hCG Beta Gene Research Articles

Serum levels of intact human chorionic gonadotropin (HCG) and its free alpha and beta subunits, in relation to maternal thyroid stimulation during normal pregnancy.

The main objective of the present study was to present additional evidence of the potentially important thyrotropic role of hCG to regulate the maternal thyroid gland during normal pregnancy. Sequential determinations (first and last trimesters) of intact hCG, free alpha and beta-hCG subunits concentrations (using monoclonal IRMAs), and assessment of parameters of thyroid function and thyroid volume were carried out in 62 pregnant women who exhibited during the first trimester of gestation low TSH levels (< or = 0.20 mU/L), and compared to 276 pregnant women with normal TSH levels. The prevalence of having low serum TSH represented 18% of all pregnancies, with almost one half of cases who transiently had undetectable TSH levels. Lowering of TSH was associated with high hCG levels, and occurred primarily during the first trimester. About 10% of women with low TSH presented transient gestational thyrotoxicosis, frequently associated with vomiting. In comparison to control subjects, women with a suppressed serum TSH had significantly and markedly higher intact hCG and free beta-hCG subunit concentrations. The results suggest that TSH reduction may result from a relative oversecretion of both intact hCG and free beta-hCG subunits, compatible with three hypotheses: a) transient overexpression of the beta-hCG gene, leading to enhanced production of hCG heterodimer; b) increased glycosylation of circulating hCG, with in turn a prolonged half life; c) larger syncytiotrophoblast mass with increased hCG production.(ABSTRACT TRUNCATED AT 250 WORDS)

Disulfide bond mutations affect the folding of the human chorionic gonadotropin-beta subunit in transfected Chinese hamster ovary cells

Previous kinetic studies have characterized the intracellular folding pathway of the human chorionic gonadotropin (hCG)-beta subunit in which each of the folding intermediates can be biochemically identified based on the formation of disulfide (S-S) bonds: p beta 1-early--> p beta 1-late--> p beta 2-free--> p beta 2-combined--> native hCG-alpha beta. Based on these data, we postulated that hCG-beta folding coincides with the formation of specific S-S bonds. We have now tested this hypothesis employing Chinese hamster ovary cells transfected with mutated hCG-beta genes in which the Cys residues required for the formation of the final four (of six total) S-S bonds were replaced by Ala. When the Cys residues required for the third hCG-beta S-S linkage to form (bond 9-90) were substituted, folding did not proceed beyond the earliest detectable folding intermediate (p beta 1-early). In the absence of the subsequently formed S-S bond (bond 23-72), p beta 1-early was converted into a second folding intermediate (p beta 1-late), but conversion to the next intermediate (p beta 2-free) was inhibited. When either of the final two S-S bonds (the carboxyl-terminal 93-100 or 26-110 bonds) were removed, conversion of p beta 1-late to p beta 2-free was detected, but conversion of p beta 2-free to the last folding intermediate (p beta 2-combined) was not observed. These data support the hypothesis that individual S-S bonds are involved in discrete steps in the hCG-beta folding pathway.

Site-directed mutagenesis defines a domain in the gonadotropin alpha-subunit required for assembly with the chorionic gonadotropin beta-subunit.

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that share a common alpha-subunit, but differ in their hormone-specific beta-subunits. Using site-directed mutagenesis and gene transfer, we studied the region in the common alpha-subunit that has been implicated in the assembly with the beta-subunits. The wild-type or mutated alpha-gene was cotransfected into Chinese hamster ovary cells with the wild-type hCG beta gene. Deletion of the sequence Pro38-Thr39-Pro40 or a change in Tyr37 or Thr39 in the alpha-subunit eliminated or reduced combination with the beta-subunit. Deletion of the sequence Leu41-Arg42-Ser43 had little effect on hCG dimer formation. Disruption of the disulfide bone in the carboxyl end of the subunit did not affect assembly, which suggests that the disulfide bond of Cys59 and Cys87 is not critical for dimer formation. Based on our data and the previously published results from several laboratories, the region encompassed by amino acids 37-40 is a key determinant in initiating and maintaining alpha:beta assembly.

The Biological Role of the Carboxyl-Terminal Extension of Human Chorionic Gonadotroin β-Subunit

hCG is a member of a family of glycoprotein hormones which share a common alpha-subunit, but differ in their hormone-specific beta-subunits. The CG beta-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG beta on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG alpha and hCG beta expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the beta-subunit O-linked oligosaccharide units. In addition, a mutant hCG beta gene (CG beta delta T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG alpha gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG beta (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind to the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show that the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG beta and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.

The Transcriptional Response of the Human Chorionic Gonadotropin β-Subunit Gene to cAMP is Cycloheximide Sensitive and is Mediated bycis-Acting Sequences Different from that Found in the α-Subunit Gene

Cyclic AMP stimulates transcription of the genes encoding the alpha- and beta-subunits of human CG (hCG). Although the cis-acting cAMP response element (CRE) of the alpha-subunit gene has been extensively characterized, relatively little is known about the putative response element of the hCG beta gene. Here, we demonstrate that cAMP stimulation of hCG beta gene transcription requires ongoing protein synthesis, whereas cAMP stimulation of alpha-subunit gene transcription does not. These results suggest that cAMP-stimulated transcription of the alpha and hCG beta genes may involve different cis-acting elements and trans-acting factors. Accordingly, we have constructed a series of deletion mutants consisting of fragments of the hCG beta promoter-regulatory region linked to the bacterial chloramphenicol acetyl transferase gene. To potentiate detection of a CRE, we constructed vectors containing the Rous sarcoma virus enhancer to augment transcriptional activity of the hCG beta-promoter. We also used vectors designed to determine whether regions containing a putative CG beta CRE could confer cAMP responsiveness to a heterologous promoter. These constructs were evaluated for activity by transfection into choriocarcinoma (BeWo) cells. Transient expression of these vectors identifies a broad region which renders transcription of the chloramphenicol acetyl transferase gene responsive to cAMP. Surprisingly, division of the hCG beta 5'-flanking and untranslated regions into smaller fragments led to loss of cAMP responsiveness, suggesting that the hCG beta CRE may be comprised of multiple domains dispersed across the proximal 650 base pairs of CG beta 5'-flanking and untranslated sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the asparagine-linked oligosaccharides of the alpha subunit in the secretion and assembly of human chorionic gonadotrophin.

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunit. Site-directed mutagenesis of the two asparagine-linked glycosylation sites of hCG alpha was used to study the function of the individual oligosaccharide chains in secretion and subunit assembly. Expression vectors for the alpha genes (wild-type and mutant) and the hCG beta gene were constructed and transfected into Chinese hamster ovary cells. Loss of the oligosaccharide at position 78 causes the mutant subunit to be degraded quickly and less than 20% is secreted. However, the presence of hCG beta stabilizes this mutant and allows approximately 45% of the subunit in the form of a dimer to exit the cell. Absence of carbohydrate at asparagine 52 does not perturb the stability or transport of the alpha subunit but does affect dimer secretion; under conditions where this mutant or hCG beta was in excess, less than 30% is secreted in the form of a dimer. Mutagenesis of both glycosylation sites affects monomer and dimer secretion but at levels intermediate between the single-site mutants. We conclude that there are site-specific functions of the hCG alpha asparagine-linked oligosaccharides with respect to the stability and assembly of hCG.

Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells.

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunits. The beta subunit of hCG (hCG beta) is unique among the beta subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either the hCG beta gene alone or together with the hCG alpha gene were transfected into a mutant Chinese hamster ovary cell line, IdID, which exhibits a reversible defect in O-glycosylation. Our results reveal that hCG beta can be secreted normally in the absence of its O-linked oligosaccharides. hCG beta devoid of O-linked carbohydrate can also combine efficiently with hCG alpha and be secreted as an intact dimer. We conclude that in Chinese hamster ovary cells, the hCG beta O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.

Ectopic β-Human Chorionic Gonadotropin Production by a Human Hepatoma Cell Line (FOCUS): Isolation and Immunochemical Characterization*

We have established a human hepatocellular carcinoma cell line designed FOCUS that produces and secretes the beta-subunit of hCG. In the study of beta hCG production by FOCUS cells, we have developed and employed a series of monoclonal immunoradiometric assays (IRMAs) that detect epitopes unique to beta hCG, alpha hCG, hCG, and sequence-specific regions of the carboxyl-terminal peptide of beta hCG. The cells secrete approximately 15 ng beta hCG/10(6) cells and per 24 h; however, we were unable to detect either hCG or the alpha-subunit. The ectopic beta hCG was subsequently affinity purified from the culture medium and partially characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The mol wt of ectopic beta hCG was approximately 35,000 daltons. Immunochemical analysis of ectopic beta hCG by sequence-specific monoclonal antibodies revealed that epitopes corresponding to amino acid sequences 109-115, 121-145, 134-140, and 139-145 of the carboxyl-terminal peptide were present. FOCUS cells also demonstrate an intracytoplasmic localization of beta hCG by immunoperoxidase-staining techniques. Taken together, these findings suggest that FOCUS cells produce and secrete only beta hCG and that thus far, its physical properties appear indistinguishable from those of the native subunit. This unique cell line will be useful to study beta hCG gene(s) regulation as well as the mechanisms of ectopic beta hCG production and secretion.

Cosmid mapping of the human chorionic gonadotropin beta subunit genes by field-inversion gel electrophoresis.

A cosmid clone containing the entire hCG beta gene cluster has been isolated. The restriction map of this clone has been determined by an indirect-end-label FIGE (field inversion gel electrophoresis) method. Analysis of this cosmid clone shows that there are 6 hCG beta genes in human genomic DNA. A previously uncloned portion of the hCG beta cluster, termed the "gap" region, has been shown not to contain any sequences homologous to the hCG beta cDNA. The restriction mapping method employed in this study takes advantage of the superior resolution of FIGE for high molecular weight DNA fragments in the size range 15-50 kb. This method is broadly applicable and permits rapid and accurate restriction mapping for extended regions of genomic DNA that have been cloned into cosmid or lambda vectors.

A map of the hCG beta-LH beta gene cluster.

The beta-subunit of human chorionic gonadotropin (hCG beta) is reportedly encoded by as many as seven non-allelic genes or pseudogenes. Previous studies have identified a cluster of three hCG beta gene copies and the single-copy lutropin-beta subunit (LH beta) gene, but overlap of this cluster with two additional pairs of hCG beta genes has not been demonstrated, despite the isolation of 18 genomic clones. To define the number and organization of non-allelic hCG beta gene copies, genomic Southern blot analyses were performed using hCG beta cDNA and gene-flanking unique-sequence probes. The data show the linear arrangement of six genes (or pseudogenes) and show that the hCG beta-IH beta gene cluster is present in a single 58-kilobase EcoRI fragment. Our map of the cluster indicates which of the cloned hCG beta genes reflect somatic genotypes rather than recombinant artifacts and will thus permit investigation of factors regulating expression during gestation.

Expression of Chorionic Gonadotropinα- andβ-Genes in Normal and Neoplastic Human Tissues: Relationship to Deoxyribonucleic Acid Structure*

We have investigated whether the expression of hCG genes can be attributed to changes in the structure of the alpha- and beta hCG genes, such as rearrangements, duplications, or methylation patterns. Various tissues and cell lines were studied: two term placentae, three trophoblastic tumor cell lines, two tumor cell lines ectopically producing alpha-subunit, normal cells not producing hCG or subunits, and a nonproducing malignancy. Gene structure was explored by restriction enzyme analysis and Southern blotting of DNA, using as probes 32P-labeled plasmids containing alpha- and beta hCG cDNAs. Similarly, methylation was evaluated using the restriction enzymes Msp I, Hpa II, and Hha I, each sensitive to a different pattern of cytosine methylation. No structural changes were observed in alpha- and beta hCG genes, although certain polymorphisms were observed. Analysis of methylation patterns revealed variation of the methylated cytosines; however, no clear correlation was seen between overall methylation or a specific pattern of methylation of these genes and their expression. Although specific methylated nucleotides of regulatory importance may not have been detected by our methods, we can still conclude that neither DNA structural alterations nor patterns of cytosine methylation appear to be major determinants of hCG expression.

Linkage of human chorionic gonadotrophin and placental lactogen biosynthesis to trophoblast differentiation and tumorigenesis

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotrophin (hCG) and placental lactogen (hPL). Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their mRNAs in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha/beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and beta in the syncytial-like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues which contain cytotrophoblastic elements.

The role of trophoblast differentiation in the control of the hCG and hPL genes.

Normal trophoblast of the human placenta elaborates at least two major protein hormones, hCG and hPL. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their mRNAs in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and hCG beta mRNAs can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and hCG beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and hCG beta in the syncytial-like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villus formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues which contain cytotrophoblastic elements.

Only three of the seven human chorionic gonadotropin beta subunit genes can be expressed in the placenta.

Human chorionic gonadotropin (hCG) is a placental hormone essential for the maintenance of pregnancy. While the alpha subunit of this hormone is encoded by a single gene, the beta subunit is encoded by a complex family of seven very similar genes or pseudogenes. Two approaches have been taken to establish which of these genes are functional. First, we have used two restriction enzyme site polymorphisms to correlate 15 independently isolated beta hCG cDNA clones with their corresponding genes. Second, we have used transient expression in COS cells to assay for correctly-initiated transcription from six of the seven beta hCG gene promoters. From these data, we conclude that, at most, only three of the seven beta hCG genes are expressed in the placenta. Comparison of the sequences of a functional and a non-functional beta hCG gene reveals no obvious differences, such as promoter changes, that could account for this differential expression.

Cytological distribution of chorionic gonadotropin subunit and placental lactogen messenger RNA in neoplasms derived from human placenta.

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotropin (hCG), and placental lactogen (hPL). There are several gestational trophoblastic diseases of the placenta called hydatidiform mole, invasive mole, and choriocarcinoma. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their messenger RNA (mRNA) in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and -beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and -beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and -beta in the syncytial-like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues that contain cytotrophoblastic elements.