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At least 19 recordsLinked to original sources
PubMed1989

Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin beta subunit.

Human chorionic gonadotropin (hCG) consists of two noncovalently joined alpha and beta subunits similar to the other glycoprotein hormones. To study the function of the individual disulfide bonds in subunit assembly and secretion, site-directed mutagenesis was used to convert the 12 cysteine (Cys) residues in the beta subunit of hCG to either alanine or serine. Both cysteines of proposed disulfide pairs were also mutated. These mutant hCG beta genes were transfected alone or together with the wild-type alpha gene into Chinese hamster ovary cells. Only 3-10% assembly could be achieved with derivatives containing single Cys mutations at positions 26, 110, 72, and 90, whereas no assembly was detected with the other 8 mutants. However, double mutations of pairs 26-110 or 23-72 showed increased dimer formation (11 and 36%, respectively). The secretion rate of individual mutants varied significantly. Whereas the Cys-23 and 72 mutants were secreted normally (t1/2 = 140-190 min), the Cys-26 mutant was secreted faster (t1/2 = 70 min), and the other 9 mutants were secreted slower (t1/2 = 280-440 min); mutations of both Cys at 26 and 110 caused much faster secretion (t1/2 = 34 min). Although the secretion rate of these mutants differed, they were quantitatively recovered in the medium except for mutant Cys-88, Cys-23-72, and Cys-34-88 (40, 55, and 10% secreted, respectively). Thus, interruption of any disulfide bond in the hCG beta subunit alters the structure sufficiently to block dimer formation and in some cases slow secretion, although the stability for most of the mutant hCG beta subunits is not greatly affected. The data indicate that interruption of any hCG beta disulfide bond generates different structural forms that are unable to assemble with the alpha subunit, and that the structural requirements for stability and assembly are different.

Animals↗
PubMed1994

Multiple promoter elements in the human chorionic gonadotropin beta subunit genes distinguish their expression from the luteinizing hormone beta gene.

The beta subunit of human chorionic gonadotropin (CG beta) is encoded by a cluster of six genes, which have developed through gene duplication from an ancestral LH beta gene. Despite approximately 90% sequence homology between the CG beta and LH beta promoters, the CG beta gene is expressed in the placenta, whereas the LH beta promoter is active only in the pituitary. The CG beta gene uses a TATA-less promoter that is located upstream of the transcriptional start site used by the homologous LH beta gene. The purpose of this study was to use the high degree of homology among members of the CG beta gene cluster and between the CG beta and LH beta promoters to localize regulatory elements that confer CG beta expression in the placenta. The 5'-flanking regions of the different CG beta genes were cloned and expressed in JEG-3 placental cells. Naturally occurring sequence variations were correlated with promoter activity and used to identify candidate regulatory elements. Exchanges of homologous sequences in the CG beta 5 and LH beta proximal identified three separate regions between -362 and +104 that are necessary for full basal expression of the CG beta promoter. Site-directed mutagenesis of four evolutionarily divergent sequences near the CG beta transcription start site confirmed the importance of multiple distinct regulatory elements as each of these mutations resulted in an 80% decrease in promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Genetic immunization with the free human chorionic gonadotropin beta subunit elicits cytotoxic T lymphocyte responses and protects against tumor formation in mice.

The free beta subunit of human chorionic gonadotropin (hCG beta) is produced and secreted by human lung, bladder, and pancreatic tumors. We attempted to generate cytotoxic T lymphocytes (CTL) with activity against free hCG beta-producing tumors by genetic immunization using a construct containing a beta subunit expressing cDNA. To assess CTL activity in vivo, a cloned syngeneic SP2/O myeloma call line was established that constitutively expresses the free hCG beta protein. Inoculation of this cell line into BALB/c mice produced large tumors within 2 weeks. However, mice immunized with the free hCG beta expression construct demonstrated a marked reduction of tumor size and weight compared with animals immunized with mock DNA ("empty" plasmid). Indeed, 30% of immunized mice were tumor-free after 3 months and thus considered long-term survivors. Inhibition of tumor growth was strongly associated with the level of CTL activity present in CD8+ cells derived from the spleen. In addition, immunized mice developed high titer anti-hCG beta antibodies that neutralized the biologic effects of the intact hCG glycoprotein hormone on its cellular receptor as well. These results illustrate that substantial cellular and humoral immune responses to the free hCG beta subunit may be generated by DNA immunization. This study thus presents a potential approach to inhibiting growth of human tumor cells that produce and secrete the free hCG beta protein.

Animals↗
PubMed1996

Identification and selective destruction of shared epitopes in human chorionic gonadotropin beta subunit.

The feasibility of producing epitope-specific antigens by mutation of the gene is demonstrated, the aim being to eliminate unwanted surface epitopes yet allowing the natural folding of the protein to maintain the desired epitope(s). The model protein is the beta subunit of human chorionic gonadotropin (hCG beta) which previously has been used as an immunological contraceptive vaccine but has extensive cross-reaction with human luteinizing hormone. Of a series of mutants made, the mutant with substitutions of Glu for Arg 68, Ser for Arg 74, His for Gly 75 and His for Val 79, lost the ability to react with a panel of cross-reacting monoclonal antibodies while retaining the discontinuous and linear epitopes specific to the holo-hormone. In addition, allocation of amino acid residues to established epitope clusters could be made: residues 24, 25, 68 and 71 probably contribute to the cluster termed beta 3, residues 20, 21, 22, 75 and 77 to cluster beta 6 and residue 68 to clusters beta 2, beta 4 and beta 5.

Chorionic Gonadotropin, beta Subunit, Human↗
PubMed1991

Quantitation of whole molecule human chorionic gonadotropin and free beta-human chorionic gonadotropin subunit in the Abbott IMx analyser.

An assay for the quantitation of the serum levels of free and whole molecule-associated beta-subunit of human chorionic gonadotropin on the Abbott IMx analyzer is described. The assay detects human beta-chorionic gonadotropin with a sensitivity of approximately 0.5 IU/l while showing no cross-reactivity with follitropin (1000 IU/l) or thyrotropin (2.0 IU/l), and 0.02% cross-reactivity with lutropin (1000 IU/l). Haemoglobin (7.50 milligrams), bilirubin (0.50 milligrams), and triacylglycerols (10.6 milligrams) did not interfere with the assay. Pooled within-run, between-run, and total assay CVs were less than or equal to 5.2%, and less than or equal to 2.7%, and less than or equal to 6.3%, respectively. Values obtained with this assay correlated well (r = 0.98, n = 228) with those values obtained using the Hybritech TandemR-R HCG (Total Beta-HCG) IRMA. The normal range of the assay was found to be less than or equal to 5 IU/l (n = 311). The assay protocol provides results for up to 23 serum samples in approximately 47.5 minutes with the ability to report the human beta-chorionic gonadotropin concentrations of 5 specimens in approximately 15.5 minutes. We conclude that this is an acceptable assay for monitoring human beta-chorionic gonadotropin levels associated with normal pregnancy, reproductive pathology, and reproductive technology such as in vitro fertilization or embryo transfer.

Chorionic Gonadotropin↗
PubMed1984

Posttranslational modification of the carboxy-terminal region of the beta subunit of human chorionic gonadotropin.

The beta subunit of human chorionic gonadotropin (hCG) contains at its carboxy terminus an extension of 29 amino acids not found in the beta subunits of the other glycoprotein hormones. This region provides the sites of attachment of four serine-linked oligosaccharide chains. We have examined the synthesis of this subunit in a cell-free translation system derived from Krebs II ascites tumor cells. The primary translation product was found to undergo a temperature-dependent posttranslational modification which resulted in an increase in apparent molecular weight of 2000 on sodium dodecyl sulfate gel electrophoresis. This modification was specific for the beta subunit of hCG, since no changes were observed for the beta subunit of bovine luteinizing hormone or for the alpha subunits of either hormone. The increase in molecular weight occurred in the absence of microsomal membranes and was not due to the addition of N-linked carbohydrate. An identical shift was observed when pre-hCG beta was incubated with extracts of human placenta. The site of modification was localized by fingerprint analysis to a carboxy-terminal tryptic peptide which contains two of the four O-glycosylated serine residues in the mature form of the subunit. The modified protein was resistant to oligosaccharidase digestion and beta-elimination, indicating that it does not contain O-linked oligosaccharides of the type found on mature hCG beta. These results demonstrate that a specific modification of the carboxy-terminal segment of hCG beta synthesized in vitro occurs in the absence of O-linked glycosylation.

Amino Acid Sequence↗
PubMedDate not supplied

Doppler velocimetry of the umbilical artery as a predictor of outcome in pregnancies characterized by elevated beta-subunit human chorionic gonadotropin.

OBJECTIVE: Women with unexplained elevated maternal serum beta-subunit human chorionic gonadotropin (beta-HCG) are at an increased risk for adverse pregnancy outcome, most likely due to placental abnormalities. Such abnormalities may also result in disturbed blood flow through placental vessels. The purpose of this study was to assess whether Doppler velocimetry of the umbilical artery has a predictive value for pregnancy outcome in patients with unexplained elevated maternal serum beta-HCG. STUDY DESIGN: The study group included 63 patients, in whom the only finding was elevated maternal serum beta-HCG. Systolic/diastolic (S/D) ratios were calculated using a continuous wave Doppler measurement of the umbilical artery, performed beginning at 22 weeks of gestation and followed at 6- to 8-week intervals. Serial results for each individual were incorporated into a single 'velocimetry score', calculated as the rate of abnormal velocimetry measurements. RESULTS: beta-HCG was found to be associated with poor pregnancy outcome: including intrauterine growth restriction (IUGR) (19%), pregnancy-induced hypertension (PIH) (14%), and preterm labor (PTL) (19%). Patients were then divided into 2 groups according to their velocimetry score: group A, VS < or = 80 (n = 47), and group B, VS > 80 (n = 16). A low velocimetry score was associated with a higher rate of IUGR, PIH, and a significantly higher rate of PTL. CONCLUSIONS: Umbilical artery Doppler velocimetry may serve as a predictor of pregnancy outcome in the high-risk group characterized by unexplained elevated beta-HCG.

Biomarkers↗
PubMed1978

Location of major antigenic sites of the beta subunit of human chorionic gonadotropin.

Purified beta subunit of human chorionic gonadotropin (hCG) was partially reduced with beta-mercaptoethanol, carboxymethylated, and digested with chymotrypsin. The peptides were isolated by high-voltage electrophoresis and paper chromatography. Five major disulfide-containing peptides were isolated, and their location in the parent molecule was established by amino acid composition and amino- and carboxy-terminal analyses. All of these peptides inhibited the binding of 125I-labeled hCG by anti-beta hCG serum. The inhibitory effect of these peptides was lost when their disulfide bonds were reduced and alkylated. Synthetic carboxy-terminal peptides were not inhibitory. Based on these data it is concluded that a major antigenic site of hCG resides in the region of residues 21-23 with a disulfide bond connecting cysteine-23 or -26 with the cysteines at positions 72 or 110.

Amino Acid Sequence↗
PubMedDate not supplied

Evolution of the genes for the beta subunits of human chorionic gonadotropin and luteinizing hormone.

Nucleotide sequence comparisons of the single gene for the human luteinizing hormone gene beta subunit with two of the seven genes for the human chorionic gonadotropin beta subunit suggest that the beta human chorionic gonadotropin genes have evolved from an ancestral beta luteinizing hormone gene by a series of selected changes with very little neutral drift. Moreover, the 24 amino acid carboxy-terminal extension of the human chorionic gonadotropin beta subunit appears to have arisen by a single base deletion that incorporated the 3'-untranslated region of the ancestral beta luteinizing hormone gene into the coding region.

Base Sequence↗
PubMedDate not supplied

Bioneutralization capacity of the antibodies generated in women by the beta subunit of human chorionic gonadotropin (beta hCG) and beta hCG associated with the alpha subunit of ovine luteinizing hormone linked to carriers.

Data is presented on the bioneutralization capacity per unit immunoactivity of 30 serum samples of women immunized with the beta-subunit of human chorionic gonadotropin (beta-hCG) or beta hCG associated noncovalently with the alpha-subunit of ovine luteinizing hormone (alpha-oLH), to form a heterospecies dimer (HSD), which were linked to carriers. The bioassays utilized were inhibition of radioiodinated hCG binding to rat testicular receptors in vitro and the inhibition of hCG induced testosterone production in mice. In both assays, antisera of women immunized with the HSD had a bioactivity to immunoactivity ratio that was about 25% higher, on an average, than antisera of women immunized with beta-hCG, suggesting a better bioneutralization capacity of sera raised by the HSD in women.

Adult↗
PubMed1981

Development and characterization of a monoclonal antibody which distinguishes the beta-subunit of human chorionic gonadotropin (beta hCG) in the presence of the hCG.

A monoclonal antibody specific for the beta-subunit of human chorionic gonadotropin (beta hCG) has been developed which has a cross reactivity of 0.23% with intact hCT. There was no cross reactivity with other glycoprotein pituitary hormones in the range in which they might be expected to be present in serum. The beta-subunits of hFSH, hTSH and hLH did not inhibit binding of [125I]iodo-beta hCG to the antibody at up to 250 ng/tube. The dissociation constant (Kd) with [125I]iodo-beta hCG was 3.3 x 10(-10), and the reaction had reached equilibrium within 1 h.

Animals↗
PubMed2004

[Expression and purification of three fusion proteins containing a single B-cell epitope (beta5, beta9 or beta8) of human chorionic gonadotropin beta subunit].

The possibility of using a subunit or fragment of human chorionic gonadotropin (hCG) as an immunogen for birth control has been actively explored for many years. This protein homone is produced by the fertilized egg and is required for implantation of the blastocyst into the maternal uterus and the maitenance of pregnancy. In previous studies, several bio-synthesized hCG chimeric peptides (CP) that contain three linear B-cell epitopes (beta5, beta9 and beta8) of beta-hCG subunit together with various foreign 'promiscuous' T-cell epitopes were constructed and expressed as potential new hCG vaccine immunogens. In order to detect antibodies to each of the individual B-cell epitopes present in the animal antiserum raised against the hCG CPs, we decided to construct three recombinant proteins, each contains a single target B-cell epitope (betaE) of beta-hCG. Two sets of DNA fragments were chemically synthesized encoding the beta5, beta9 and beta8 epitopes (betaE) 45 approximately 52, 113 approximately 116 or 133 approximately 144 of beta-hCG subunit and were inserted into the downstream of streptavidin (Stv) gene in pTSA18 separately, with or without an extra TAA codon at the 3'-terminals of the genes. SDS-PAGE analysis revealed that only Stv-betaE (-beta5, -beta9 or -beta8) fusion genes set with the TAA codon can be expressed in E. coli BL21 (DE3) pLysS strain at high level after 1mM IPTG induction for 4 hours. Additionally, these fusion proteins can all be recognized by specific polyclonal antiserum (RS-4157) generated upon immunization with the loop peptide 38 approximately 57 of beta-hCG, monoclonal antibody (mAb) FB12 to beta9 epitope and mAb OT3A that specially recognizes reporter sequence 133 approximately 139 of beta8 epitope 137 approximately 144. Each of the proteins can be purified to 95% relative homogeneity using an improved method of preparative gel polyacrylamide gel electrophoresis. The yields were 5 mg per 1 L culture. The three target Stv-betaE fusion proteins will be useful in determining the immunogenicity of designed hCG CPs and hCG vaccines, including hCG DNA vaccines.

Chorionic Gonadotropin, beta Subunit, Human↗
PubMed2002

Detection of beta-subunit human chorionic gonadotropin mRNA in the peripheral blood of patients with nonmetastatic gestational trophoblastic disease.

OBJECTIVE: The aim of this study was to detect beta-subunit human chorionic gonadotropin (beta(h)CG) mRNA in the peripheral blood samples of patients with nonmetastatic gestational trophoblastic disease (GTD) undergoing hysterectomy. METHODS: Heparinized peripheral blood samples were obtained from four patients with nonmetastatic GTD before, during, and after hysterectomy. The beta(h)CG mRNA expression was examined by reverse transcriptase-polymerase chain reaction using beta(h)CG primers. The expression of beta(h)CG mRNA was quantified using a densitometer. RESULTS: Beta(h)CG expression was detected in all patients before and during hysterectomy. The expression of beta(h)CG mRNA during operation was so high that it could not be quantified using densitometer. The expression decreased rapidly after operation. CONCLUSIONS: Disseminated trophoblastic cells are present in the peripheral blood even in cases without metastasis. Trophoblastic cells circulating in the peripheral blood can be reduced by surgical intervention.

Adult↗
PubMed1990

Expression of alpha- and beta-human chorionic gonadotropin subunits in cultured human cells.

We surveyed several human cell lines for production of alpha- and beta-human chorionic gonadotropin (hCG) under a variety of conditions known to induce gene expression. alpha- and beta-hCG subunits were monitored in culture media by specific radioimmunoassays and were shown to be quite sensitive to serum refeeding and growth state of all cell types studied. The permanent line JEG-3 secreted both alpha- and beta-subunits whereas HeLa cells secreted only the alpha-subunit. Production of both subunits was augmented in these permanent cell lines, for each growth state, by pretreating cells with 5-azacytidine; in contrast, spontaneous beta-hCG production by normal human fibroblasts (four of six strains) was only rarely increased after 5-azacytidine treatment, and more often was suppressed by 30 to 40%. Three of five strains from inherited chromosomal breakage syndromes produced immunoassayable beta-hCG spontaneously, two of which increased secretion upon treatment with either UV or mitomycin C. Surprisingly, one normal cell strain of fetal origin was induced to secrete alpha-hCG, but not beta-hCG, after UV irradiation. JEG-3 and HeLa cells produced detectable cognate mRNA for alpha- or beta-hCG subunits or both by Northern and S1 nuclease protection analyses, whereas such transcripts from untransformed human fibroblasts were consistently below detectable levels. Quantitation of beta-hCG mRNA by RNA:RNA annealing kinetics indicates that even the fibroblast strain producing the highest secreted beta-hCG levels contained cognate mRNAs at only approximately 0.1 per cell. We conclude that hCG expression in human fibroblasts is strongly repressed at the transcriptional level, although a variety of conditions (growth state, serum refeeding, cell senescence, or DNA damage) can affect the level of "leaky" expression, at least in some responding fraction of cells.

Cell Division↗
PubMed1995

Expression of human chorionic gonadotropin beta subunit genes in superficial and invasive bladder carcinomas.

Increased serum levels of human chorionic gonadotropin beta subunit (hCG beta) were described previously in patients with bladder cancer. To obtain insight into such production of hCG beta, the expression of hCG beta 7, 8, 5, and 3 genes in bladder carcinomas and normal urothelia was investigated by reverse transcription PCR. Surprisingly, hCG beta mRNAs were detected in both normal urothelial and carcinomatous cells. However, tumor progression was characterized by different patterns of transcription of the hCG beta genes; the beta 7 gene was the only gene transcribed in normal urothelia and Ta tumors included in this study, whereas in addition to beta 7, genes beta 5, 8, and 3 were transcribed in T1 to T4 tumors. Moreover, transcription levels of the latter three genes increased with the stage of the disease. These observations showed that dramatic modifications in the expression of hCG beta genes accompany progression of bladder carcinomas.

Base Sequence↗
PubMed1993

Replacement of the invariant tyrosine in the CAGY region of the human chorionic gonadotropin beta subunit.

The mammalian glycoprotein hormone beta subunits contain a highly conserved amino acid sequence, Cys-Ala-Gly-Tyr-Cys (residues 34-38 of human chorionic gonadotropin beta), that is denoted as the 'CAGY region'. Using site-directed mutagenesis we have replaced Tyr-37 in hCG beta, i.e., the invariant Tyr in all known mammalian CG, LH, FSH, and TSH beta subunits, with two hydrophobic amino acid residues, Phe and Leu. The resultant mutant forms were characterized for alpha subunit binding and the resulting heterodimers were analyzed for biological activity using two in vitro assays with transformed murine Leydig cells (MA-10). Chinese hamster ovary cells containing a stably integrated gene for bovine alpha were transiently transfected with a eukaryotic expression vector containing a Rous sarcoma viral promoter and the wild-type and mutant cDNAs. The hCG beta(Phe-37) mutant bound to alpha essentially to the same extent as hCG beta wild-type, while the hCG beta(Leu-37) mutant formed somewhat less heterodimer. The heterologous heterodimeric mutant and wild-type gonadotropins were equipotent in a competitive binding assay with [125I]hCG. In a steroidogenic assay, the mutant hormones were active, but they appeared slightly less potent than the wild-type form. Thus, this invariant Tyr can be replaced with another aromatic amino acid residue or with a hydrophobic, but not aromatic amino acid residue in hCG beta without any dramatic effect on function. These results indicate that Tyr-37 in hCG beta, while not obligatory, may participate, either directly or indirectly, in subunit assembly and that the hydroxyl group may function in a modulatory role in signaling.

Amino Acid Sequence↗
PubMed1984

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.

Animals↗