A novel form of ectopic human chorionic gonadotrophin β-subunit in the serum of a woman with epidermoid cancer

1985 ◽  
Vol 107 (3) ◽  
pp. 403-408 ◽  
Author(s):  
S. B. Nagelberg ◽  
L. A. Cole ◽  
S. W. Rosen
Keyword(s):  
Molecular Weight ◽  
Unknown Origin ◽  
Apparent Molecular Weight ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Size Difference ◽  
Gel Chromatography ◽  
Β Subunit ◽  
Peanut Agglutinin ◽  
Pregnancy Urine

ABSTRACT A novel form of free human chorionic gonadotrophin β-subunit (hCGβ) was found in serum from ElBre, a woman with epidermoid carcinoma of unknown origin. ElBre hCGβ was larger than standard (pregnancy urine) hCGβ when analysed by gel chromatography (apparent molecular weight 54 000 vs 44000). This size difference appeared to be due to a larger carboxyterminal extension (CTE) of ElBre hCGβ since thermolysin cleavage of the CTE from standard hCGβ and Elbre hCGβ yielded core products of the same size. Oligosaccharides, O-linked to serine or threonine, were present in ElBre hCGβ, presumably on its CTE as judged by the complete binding of desialylated ElBre hCGβ to immobilized peanut agglutinin (this lectin is specific for terminal galactose linked β1 → 3 to N-acetylgalactosamine, a disaccharide exposed after desialylation of the O-linked oligosaccharides of standard hCGβ). ElBre hCGβ, however, was incompletely recognized by antisera specific for the CTE of standard hCGβ, especially the carbohydrate-sensitive antiserum R141. The O-linked oligosaccharides of standard hCGβ are heterogeneous in size; 13% are of the largest (hexasaccharide) form. In contrast, over 50% of the O-linked oligosaccharides in hCGβ from the JAr choriocarcinoma cell line are hexasaccharides. Like desialylated ElBre hCGβ, desialylated JAr hCGβ bound completely to peanut agglutinin, but was incompletely recognized by antisera to the hCGβ-CTE. Furthermore, JAr hCGβ was intermediate in size between standard hCGβ and ElBre hCGβ when analysed by gel chromatography (apparent molecular weight 49 000). Thus, we propose that ElBre hCGβ had an even higher proportion of large O-linked oligosaccharides than had JAr hCGβ. Moreover, the N-linked oligosaccharides of ElBre hCGβ differed from standard hCGβ; only 55% of ElBre hCGβ bound to Concanavalin A versus 89% of standard hCGβ. These data further support the concepts of aberrant glycosylation by neoplastic tissues and carbohydrate heterogeneity of hCGβ produced by various tissues. J. Endocr. (1985) 107, 403–408

Molecular heterogeneity of the β-core fragment of human chorionic gonadotrophin

1993 ◽  
Vol 139 (3) ◽  
pp. 519-532 ◽  
Author(s):  
S. F. de Medeiros ◽  
F. Amato ◽  
C. D. Matthews ◽  
R. J. Norman
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Apparent Molecular Weight ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Molecular Heterogeneity ◽  
Hplc Separation ◽  
Con A ◽  
Peripheral Tissues ◽  
Core Fragment

ABSTRACT We have analysed the structure and composition of the β-core fragment of human chorionic gonadotrophin (βC-hCG) from fresh urine specimens obtained from pregnant women and compared our findings with those previously proposed by other groups using different protocols. SDS-PAGE separation of reduced βC-hCG demonstrated two major bands with apparent molecular weights of Mr 8900 and Mr 7500. The molecular weight of the agalacto βC-hCG was estimated to be Mr 10 218 from the amino acid analysis after high-performance liquid chromatography (HPLC) separation. Moreover, HPLC separation of its reduced and S-carboxymethylated peptides resulted in three peaks, but only two of them could be sequenced and demonstrated to be the previously reported β6-40 (Mr 5000) and β55-92 (Mr 5300) peptides of the βhCG subunit. The results showed that 56-78% of βC-hCG molecules of molecular weight Mr 12 800 were able to bind Concanavalin A (Con A). While most were lacking all the peripheral monosaccharides and terminated in mannose, some retained other sugar residues on their antennae. Direct carbohydrate analysis showed the following molar content normalized to six mannose molecules: galactose 2·8, glucosamine 5·3, galactosamine 0·3, fucose 1·7 and sialic acid 3·0. Approximately 22–44% of the βC-hCG molecules did not bind Con A (Con A non-reactive forms), of which 88% were totally deprived of sugar units and had an apparent molecular weight of approximately Mr 10 000, and 12% were weakly reactive to Con A and reactive to anion exchange (negatively charged forms), being incompletely trimmed of their oligosaccharide chains. Comparison of our results with those of two other groups have indicated that the differences noted among preparations are due to either the source or the methods used to purify and characterize this fragment. In addition, our results showed significant microheterogeneity on the N-linked oligosaccharide moieties with some molecules apparently having no sugar molecules. These results have implications for the origins of βC-hCG, suggesting secretion of some molecules without sugar chains and in other cases possible metabolism of hCG in the peripheral tissues. Journal of Endocrinology (1993) 139, 519–532

Distribution of the β-core human chorionic gonadotrophin fragment in human body fluids

1992 ◽  
Vol 135 (1) ◽  
pp. 175-188 ◽  
Author(s):  
S. F. de Medeiros ◽  
F. Amato ◽  
D. Bacich ◽  
L. Wang ◽  
C. D. Matthews ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Intramuscular Injection ◽  
Body Fluids ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Pregnancy Urine ◽  
In Pregnancy

ABSTRACT The origins of a fragment of the human chorionic gonadotrophin (hCG) molecule, β-core (βC-hCG) were studied by analysis of βC-hCG concentrations in biological fluids. In addition, the ability of the placenta to produce the fragment and the metabolism of hCG to βC-hCG by human granulosa cells was determined in tissue culture. Finally the conversion of exogenous hCG to βC-hCG was studied in vivo. The fragment was present in pregnancy urine as well as that from premenopausal and postmenopausal subjects. The highest concentrations were found in pregnant women. Ratios of βC-hCG to intact hCG were higher in pregnancy urine when radioimmunoassay (RIA) was used compared with immunoradiometric assay (IRMA) (0·67 and 0·37 respectively). Concentrations of βC-hCG were higher in postmenopausal urine than in premenopausal specimens. A significant amount of a high molecular weight βC-hCG immunoreactive material was found in serum samples after size separation, and the molar ratio of βC-hCG/hCG was estimated as 0·019. Amniotic fluid also contained small quantities of two forms of immunoreactive βC-hCG and the ratio of 0·01 for authentic βC-hCG/hCG increased to 0·026 when the high molecular weight form was considered. Cultured trophoblastic tissue released material with βC-hCG immunoreactivity in the medium and chromatographic separation revealed that the majority of this material was of higher molecular weight compared with the authentic βC-hCG form. βC-hCG was the principal glycoprotein found in follicular fluid after hyperstimulated folliculogenesis and intramuscular injection of 5000 IU hCG. We also demonstrated that 26% of follicular fluid samples (n = 50) were positive for βC-hCG; levels ranged from 5·2 to 23·0 pmol/l (13·1 ±5·7); s.d.) when a specific IRMA was used. The RIA could detect βC-hCG in 48 samples (96%), levels ranging from 7·0 to 28·5 pmol/l (19·4±5·2). Moreover, granulosa cells cultured in the presence of hCG were able to degrade the intact molecule to both high molecular weight and authentic immunoreactive forms of βC-hCG. After gel filtration, material of molecular weight over a wide range and immunoreactive for βC-hCG was present in human seminal plasma. Assaying 74 samples of this fluid by IRMA, βC-hCG was detected in 42 (56·7%), levels ranging between 5·5 and 59·5 pmol/l (24·9± 15·2). Following intramuscular injection of 1500 IU hCG into male volunteers, the levels of βC-hCG in urine increased by approximately 220% during the first 24 h (P= 0·036 for βC-hCG levels at 2 h and 24 h), decreasing thereafter to undetectable levels in the next 72 h. However, in serum, βC-hCG immunoactivity remained under the limit of detection of the assay at all times. We concluded that (1) the βC-hCG fragment is widely distributed in body fluids and a dissociable high molecular weight material immunoreactive for βC-hCG is found in some biological compartments; (2) granulosa-lutein cells are able to degrade intact hCG to a small βC-hCG immunoreactive fragment; (3) trophoblastic cells synthesize and release different size material with βC-hCG immunoreactivity; (4) intramuscular injection of hCG is followed by increased βC-hCG immunoreactivity in urine; and (5) our results support previous studies indicating the peripheral metabolism of intact hCG to βC-hCG as the principal source for this fragment but raise the possibility that a high molecular weight-associated form, probably bound to a specific protein, may be produced by some tissues. Journal of Endocrinology (1992) 135, 175–188

COMPARISON OF BIOASSAY AND IMMUNOASSAY OF HUMAN CHORIONIC GONADOTROPHIN IN URINE

1965 ◽  
Vol 50 (3) ◽  
pp. 335-344 ◽  
Author(s):  
Rudi Borth ◽  
Michel Ferin ◽  
Annette Menzi
Keyword(s):  
Biological Activity ◽  
Total Variation ◽  
Regression Line ◽  
Haemagglutination Inhibition ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Factorial Experiment ◽  
Serological Result ◽  
Pregnancy Urine ◽  
Acceptable Agreement

ABSTRACT In 39 samples of pregnancy urine, the concentration of human chorionic gonadotrophin (HCG) was estimated biologically by the ovarian hyperaemia reaction in rats, and serologically by the passive haemagglutinationinhibition technique. The results of the bioassays varied from 3 to 150 IU/ml, those of the immunoassays from 5 to 640 IU-eq./ml, and the correlation between the two (calculated for their logarithms) accounted for only 17 per cent of the total variation (r2 = 0.169, P ≈0.01). If the biological activity were estimated from a serological result and the appropriate regression line, the fiducial interval for P = 0.05 would extend from 17 to 610 per cent of the estimate. In a factorial experiment using three anti-HCG sera, three standard and three sensitizing preparations of HCG, the sensitivity of the serological system (expressed as the endpoint concentration in IU of HCG) varied considerably between the 27 combinations of the 3 factors, but there was no interaction between the latter. From these data and those of other authors, it is concluded that immunoassays based on haemagglutination inhibition cannot replace bioassays in the estimation of HCG, as distinct from its hypothetical metabolites or other related antigens, unless specificity has been demonstrated. The well-documented reliability of serological pregnancy tests is, of course, not in dispute. Attention is drawn to the fact that »statistically significant« correlation does not guarantee analytically acceptable agreement between two methods of assay.

CHARACTERIZATION OF THE HUMAN CHORIONIC GONADOTROPHIN FRACTIONS IN PREGNANCY URINE

1978 ◽  
Vol 89 (3) ◽  
pp. 612-624 ◽  
Author(s):  
D. Puett ◽  
A. Kenner ◽  
R. Benveniste ◽  
D. Rabinowitz
Keyword(s):  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Pregnancy Urine ◽  
In Pregnancy

IgE response in schistosomiasis japonica: characterization of a cercarial allergen in comparison with purified egg allergens

1997 ◽  
Vol 71 (3) ◽  
pp. 221-226 ◽  
Author(s):  
M. Owhashi ◽  
Y. Horii ◽  
A. Ishii
Keyword(s):  
Molecular Weight ◽  
Apparent Molecular Weight ◽  
Cross Reactivity ◽  
Schistosomiasis Japonica ◽  
Gel Chromatography ◽  
Con A ◽  
The Cross ◽  
Ige Response ◽  
The Relationship

AbstractThe relationship between the cercarial allergen and two previously isolated egg allergens (J1, J2) of Schistosoma japonicum was examined especially in terms of the cross-reactivity between them. Semi-purified cercarial allergen (JAC) was obtained from the crude extract of S. japonicum cercariae by gel chromatography on Sephadex G-200. The apparent molecular weight of JAC was estimated approximately as 60–100 kDa. JAC could bind to Con A-Sepharose, indicating its glycoprotein nature. Three groups of BALB/c mice were immunized with JAC, J1 or J2 using A1(OH)3 as adjuvant, and the cross-reactivity of each antiserum was examined by PCA. Anti-JAC, anti-Jl or anti-J2 serum was highly specific to the corresponding antigen. When IgE-ELISA of S. japonicum patient sera was performed using JAC, J1 or J2 as an antigen, the correlation between anti-J1 and anti-J2 (r = 0.78) was high, whereas the correlation between anti-JAC and anti-J1 (r = 0.27) or between anti-JAC and anti-J2 (r = 0.12) was low. These results suggest that most IgE epitopes on cercarial allergen are independent from those on egg allergens in S. japonicum.

A single amino acid residue replacement in the β subunit of human chorionic gonadotrophin results in the loss of biological activity

1992 ◽  
Vol 8 (1) ◽  
pp. 87-89 ◽  
Author(s):  
F. Chen ◽  
D. Puett
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Β Subunit ◽  
Single Amino Acid Residue

ABSTRACT The heterodimer, human chorionic gonadotrophin (hCG), contains an a subunit that is common to the glycoprotein hormones and a hormone-specific β subunit. A comparison of all known β amino acid sequences shows that an aspartic acid at position 99 (with the numbering scheme for hCG-β) is one of the seven non-Cys invariant residues. Using site-directed mutagenesis we have replaced hCG-β Asp99 with Arg. Chinese hamster ovary cells, containing a stably integrated gene for bovine a subunit, were transiently transfected with plasmids containing wild-type and mutant hCG-β cDNAs. The Arg99 β mutant associated with the a subunit, but the resulting heterodimer failed to enhance intracellular cyclic AMP production in a gonadotrophin-responsive transformed murine Leydig cell line. Thus, a single amino acid residue replacement in this glycosylated heterodimer containing 237 amino acid residues is sufficient to abolish activity.

scholarly journals Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin β-subunit

2012 ◽  
Vol 18 (8) ◽  
pp. 379-390 ◽  
Author(s):  
Liina Nagirnaja ◽  
Česlovas Venclovas ◽  
Kristiina Rull ◽  
Kim C. Jonas ◽  
Hellevi Peltoketo ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Missense Mutations ◽  
Β Subunit

Total amounts of circulating human chorionic gonadotrophin α and β subunits can be assessed throughout human pregnancy using immunoradiometric assays calibrated with the unaltered and thermally dissociated heterodimer

1994 ◽  
Vol 140 (3) ◽  
pp. 513-520 ◽  
Author(s):  
A-M Nagy ◽  
D Glinoer ◽  
G Picelli ◽  
J Delogne-Desnoeck ◽  
B Fleury ◽  
...  
Keyword(s):  
Early Pregnancy ◽  
Thermal Dissociation ◽  
Heat Exposure ◽  
Free Fraction ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Β Subunit ◽  
Human Pregnancy ◽  
Molar Basis ◽  
Α And Β Subunits

Abstract The aim of the present study was to determine the variations in the balance between total (free plus combined) circulating α and β subunits of human chorionic gonadotrophin (hCG) throughout human pregnancy. The equivalence between the International Units (IU) of hCG (IRP 75/537) and those assigned to the α (IRP 75/569) and β (IRP 75/551) free subunits was experimentally determined by using intact and thermally dissociated hCG. Heat exposure (2 min at 100 °C) of hCG preparations resulted in a complete dissociation of hCG into free, soluble and intact α and β subunits. The hCG and α and β subunit contents of unaltered and heated hCG preparations were assessed by specific immunoradiometric assays. The amount of immunoreactive subunits dissociated by heat from hCG could then be evaluated on a molar basis. Circulating hCG and its free α and β subunits were immunoassayed in 836 blood samples collected from healthy pregnant women at different gestational ages. After conversion of hCG and its subunits into a common IU system, the gestational profiles of the total amounts (free plus combined) of α- and βhCG subunits increased together and peaked at 9–10 weeks of gestation. Thereafter, total α and β subunits decreased and subsequently remained stable until term. The decline in total αhCG subunit was less marked than that of total βhCG subunit. The α- to βhCG ratio was equimolar until 10 weeks of gestation when it increased almost fourfold until term (P<0·0001). Finally, the free fraction of the total circulating αhCG subunit represented 5–7% in early pregnancy but reached 60–70% in the last trimester (P<0·0001). In contrast, the free fraction of the total βhCG subunit decreased slightly from 4–5% of total β subunit in early pregnancy to 2–3% at term (P<0·0001). The present study suggests that thermal dissociation of hCG is a useful method with which to calibrate immunoassays on a molar basis in order to assess circulating levels of the heterodimer and its subunits. Journal of Endocrinology (1994) 140, 513–520

The molecular basis for epitopes on the free β-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCGβ-core fragment

1994 ◽  
Vol 141 (1) ◽  
pp. 153-162 ◽  
Author(s):  
S Dirnhofer ◽  
S Madersbacher ◽  
J-M Bidart ◽  
P B W Ten Kortenaar ◽  
G Spöttl ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Basis ◽  
Tertiary Structure ◽  
Solid Phase ◽  
Critical Role ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Antigenic Determinants ◽  
Β Subunit ◽  
Carboxyl Terminal

Abstract The molecular basis for antigenic determinants on the free β-subunit of human chorionic gonadotrophin (hCGβ), its carboxyl-terminal peptide (hCGβCTP) and the hCGβcore fragment (hCGβcf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCGβcf preparation, a synthetic peptide corresponding to the hCGβCTP (β109–145), overlapping synthetic peptides spanning the entire amino acid sequence of hCGβ, and a reduced and alkylated hCGβ preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a solublephase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCGβ, nine different epitopes (β1–β9), arranged in three spatially distinct domains, could be distinguished. Epitopes β1–β7 were located in a single large domain on both hCGβ and the hCGβcf whereas hCGβCTP contained two topographically distant determinants, designated β8 and β9 respectively. All but the two epitopes located on hCGβCTP (β8 and β9) were destroyed by reducing and alkylating hCGβ, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCGβCTP is linear. At a molecular level, amino acid residues spanning hCGβ 45–52, hCGβ 137–144 and hCGβ 113–116 contributed to the formation of epitopes β5, β8 and β9 respectively. We have also shown that the hCGβcf represents the immunodominant part of the free β-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence β45–52. The hCGβCTP does not play a critical role in the immunologically important tertiary structure of hCGβ and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues β113–116 and β137–144 respectively. Journal of Endocrinology (1994) 141, 153–162

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