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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↗
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↗
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↗
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↗
PubMed1992

Intracellular folding pathway of human chorionic gonadotropin beta subunit.

Few experimental models have been used to investigate how proteins fold inside a cell. Using the formation of disulfide bonds as an index of conformational changes during protein folding, we have developed a unique system to determine the intracellular folding pathway of the beta subunit of human chorionic gonadotropin (hCG). Three folding intermediates of the beta subunit were purified from [35S]cysteine-labeled JAR choriocarcinoma cells by immunoprecipitation and by reverse-phase high performance liquid chromatography (HPLC). To identify unformed disulfide bonds, nonreduced folding intermediates were treated with trypsin to liberate non-disulfide-bound, [35S]cysteine-containing peptides from the disulfide-linked peptides. Released peptides were purified by HPLC and identified by amino acid sequencing. The amount of a peptide that was released indicated the extent of disulfide bond formation involving the cysteine in that peptide. Of the six disulfide bonds in hCG-beta, bonds 34-88 and 38-57 form first. The rate-limiting event of folding involves the formation of the S-S bonds between cysteines 23 and 72 and cysteines 9 and 90. Disulfide bond 93-100, the formation of which appears to be necessary for assembly with the alpha subunit of the hCG heterodimer, forms next. Finally, disulfide bond 26-110 forms after assembly with the alpha subunit, suggesting that completion of folding of the COOH terminus in the beta subunit occurs after assembly with the alpha subunit.

Amino Acid Sequence↗
PubMed1992

Free human chorionic gonadotropin beta subunit in gonadal and nongonadal neoplasms.

The diagnostic value of elevated human chorionic gonadotropin (hCG) and its free alpha (hCG alpha) and beta (hCG beta) subunit serum levels as specific tumor markers for nongonadal malignancies is controversial. In the present report, different monoclonal based immunoradiometric assays specific for hCG and its free hCG alpha and hCG beta subunits have been used to reevaluate the presence of these molecules in the serum of patients with a wide variety of tumors. Serum samples from patients with newly diagnosed, persistent, or recurrent malignancies of either known (n = 717) or unknown (n = 32) primary site, healthy blood donors (n = 309), and nonmalignant disease controls (n = 86) were studied using four highly specific and sensitive monoclonal based immunoradiometric assays to hCG and its free subunits. Low level hCG elevations (less than 1000 pg/ml) were found to be common in cancer patients, normal subjects, and disease controls. However, serum levels greater than 1000 pg/ml were highly diagnostic of gonadal tumors and specifically identified nonseminomatous testicular tumors. Significant serum elevations of free hCG alpha subunit (as high as 3000 pg/ml) were found in approximately 96% of cancer patients, normal individuals, and disease controls. In contrast, free hCG beta subunit levels (greater than or equal to 100 pg/ml) were detected in 70 and 50% of patients with nonseminomatous and seminomatous testicular cancers, respectively, and in 47% of bladder, 32% of pancreatic, and 30% of cervical carcinomas. All normal subjects and disease controls had free hCG beta levels less than 100 pg/ml. Thus, the detection of the free hCG beta subunit in serum of nonpregnant subjects was highly diagnostic of malignancy in general and specifically defines a subgroup of aggressive nongonadal malignancies.

Adult↗
PubMed1990

Role of disulfide bond formation in the folding of human chorionic gonadotropin beta subunit into an alpha beta dimer assembly-competent form.

The malignant trophoblastic cell line JAR was used as a model system to study protein folding in intact cells. We have used this model previously to identify conformational intermediates in the production of an assembly-competent form of the human chorionic gonadotropin beta subunit (Ruddon, R. W., Krzesicki, R. F., Norton, S. E., Beebe, J. S., Peters, B. P., and Perini, F. (1987) J. Biol. Chem. 262, 12533-12540). The earliest biosynthetic precursor of the human chorionic gonadotropin beta subunit detectable in JAR cells pulse labeled for 2 min is p beta 1, a form that lacks half of the six intrachain disulfide bonds observed in the fully processed dimer form of beta and that does not combine with the alpha subunit. p beta 1 is rapidly (t1/2 approximately 4 min) converted into p beta 2, which has a full complement of intrachain disulfide bonds and does combine with the alpha subunit. In this study, we have identified the three late forming disulfide bonds involved in the transition of p beta 1 into the assembly-compete form, p beta 2. The last three disulfide bonds to form are those between cysteines 9 and 90, 23 and 72, and 93 and 100. These were identified in JAR cell lysates that had been pulse labeled with [35S]cysteine for 2 or 5 min followed by trapping of the cysteine thiols with iodoacetic acid before immunopurification of the beta subunit forms. Immunopurified p beta 1 was treated with trypsin under nonreducing conditions to liberate [35S]cysteine-containing peptides from the disulfide-linked beta core polypeptide. These tryptic peptides were then separated by high performance liquid chromatography and sequenced to determine the location of the carboxymethyl-[35S]cysteine residues. The three late forming disulfide bonds are most likely the ones involved in stabilizing the conformation of the beta subunit that is required for combination with alpha to form the biologically functional alpha beta heterodimer.

Amino Acid Sequence↗
PubMed1995

The role of human chorionic gonadotropin beta subunit elevation in small-cell lung cancer patients.

Human chorionic gonadotropin (HCG)-like immunoreactivity has been found in many non-trophoblastic tumours, but the biological behaviour of HCG-producing cells has not been clarified yet. The aim of the study was to estimate the frequency of serum HCG beta subunit (s beta HCG) elevation in patients with small-cell lung cancer (SCLC) and to assess its possible prognostic role in this type of tumour. An attempt was also made to reclassify the histology in selected cases to see whether the elevated (s beta HCG) level is connected with any special subtype of small-cell lung cancer. A total of 156 SCLC patients entered the study: 93 men, 63 women, median age 58 years. s beta HCG activity was measured by immunoenzyme assay (Abbott EIA beta HCG 15-15) before treatment. s beta HCG elevation (above 5 mIU/ml) was found in 21 of 156 patients (14%). Response to treatment after chemotherapy (complete and partial response) was obtained in only 48% of those patients in whom elevated s beta HCG was found, in comparison to the 73% response rate observed in the remaining patients. Only 5% of patients with elevated s beta HCG survived 2 years, in comparison to 21% surviving for 2 years among the remaining patients. The prognostic significance of elevated s beta HCG and extent of disease were independent of each other (Cox's proportional-hazard model). Thus s beta HCG elevation in SCLC seems to be a marker of more resistant tumours and of poor prognosis. We have not found any connection between the subtype of small-cell lung cancer and elevated s beta HCG. Elevated s beta HCG was found in 2 out of 11 patients with oat-cell carcinoma, in 3 out of 10 patients with an intermediate cell type and in 5 out of 13 patients with small-cell lung cancer in which the assessment of the subtype was not possible.

Adult↗
PubMed1977

Plasma beta-subunit human chorionic gonadotropin assay in molar pregnancy and choriocarcinoma.

Serum plasma levels of the beta-subunit of human chorionic gonadotropin (HCG-beta) were measured by radioimmunoassay in 24 women with molar pregnancies and five with choriocarcinoma. In the patients with molar pregnancy, plasma HCG-beta concentrations became undetectable within 8 to 173 days. In the patients with choriocarcinoma, plasma HCG-beta levels declined rapidly with appropriate chemotherapy. Serial plasma HCG-beta assay in this series accurately determined remission and detected early recurrences during follow-up. All patients are in remission with a mean follow-up of 22 months for the patients with choriocarcinoma.

Choriocarcinoma↗