similar (200–250 mIU/mL) from days 3 to 7 post-transfection. The molecular weight of rec-eCGβ/α, rec-eCGβ/αΔ56 and rec-eCG β-D/α were in the ranges of 40–45, 37–42, and 34–36 kDa, respectively. Treatment with peptide-N-glycanase F markedly decreased the molecular weight to approximately 5–10 kDa. Rec-eCGβ/αΔ56 exhibited markedly downregulated LH-like activity. The signal transduction activity of both double mutants was completely impaired. This indicated that the glycosylation site at Asn56 of the α-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCGβ-D/α exhibited markedly downregulated LH-like and FSH-like activities.ConclusionsRec-eCGβ/α exhibits potent biological activity in cells expressing rLH/CGR and rFSHR. The findings of this study suggest that the LH-like and FSH-like activities of eCG are regulated by the N-linked glycosylation site at Asn56 of the eCG α-subunit and/or by the O-linked glycosylation sites of the eCG β-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.

A Novel Follitropin Analog Inhibits Follitropin Activity In Vitro.

Follitropin (FSH) is a heterodimeric protein composed of an α subunit that is shared with the glycoprotein hormone family, including lutropin (LH), thyrotropin (TSH), human choriogonadotropin (hCG), and a unique β specific subunit. Both α and FSHβ subunits contain two sites of N-linked oligosaccharides, which are important for its function. FSH has a crucial function in the reproductive process in mammals. However, there are some clinical conditions, such as menopausal osteoporosis or adiposity, associated with increased FSH activity. Moreover, in some cases, carcinogenesis is evidently associated with activation of FSH receptor. Therefore, developing a follitropin antagonist might be beneficial in the treatment of these conditions. Here, we describe a novel, engineered, non-glycosylated single-chain FSH variant, prepared by site-directed mutagenesis and fusion of the coding genes of the α and β subunits. The designed variant was expressed in Chinese hamster ovary (CHO) cells and successfully secreted into the culture medium. We found that the non-glycosylated single-chain FSH analog binds with high affinity to FSH receptor and efficiently inhibits FSH activity in vitro. This variant acts at the receptor level and has the potential to serve as a follitropin antagonist for clinical applications in the future.

Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry

The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGβ, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGβ glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGβ glycoforms, which allowed for the first time the detection of 33 hCGβ glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.

Expression Analysis of Recombinant Equine Chorionic Gonadotropin in Three Host Systems: E. coli BL21C, Sf insect cell lysate and COS-1 mammalian cells

Mammalian cells are the recommended host for recombinant eukaryotic protein production aimed at incorporation of post-translational modifications for downstream applications. The bacterial system and insect cells are widely used because of ease of technical methodology, economics of production, purification and yield of final protein. The present research objective was expression of recombinant reproductive hormones of animal origin and study of their immunogenic potential for reproductive applications. The equine Chorionic Gonadotropin (eCG) is one of the most heavily glycosylated protein amongst all glycoprotein hormone family. Hence, experiments were carried out to observe its expression in the three most popular host systems and it led to comparative studies for their post-translational modifications. The Pregnant Mare Serum Gonadotropin (PMSG, also called as eCG) gene was cloned in TOPO-TA vector, pIX 4.0 and pTARGET vectors accordingly and expression analysis in E. coli BL21C, Sf insect cell lysate and COS-1 cells was carried out. We observed diverse sizes of recombinant proteins in SDS-PAGE analysis which indicated post-translational modification in mammalian expression system towards the linking of tags as well as side chains in respective host cells. Basic diagnostic immunogenicity tests showed encouraging results, however, no significant in vivo and in vitro activity was observed for the expressed reCG in all the employed host systems.

Discovery of BAY-298 and BAY-899: Tetrahydro-1,6-naphthyridine-Based, Potent, and Selective Antagonists of the Luteinizing Hormone Receptor Which Reduce Sex Hormone Levels in Vivo.

The human luteinizing hormone receptor (hLH-R) is a member of the glycoprotein hormone family of G-protein-coupled receptors (GPCRs), activated by luteinizing hormone (hLH) and essentially involved in the regulation of sex hormone production. Thus, hLH-R represents a valid target for the treatment of sex hormone-dependent cancers and diseases (polycystic ovary syndrome, uterine fibroids, endometriosis) as well as contraception. Screening of the Bayer compound library led to the discovery of tetrahydrothienopyridine derivatives as novel, small-molecule (SMOL) hLH-R inhibitors and to the development of BAY-298, the first nanomolar hLH-R antagonist reducing sex hormone levels in vivo. Further optimization of physicochemical, pharmacokinetic, and safety parameters led to the identification of BAY-899 with an improved in vitro profile and proven efficacy in vivo. BAY-298 and BAY-899 serve as valuable tool compounds to study hLH-R signaling in vitro and to interfere with the production of sex hormones in vivo.

Ultrasensitive Electrical Detection of Follicle-Stimulating Hormone Using a Functionalized Silicon Nanowire Transistor Chemosensor.

The follicle-stimulating hormone (FSH) is a hormone that belongs to a family of glycoprotein hormones. Determination of FSH can help in interpreting various factors that include physiology of the reproductive system, fertility maintenance, and identification or treatment of reproductive disorders. Sialic acids are derivatives of neuraminic acids with negative charges, present at the end of the sugar chains and further linked to the cell surfaces and glycoproteins. The direct measurement of FSH in a human body can be recorded by developing a sensor probe that responds particularly to sialic acids over the other hormones. However, existing diagnostic methods still suffer from many difficulties in terms of complicated handling techniques, expensive instrumentation, etc. Development of accurate, rapid, and low-cost FSH detection chemosensors is important to meet these demands. Herein, we utilized a novel sensing method for accurate and fast FSH detection using a metal-oxide semiconductor silicon nanowire field effect transistor (SiNW-FET) device. This is the first report to demonstrate the boronic acid-functionalized SiNW-FET device in FSH detection. FSH detection has been successfully determined using an assay buffer solution with 0.72 fM detection limit as well as using 20% serum with 1.1. fM detection limit. We also investigated the specificity with other gonadotropins/glycosylated serum proteins. The current measurements on FSH concentrations at different time intervals were also studied. The sensitive, cheap, and miniaturized SiNW-FET device can serve as an effective sensing approach for rapid screening of FSH and menopause diagnosis.

The gonadotropin system, lessons from animal models and clinical cases.

This review article centers upon family of gonadotropin hormones which consists of two pituitary hormones - follicle-stimulating hormone (FSH) and luteinizing hormone (LH) as well as one non-pituitary hormone - human chorionic gonadotropin (hCG) secreted by placenta, and their receptors. Gonadotropins play an essential role in proper sexual development, puberty, gametogenesis, maintenance of pregnancy and male sexual differentiation during the fetal development. They belong to the family of glycoprotein hormones thus they constitute heterodimeric proteins built of common α subunit and hormone-specific β-subunit. Hitherto, several mutations in genes encoding both gonadotropins and their receptors have been identified in humans. Their occurrence resulted in a number of different phenotypes including delayed puberty, primary amenorrhea, hermaphroditism, infertility and hypogonadism. In order to understand the effects of mutations on the phenotype observed in affected patients, detailed molecular studies are required to map the relationship between the structure and function of gonadotropins and their receptors. Nonetheless, in vitro assays are often insufficient to understand physiology. Therefore, several animal models have been developed to unravel the physiological roles of gonadotropins and their receptors.

Gonadotropins and their receptors: coevolution, genetic variants, receptor imaging, and functional antagonists

Gonadotropins belong to the family of dimeric glycoprotein hormones and regulate gonadal physiology mediated by G protein-coupled, seven-transmembrane receptors. These glycoprotein hormones are widely used in the clinic to promote ovarian follicle development and for treating some cases of male infertility. We traced the coevolution of dimeric gonadotropin hormones and their receptors, together with thyrotropin and its receptor. We updated recent findings on human genetic variants of these genes and their association with dizygotic twining, polycystic ovarian syndrome, primary ovarian insufficiency, male-limited precocious puberty, and infertility. In addition to the known physiological roles of gonadotropin-receptor signaling in gonadal tissues, we also discussed emerging understanding of extragonadal functions of gonadotropins in bones and adipose tissues, together with recent advances in in vivo imaging of gonadotropin receptors in live animals. Recent development of gonadotropin receptor agonists and antagonists were summarized with an emphasis on the development of functional antagonists for FSH receptors to alleviate osteoporosis and obesity associated with menopause.

HCG: Biological Functions and Clinical Applications.

Human chorionic gonadotropin (hCG) is produced primarily by differentiated syncytiotrophoblasts, and represents a key embryonic signal that is essential for the maintenance of pregnancy. hCG can activate various signaling cascades including mothers against decapentaplegic homolog 2 (Smad2), protein kinase C (PKC), and/or protein kinase A (PKA) in several cells types by binding to luteinizing hormone/chorionic gonadotropin receptor (LHCGR) or potentially by direct/indirect interaction with transforming growth factor beta receptor (TGFβR). The molecule displays specialized roles in promoting angiogenesis in the uterine endothelium, maintaining myometrial quiescence, as well as fostering immunomodulation at the maternal-fetal interface. It is a member of the glycoprotein hormone family that includes luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH). The α-subunit of hCG displays homologies with TSH, LH, and FSH, whereas the β subunit is 80–85% homologous to LH. The hCG molecule is produced by a variety of organs, exists in various forms, exerts vital biological functions, and has various clinical roles ranging from diagnosis and monitoring of pregnancy and pregnancy-related disorders to cancer surveillance. This review presents a detailed examination of hCG and its various clinical applications.

PHYLOGENETIC ANALYSIS OF THE HUMAN THYROID-STIMULATING HORMONE

Background: Thyroid-stimulating hormone (TSH) is a member of the vertebrate glycoprotein hormone family [1]. It’s secreted from pituitary cells as heterodimers composed of an alpha and a beta- subunit. The thyrotrophic cells that secrete TSH are preferentially located in the anteromedial and anterolateral portions of the pituitary. Objective: We performed a phylogenetic analysis of the TSH, and phylogram of the maximum likelihood relations between TSH coding sequences of five representative species. Methods: We extracted the available DNA and protein sequences for TSH from the NCBI databank. Searched for regions presenting sequence similarities to the constituent domains of TSH – alpha and beta-subunits - with the Blastall command ftp://ftp.ncbi.nlm.nih.gov/blast/db/ website, pairs of sequences were compared on the basis of their global alignment with the Myers & Millers algorithm manpages.ubuntu. com/manpages/karmic/man1, and the phylogenetic reconstructions were performed online by using a maximum likelihood method with PhyMyL 3.0 software on the website file (http://www.phylogeny.fr/). Results: The comparison of the phylogenetic trees that we obtained compared with those studies previously published revealed similar subclusters with high protein homology. Conclusion: This study demonstrates that human TSH is structurally related to TSH of the species analysed, Bos taurus, Mus musculus, Canis lupus familiaris, and Cyprinus carpio, respectively.

Hormone-like peptides in the venoms of marine cone snails

The venoms of cone snails (genus Conus) are remarkably complex, consisting of hundreds of typically short, disulfide-rich peptides termed conotoxins. These peptides have diverse pharmacological targets, with injection of venom eliciting a range of physiological responses, including sedation, paralysis and sensory overload. Most conotoxins target the prey’s nervous system but evidence of venom peptides targeting neuroendocrine processes is emerging. Examples include vasopressin, RFamide neuropeptides and recently also insulin. To investigate the diversity of hormone/neuropeptide-like molecules in the venoms of cone snails we systematically mined the venom gland transcriptomes of several cone snail species and examined secreted venom peptides in dissected and injected venom of the Australian cone snail Conus victoriae. Using this approach we identified several novel hormone/neuropeptide-like toxins, including peptides similar to the bee brain hormone prohormone-4, the mollusc ganglia neuropeptide elevenin, and thyrostimulin, a member of the glycoprotein hormone family, and confirmed the presence of insulin. We confirmed that at least two of these peptides are not only expressed in the venom gland but also form part of the injected venom cocktail, unambiguously demonstrating their role in envenomation. Our findings suggest that hormone/neuropeptide-like toxins are a diverse and integral part of the complex envenomation strategy of Conus. Exploration of this group of venom components offers an exciting new avenue for the discovery of novel pharmacological tools and drug candidates, complementary to conotoxins.

Production and characterization of a monoclonal antibody against eel glycoprotein hormone

The glycoprotein hormone family consists of luteinizing hormone (LH), follicle‐stimulating hormone (FSH), and thyroid‐stimulating hormone (TSH), which are secreted by the pituitary gland in all mammalian species, and chorionic gonadotropin (CG) secreted by placental trophoblast cells in primates and equids. These hormones consists of non‐covalently associated α and β subunits. Within a species, the amino acid sequence of the α subunit is identical across all glycoprotein hormones and is encoded by a single gene. The α/β dimer is the active form of the hormone, and biological specificity is conferred by the β subunit. Also in fish, the duality of gonadotropin hormone (GTH), GTH‐1 (FSH‐like GTH), and GTH‐II (LH‐like GTH) is found in certain teleost. In this study, to understand the fundamental mechanisms involved in teleost reproduction and to prove a broader basis for comparative study of teleost GTHs, we produced tethered rec‐eelFSH protein, the cDNA encoding the full‐length eel FSH β‐subunit (signal sequence of 22 amino acid residues and the mature protein of 105 amino acid residues) was fused with the mature protein (93 amino acids) of eel α‐subunit, immunized and A positive hybridoma was selected by ELISA using eel FSH. Anti‐eel glycoprotein mAbs was purified from culture supernatants by affinity chromatography. Six monoclonal antibodies for eel FSH were produced and 2 clones (#11, #5) of these antibodies were bound with α‐subunit of eel glycoprotein. And the other one (#14) was bound with FSHβ. Recombinant FSHβ/α also produced into the CHO cells. Its molecular weight was about 34 kDa. The deglycosylated rec‐FSHβ/α was about 25 kDa. This mAb can specially recognize eel glycoprotein and may serve as a component of eel glycoprotein ELISA kit. (This work was supported by NFRDI. Eun Bi Seo was supported by a scholarship from the BK21 Plus Program (31Z20130012928). the Ministry of Education, Science and Technology, korea)

Expression and activity of single‐chain recombinant eel follicle stimulating hormone in mammalian cells (792.2)

The glycoprotein hormone family consists of luteinizing hormone (LH), follicle‐stimulating hormone (FSH), and thyroid‐stimulating hormone (TSH), which are secreted by the pituitary gland in all mammalian species, and chorionic gonadotropin (CG), which is secreted by placental trophoblast cells in primates and equids. These hormones consists of non‐covalently associated α and β subunits. Within a species, the amino acid sequence of the α subunit is identical across all glycoprotein hormones and is encoded by a single gene. The αβ dimer is the active form of the hormone, and biological specificity is conferred by the β subunit. Also in fish, the duality of gonadotropin hormone (GTH), GTH‐1 (FSH‐like GTH), and GTH‐II (LH‐like GTH) is found in certain teleost. In this study, in order to understand the fundamental mechanisms involved in teleost reproduction and to proved a broader basis for comparative studt of teleost GTHs, we isolated a cDNA encoding the α and β subunit of GHT‐1 from eel and produced it's recombinant protein from E.coli and CHO cells. To obtain tethered eelFSH, the cDNA encoding the full‐length eel FSH β‐subunit (signal sequence of 22 amino acid residues and the mature protein of 105 amino acid residues) was fused with the mature protein (93 amino acids) of the α‐subunit by overlapping PCR mutagenesis. Subsequently, PCR fragement was inserted into the pCR2.1 clon ing vector and sequenced. After then, it was digested with the EcoRI and SalI enzymes and ligated into the eukaryotic expression vector pcDNA3. The gene was completely sequenced to confirm the presence of the Kozak site and, myc‐tag and to role out the possibility of any PCR erros. Eel FSH protein was expressed in the E. coli system using the pREST expression plasmid vector, and the protein was purified by NI‐NTA column. Eel FSH protein was detected at the predicted molecular weight of 24.3 kDa by SDS‐PAGE in E.coli and 32‐45 kDa in CHO cell lines.Grant Funding Source: This work was supported by NFRDI

Naturally Occurring Follicle-Stimulating Hormone Glycosylation Variants

Follicle-stimulating hormone (FSH) is a member of the glycoprotein hormone family, which is a subfamily of the cystine knot growth factor superfamily [1,2]. The glycoprotein hormones are composed of heterodimeric glycoprotein subunits, a common α-subunit, and a hormone-specific β-subunit. While the α-subunit primary structure is identical for all glycoprotein hormones within the same species, the oligosaccharide populations differ in a hormone-specific manner [3–6]. Characterizing the oligosaccharides released from an α-subunit preparation can identify the hormone from which the subunit was derived [7]. There are 3 to 4 β-subunits in vertebrates, which combine with α-subunit to create either FSH, luteinizing hormone (LH), thyroid-stimulating hormone (TSH), or in primates and equids, chorionic gonadotropin (CG) [8]. As both glycoprotein hormone subunits are cystine knot proteins [9–11] the protein backbone is folded into a series of three loops, two relatively rigid hairpin loops on one side of the knot, designated L1 and L3, and a single, flexible loop on the other side [12], designated L2. Oligosaccharides are attached to all 3 loops in a subunit-specific pattern (Figure 1). FSH subunits possess two potential N-glycosylation sites on each subunit and all four are of the Asn-Xaa-Thr type, which exhibit very efficient carbohydrate attachment [13]. Indeed, the α-subunit is always glycosylated at both sites in all known glycoprotein hormones. Because FSH α and β subunits co-migrate during electrophoresis, it is difficult to detect missing N-glycans in this hormone. FSHβ-specific Western blots have revealed partial glycosylation in equine FSHβ, human FSHβ (hFSH β), rhesus FSH β, and Japanese macaque FSHβ [14–16]. During the past few years, we have studied partially glycosylated hFSH isolated from pituitary extracts, postmenopausal urine, and conditioned tissue culture medium containing recombinant hFSH. Each glycosylation site in hFSH is decorated with a population of N-glycans. When total glycans are removed from reduced, carboxy-methylated FSH subunits, 39–130 glycans are found in mass spectra. We have data from only one glycosylation site, αAsn52, which possessed 29 neutral core ions, and when decorated with various patterns of sialic acid grew to 109 unique glycan structures. Micro heterogeneity can affect electrophoretic mobility, for example, placental hCGα with hybrid and biantennary glycans migrated faster than pituitary hFSHα, with triantennary, biantennary and tetraantennary glycans, which complicated sorting out the hFSH variants that resulted from loss of one or more N-glycans [17].

β-Hormone chorionique gonadotrope et cancer bronchique : un piège pour les essais cliniques

Introductionβ-HCG belongs to the glycoprotein hormone family and is usually assess to exclude pregnancy for patients potentially eligible to chemotherapy, especially during clinical trials. Material and MethodsWe studied non-small-cell lung cancer (NSCLC) patients that were found with elevated serum β-HCG level during clinical trial screening. The first case is a 45-year-old woman who presented with a stage IV undifferentiated carcinoma of the lung eligible for chemotherapy. When screening the patient for a clinical trial combining platinum-based chemotherapy and targeted therapy, the plasma β-HCG level was 19 IU/L (0-5 IU/L). The second case is a 64-year-old woman presented with stage IV poorly differentiated adenocarcinoma of the lung. When screening the patient for the same clinical trial combining platinum-based chemotherapy and targeted therapy, the plasma β-HCG level was β-HCG 13 IU/L (0-5 IU/L). ResultsThe serum dosages were double-checked and confirmed elevated β-HCG level. The gynecological work-up definitely rules out an improbable pregnancy. The pathological examination was also checked and confirmed in the two cases a primary lung cancer. An immuno-histochemical reassessment of the pathological specimens with additional tests was performed: for one patient 25% of tumor cells expressed β-HCG. As pregnancies were ruled out, the two cases were extensively discussed with the promoter and the patients finally treated within this clinical trial. The treatments are ongoing. Conclusionsβ-HCG is a specific marker for trophoblastic tumors of placenta and gestational tumors. Ectopic expression of β-HCG was found in 20-40% of all common epithelial carcinoma, especially for tumors of the stomach, ovary, liver and lung. Only few cases have been reported in the literature. However, in a young patient with high serum levels of β-HCG two questions arise: is there a place for pregnancy? Are the pathology results accurate? All this could delay the appropriate management of these patients and also potentially prevents the participation of innovative therapeutic strategies. Therefore, knowing this rare but possible expression of β-HCG by lung tumors may speed out the gynecological work-up and the reevaluation of the histological samples in order to minimize the delay in the care of these patients and give them a chance to have new innovative drugs within clinical trial.

Glycoprotein hormone in the pituitary of hagfish and its evolutionary implications

The pituitary gland is present in all vertebrates, from agnathans (jawless vertebrates) to mammals, but not in invertebrates. Reproduction in gnathostomes (jawed vertebrates) is controlled by two pituitary gonadotropins (GTHs), luteinizing hormone and follicle-stimulating hormone, which are part of the pituitary glycoprotein hormone (GPH) family. Hagfishes, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Accordingly, they are of particular importance in understanding the evolution of the pituitary GPHs and their functions related to vertebrate reproduction. Nevertheless, key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Our current report has revealed the first identification of a functional GPH composed of two subunits that possess gonadotropic action at the pituitary of brown hagfish. It seems most likely that an ancestral GPH gave rise to only one GTH in hagfish pituitary and that multiplicity of GPHs arose later during the early evolution of gnathostomes. This paper briefly summarizes the latest findings on the hagfish GPH from an evolutionary point of view.

Antibody Recognition of a Human Chorionic Gonadotropin Epitope (hCGβ66–80) Depends on Local Structure Retained in the Free Peptide

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical β3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCGβ(66-80) and the closely related luteinizing hormone (LH) fragment LHβ(86-100), which differ only by a single amino acid residue. A combination of optical spectroscopic measurements and atomistic computer simulations on these free peptides reveals differences in turn type stabilized by specific hydrogen bonding motifs. We propose that these structural differences are the basis for the observed selectivity in the full protein.

EVOLUTIONARY ORIGIN OF A FUNCTIONAL GONADOTROPIN IN THE PITUITARY OF THE MOST PRIMITIVE VERTEBRATE, HAGFISH

Hagfish, which lack both jaws and vertebrae, are considered themost primitive vertebrate known, living or extinct. Hagfish havelong been the enigma of vertebrate evolution not only becauseof their evolutionary position, but also because of our lack ofknowledge on fundamental processes. Key elements of the re-productive endocrine system in hagfish have yet to be elucidated.Here, the presence and identity of a functional glycoprotein hor-mone (GPH) have been elucidated from the brown hagfish Para-myxine atami. The hagfish GPH consists of two subunits, α and β,which are synthesized and colocalized in the same cells of theadenohypophysis. The cellularand transcriptional activitiesof hag-fish GPHα and -β were significantly correlated with the develop-mental stages of the gonad. The purified native GPH induced thereleaseofgonadalsexsteroidsinvitro.Fromourphylogeneticanal-ysis, we propose that ancestral glycoprotein α-subunit 2 (GPA2)and β-subunit 5 (GPB5) gave rise to GPHα and GPHβ of the verte-brate glycoprotein hormone family, respectively. The identifiedhagfishGPHαand-βsubunitsappeartobethetypicalgnathostomeGPHαand-βsubunitsbasedonthesequenceandphylogeneticanal-yses.WehypothesizethattheidentityofasinglefunctionalGPHofthehagfish,hagfishGTH,providescriticalevidencefortheexistenceof a pituitary-gonadal system in the earliest divergent vertebratethat likely evolved from an ancestral, prevertebrate exclusivelyneuroendocrine mechanism by gradual emergence of a previouslyundescribed control level, the pituitary, which is not found inthe Protochordates.