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

A novel form of free human chorionic gonadotrophin beta-subunit (hCG beta) was found in serum from ElBre, a woman with epidermoid carcinoma of unknown origin. ElBre hCG beta was larger than standard (pregnancy urine) hCG beta when analysed by gel chromatography (apparent molecular weight 54 000 vs 44 000). This size difference appeared to be due to a larger carboxyterminal extension (CTE) of ElBre hCG beta since thermolysin cleavage of the CTE from standard hCG beta and Elbre hCG beta yielded core products of the same size. Oligosaccharides, O-linked to serine or threonine, were present in ElBre hCG beta, presumably on its CTE as judged by the complete binding of desialylated ElBre hCG beta to immobilized peanut agglutinin (this lectin is specific for terminal galactose linked beta 1----3 to N-acetylgalactosamine, a disaccharide exposed after desialylation of the O-linked oligosaccharides of standard hCG beta). ElBre hCG beta, however, was incompletely recognized by antisera specific for the CTE of standard hCG beta, especially the carbohydrate-sensitive antiserum R141. The O-linked oligosaccharides of standard hCG beta are heterogeneous in size; 13% are of the largest (hexasaccharide) form. In contrast, over 50% of the O-linked oligosaccharides in hCG beta from the JAr choriocarcinoma cell line are hexasaccharides. Like desialylated ElBre hCG beta, desialylated JAr hCG beta bound completely to peanut agglutinin, but was incompletely recognized by antisera to the hCG beta-CTE. Furthermore, JAr hCG beta was intermediate in size between standard hCG beta and ElBre hCG beta when analysed by gel chromatography (apparent molecular weight 49 000).(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of fragmentation of free and combined forms of the beta-subunit of human chorionic gonadotropin.

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.     

The biological role of the carboxyl-terminal extension of human chorionic gonadotropin [corrected] beta-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 O-linked oligosaccharide structures are striking different on pregnancy and choriocarcinoma HCG

hCG is a glycoprotein hormone which is detected in the serum and urine of pregnant women and of patients with hydatidiform mole and choriocarcinoma. The molecule contains 4 O-linked sugar chains. In an effort to identify cancer markers, the structures of these sugar units on the hCG produced in pregnancy and choriocarcinoma were compared. hCG molecules in patient urines were purified by immuno-affinity chromatography and gel filtration. beta-elimination was used to cleave the O-linked sugar units, radioactive sodium borohydride to label them, and gel filtration on Bio-Gel P4 to size them and compare their elution volumes with those of standard oligosaccharides of known structure. A trisaccharide, NeuAc alpha 2-3Gal beta 1-3GalNAc-, was found to be the principal unit attached to urinary hCG from pregnant women (10 samples). A hexasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-6)GalNAc-, which accounted for just 6% (mean, range 0-14%) of the O-linked sugar units on pregnancy hCG, was the principal unit (mean 52% of total, range 50-56%) attached to the hCG from choriocarcinoma patient urines (3 samples). These results indicate that hexasaccharide-abundant hCG is an indicator of choriocarcinoma.     

Differential occurrence of free beta and free alpha subunits of human chorionic gonadotropin (hCG) in pregnancy sera

Levels of hCG alpha beta dimer, free alpha-subunit and free beta-subunit were measured in pregnancy sera. Dimer and free alpha were quantitated by radioimmunoassays (RIAs) using specific polyclonal antisera. Free beta was quantitated both by monoclonal anti-beta RIA and by polyclonal anti-beta RIA following the complete adsorption of cross-reacting hCG by immobilized alpha-antisera. Consistent with the findings of other laboratories, hCG levels in pregnancy sera peaked at around 10 weeks after the last menstrual period (post LMP), and declined thereafter. Free alpha levels rose as hCG levels declined and accounted for 30-40% of total serum alpha in the third trimester. Although free beta accounted for only a small proportion of the total beta-subunit at the time of the hCG peak and thereafter (2.4-3.6%), in early pregnancy serum samples, 4-6 weeks post LMP, when hCG was generally first detected, an average of 16% free beta was detected. At this time, the higher the hCG level (20-2000 ng/ml), the lower the percent free beta (54-3%). Thus, the free beta portion started high and declined prior to the hCG peak; the free alpha portion increased thereafter. To explain these findings, we propose a two phase regulation of hCG dimer formation. Up to the time of the hCG peak, supplies of alpha-subunit are limiting (hence the presence of free beta). Thereafter, beta-subunit levels drop, restricting dimer formation and leaving uncombined alpha.     

Conformational intermediates in the production of the combinable form of the beta-subunit of chorionic gonadotropin

Human trophoblastic cells synthesize and secrete hCG as well as uncombined forms of the alpha- and beta-subunits of hCG. We have previously reported that the rate-limiting step in alpha beta-dimer assembly in cultured JAR choriocarcinoma cells is a conformational change in beta-subunit accompanied by the formation of intramolecular disulfide bonds. We now report on the intermediate steps in the acquisition of this combinable conformation by the beta-subunit. The earliest biosynthetically labeled form of beta detected in JAR cells is a precursor termed p beta 1 that lacks at least one of the intramolecular disulfide bonds found in mature beta-subunit, that does not combine with alpha-subunit, and that does not react with a monoclonal antibody specific for free beta. The p beta 1 precursor rapidly assumes (within 5 min) a new conformation termed p beta 2 that, in contrast to p beta 1, migrates more slowly on nonreduced sodium dodecyl sulfate-polyacrylamide gels, combines with alpha to form the hCG dimer, and reacts with the monoclonal anti-free beta antibody. Pulse-chase kinetic experiments support the following sequence of events: p beta 1----uncombined p beta 2----combined p beta 2. The transition of p beta 1 to uncombined p beta 2 involves the formation of at least one intramolecular disulfide bond coincident with the conformational shift of the p beta molecule. Furthermore, treatment of the nonreduced subunits with trypsin releases a [35S]cysteine-labeled peptide from p beta 1, but not from either form of p beta 2. This peptide presumably contains one of the two crucial cysteine residues that participate in forming the disulfide bond that distinguishes p beta 1 from the p beta 2 forms. Dimer p beta 2 differs from both p beta 1 and uncombined p beta 2 in that it contains an O-linked N-acetylgalactosamine, which represents the first step in the formation of the O-linked glycans of beta-subunit. Dimer p beta 2 is, therefore, the most fully processed and kinetically the latest of the three p beta forms that appear in JAR cell lysates. We conclude that formation of an appropriate array of intramolecular S-S bonds accompanies the acquisition of a combinable conformation of beta-subunit, and we have identified intermediate steps in the pathway leading to this conformational change. The data suggest that it is the achievement of this conformation by beta-subunit that limits the alpha beta combination reaction rather than the amount or conformation of alpha-subunit.     

Distribution of O-linked sugar units on hCG and its free alpha-subunit

hCG, a glycoprotein hormone produced by the trophoblast in pregnancy, is composed of two dissimilar subunits, alpha and beta, joined non-covalently. hCG has four O-linked sugar units, all attached to the beta-subunit. The trophoblast also produces a free form of the alpha-subunit, which unlike the alpha-component of hCG, can contain an O-linked sugar unit. The structures of the O-linked sugar units were examined. Four structures were identified on urinary hCG. A hexasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-6)GalNAc- accounting for 13%, a tetrasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc-, for 34%, a trisaccharide, NeuAc alpha 2-3Gal beta 1-3GalNAc-, for 43% and a disaccharide, NeuAc alpha 2-6GalNAc- for 10% of the total O-linked sugar structures. Similar mixtures were found on peptides containing one, three or four sugar units suggesting a random distribution among attachment sites. The distribution of O-linked sugar structures on hCG and free alpha-subunit from trophoblast explant cultures was compared. The mixture of structures attached at the single site on the free alpha-subunit paralleled that at the four sites on the hCG.     

Development of two assay systems of desialylated hCG specific for O-serine or N-asparagine linked carbohydrate chain and their clinical application

Two specific and sensitive assays have been developed for the measurement of as-hCG in this study. These assay systems are immunoradiometric assays which utilize peanut lectin, Arachis hypogaea (PNA) or caster bean lectin, Ricin communis agglutinin (RCA-I), to selectively extract as-hCG and then directly quantify with purified and radiolabeled antiserum against hCG beta COOH terminal peptide (beta-CTP). PNA is highly specific for the terminal carbohydrate linkage Gal beta-(1----3)-GalNAc which is only exposed in hCG when the O-serine linked oligosaccharide of its unique beta-CTP region are desialylated. RCA-I is also specific for the terminal carbohydrate beta-D-Galp., especially the linkage of Gal beta-(1----4)-GlcNAc, which is exposed in hCG when the N-asparagine linked oligosaccharide of its alpha and beta subunits are desialylated. The assays (PNA-R525 and RCA-I-R525) are capable of reliably and accurately measuring as little as 0.01 and 0.002 pmoles as-hCG/ml without interference from hCG, respectively (crossreactivity: 0.1% and 0.03%). The urine concentrates of a patient with choriocarcinoma and a normal pregnant woman were fractionated by Sephadex G-100 gel filtration and total hCG and as-hCG levels were measured. When the fractions were analysed by beta-CTP RIA, there were two peaks of immunoreactive hCG in the urine of a patient with choriocarcinoma. The bigger molecular fractions were hCG of native size, and 16% of which was desialylated. The smaller molecular fraction was beta-CTP, and practically all of that was desialylated. Whereas, beta-CTP could not be observed, and only 1.5% of hCG of native size was desialylated in the urine of a normal pregnant woman. Although the absolute levels of as-hCG in the urine specimens increased as total hCG increased, the proportion of as-hCG decreased as total hCG concentration increased. The proportion of as-hCG in urine specimens from choriocarcinoma patients was higher than specimens from patients with hydatidiform mole (including invasive mole) in both these lectin immunoradiometric assay (LIRMA) systems. Also, the proportion of as-hCG in urine specimens from molar patients was higher than those from normal pregnant women using PNA as extracting reagent. Whereas, when RCA-I was used, there was no significant difference between specimens from molar patients and those from normal pregnant women.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of an incompletely glycosylated form of human chorionic gonadotropin from human placenta

A small form of hCG (SP-hCG) was purified from an acetone powder preparation of human first trimester placenta by repeated gel filtration and ion exchange chromatography on a Q-Sepharose or FPLC Mono Q column. The estimated mol wt (Mr) of the small hCG by gel filtration is 43K compared to 58K for authentic hCG. The pI of SP-hCG is 10.0, suggesting deficiency of sialic acids. SP-hCG dissociates into subunits when treated with 6 M guanidine-HCl or analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two beta-subunits of SP-hCG were found with estimated Mr of 23K and 20K. Both are distinctly smaller than authentic hCG beta. A single alpha-subunit was found, with an estimated Mr of 21 K. The immunoactivity (8,900-10,000 IU/mg) of highly purified SP-hCG was comparable to that of reference hCG (CR119) determined by a RIA method using anti-hCG antibodies. The hCG/LH receptor-binding activity of SP-hCG is equivalent to that of reference hCG (CR119). Its biological activity is lower than that of reference hCG (approximately 30% or more) assayed by the in vitro stimulation of rat Leydig cells to produce testosterone and cAMP. A high dose is required to attain the same level of stimulation as reference hCG. The amino acid composition of SP-hCG is similar to that of reference hCG, whereas its hexsamine content is significantly lower. Its glucosamine content is about half that in reference hCG, while it completely lacks galactosamine. These findings suggest that SP-hCG is deficient in O-linked oligosaccharide chain in the beta-subunit, and that the N-linked oligosaccharide chains of both subunits are shortened. SP-hCG is one of the principal forms of the hormone present in first trimester placenta and may be a key intermediate in the posttranslational biosynthesis of hCG. Although it lacks O-linked sugar chains and shortened N-linked sugar chains, it possesses substantial biological activity. To have full biological activity, the hCG molecule must contain the complete complement of sugar chains.     

Human chorionic gonadotropin (hCG) beta-core fragment is produced by degradation of hCG or free hCG beta in gestational trophoblastic tumors: a possible marker for early detection of persistent postmolar gestational trophoblastic disease.

The present study was undertaken to investigate whether human chorionic gonadotropin (hCG) beta-core fragment (hCG beta cf) was directly produced by gestational trophoblastic tumors. Immunoreactivity of hCG beta cf was demonstrated in the extracts as well as in the culture media of hydatidiform mole tissues. It was also present in the extracts of choriocarcinoma tissues, and its molar concentration exceeded that of intact hCG. The presence of hCG beta cf was then confirmed by gel chromatography and Western blot analysis. Immunohistochemistry showed localization of hCG beta cf immunoreactivity to the syncytiotrophoblasts and scattered cells in the stroma of mole tissue, and to syncytiotrophoblastic cells in choriocarcinoma. Immunoreactivity of hCG beta cf was also detected in the sera of the patients with gestational trophoblastic disease, although the hCG beta cf/hCG ratio was less than one hundredth of that in the tissue extracts. Serial measurement of serum hCG beta cf levels after mole evacuation showed that they declined much more rapidly than those of hCG and became undetectable in the patients with subsequent spontaneous resolution, while hCG beta cf remained or became detectable before the rise of hCG was observed in the patients with subsequent persistent trophoblastic disease. Taken together, these results suggest that hCG beta cf is directly produced by gestational trophoblastic tumors, and monitoring of hCG beta cf in the serum after mole evacuation may be useful for early prediction of subsequent development of postmolar persistent trophoblastic disease.     

O-glycosylation of the alpha-subunit does not limit the assembly of chorionic gonadotropin alpha beta dimer in human malignant and nonmalignant trophoblast cells

Biosynthetic experiments were carried out in cultures of human malignant trophoblast cells (the JAR cell line) and in explants of normal first trimester human placental tissue to test the hypothesis that the O-glycosylation of the glycoprotein hormone alpha-subunit at Thr-39 regulates the assembly of the CG alpha beta dimer. This modification of alpha has been shown to ablate its ability to combine in vitro with the beta-subunit of bovine LH and might explain why JAR cells and placental explants secrete uncombined alpha- and beta-subunits in addition to the hCG alpha beta dimer. We have previously detected an O-linked carbohydrate chain at Thr-39 in preparations of secreted free alpha-subunit, but not dimer CG alpha from JAR culture medium. We report here evidence that the O-glycosylation of alpha does not regulate the biosynthetic assembly of the hCG dimer in cultures of JAR choriocarcinoma cells or first trimester placental explants. The intracellular precursor forms of alpha and beta that accumulate in the endoplasmic reticulum and combine in that compartment are not yet modified with O-linked carbohydrate, as determined by measurements of their [3H]galactosamine content after biosynthetic labeling of amino sugars with [3H]glucosamine. Furthermore, only half of the free alpha-subunit secreted by JAR cells and less than 10% of free alpha secreted by 10-week-old placental explants received the O-linked chain. This was shown by determining the ratio of the unglycosylated and glycosylated forms of the tryptic peptide from free alpha that contains the O-glycosylation site (residues 36-42). Based on these findings, we make the following conclusions. 1) O-Glycosylation of alpha-subunit is a late event in the secretory pathway of trophoblasts compared to the rapid combination in the rough endoplasmic reticulum of hCG subunit precursors to form alpha beta dimer. 2) Association of alpha with beta precludes the subsequent addition of the glycan to alpha at Thr-39. 3) The alpha molecules that fail to combine with beta in the endoplasmic reticulum are substrates for the later addition of O-linked carbohydrate, presumably in the Golgi complex, but only a fraction of the free alpha molecules are modified with O-linked carbohydrate.     

Subunit interaction of human chorionic gonadotropin (hCG) with rat ovarian luteinizing hormone (LH)/CG receptor.

The subunit interaction of hCG with its rat ovarian LH/CG receptor was studied by cross-linking the solubilized receptor-hormone complex with glutaraldehyde (GA), disuccinimidyl suberate (DSS) or dithiobis(succinimidyl propionate) (DSP) and analyzing the complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The hormone was labeled either in its alpha-subunit (125I-hCG) or in its beta-subunit (3H-hCG) or the label (3H) was introduced into the receptor molecule instead of the hormone. All of the labeling procedures led to the detection of only the receptor-(alpha,beta)hCG and receptor-(alpha)hCG complexes on the autoradiograms. The sizes of these complexes were 137,000 and 106,000, respectively, under reducing conditions. These results suggest that the receptor binds one hormone molecule, and that hCG interacts with the receptor mainly through its alpha-subunit. In addition, polyclonal antibodies directed against the LH/CG receptor and the alpha- and beta-subunits of hCG were used to detect the non-reduced receptor-(alpha,beta)hCG complex in immunoblotting. As antibodies directed against both the alpha-subunit and the beta-subunit were able to detect the Mr 130,000 complex, it is conceivable that both of the subunits are at least partially exposed on the receptor-hormone complex. 125I-hCG was also cross-linked to the membrane-bound receptor. The membrane-bound complex had an Mr of 144,000 under reducing conditions, i.e. approximately 7000 higher than that of the solubilized complex (Mr 137,000). This may indicate that the membrane-bound receptor is covalently modified or differs in conformation from the solubilized receptor.     

Characterization of antisera distinguishing carbohydrate structures in the beta-carboxyl-terminal region of human chorionic gonadotropin

The beta-COOH-terminal peptide region (beta CTP) of hCG is an important immunochemical domain that is specific to hCG but absent from homologous hLH. Three types of polyclonal antibodies can be elicited to the beta CTP portion of hCG. The first and most common recognizes the beta CTP amino acid sequence independent of its carbohydrate content (carbohydrate-oblivious). It can be elicited against synthetic beta CTP as well as against native or asialo beta CTP. The second type binds best to desialylated beta CTP, (galactose-requiring). The third type binds well only to sialylated beta CTP (sialic acid-requiring). All three types of antisera recognize the sialylated beta CTP as equivalent to the whole hormone on a molar basis, indicating that this peptide region of the whole hormone is neither sequestered nor conformationally altered. We have characterized the epitopes for the sialic acid-requiring and galactose-requiring beta CTP antisera. Both require elements of the primary amino acid sequence of beta hCG as well as specific elements of the carbohydrate side-chains and, therefore, are not directed solely to either peptide or carbohydrate determinants. The galactose-requiring beta CTP antiserum was generated to an ovalbumin conjugate of a desialylated form of beta CTP, beta 123-145. It binds desialylated forms 1000 times better than it binds native hCG, and binds neither synthetic beta CTP nor asialo-agalacto beta 123-141. The antiserum requires residues 123-141 and a portion of an O-serine-linked oligosaccharide chain. Its binding requirements are, thus, very different from those of the carbohydrate-oblivious beta CTP antiserum, which requires the last two residues of the hCG beta polypeptide chain and is not sensitive to the presence or absence of carbohydrate. The sialic acid-requiring beta CTP antiserum, which was generated to sialylated beta 123-145-thyroglobulin conjugate, binds well to sialylated beta CTP, but poorly to asialo beta CTP and not at all to the carbohydrate-free synthetic peptide. This antiserum requires the entire beta 123-145 sequence as well as sialic acid-terminated oligosaccharide side-chains. Since the sialylated peptide beta 115-141 binds poorly to it, this antiserum resembles the carbohydrate-oblivious anti-beta CTP; both require the COOH-terminal amino acids of hCG beta. However, the former requires intact O-linked carbohydrate moieties for binding. These antisera can be used to assess the primary protein and carbohydrate structures of beta CTP at low concentrations in urine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure of the human chorionic gonadotropin beta-subunit fragment from pregnancy urine

A major portion of the hCG immunoreactivity detectable in pregnancy urine is derived from a fragment of hCG beta. This lacks the COOH-terminal portion of hCG beta, but retains immunoreactivity with most antibodies raised against the beta-subunit of hCG. To improve clinical measurements of hCG and assess the importance of such fragments in human urine, we have isolated and determined the structure of this molecule. The hCG beta fragment was isolated from a partially purified commercial preparation of hCG (Organon) by gel filtration and immunoaffinity chromatography using monoclonal antibodies. It was found to consist of two polypeptide chains composed of residues beta-(6-40) disulfide-bridged to residues beta-(55-92). It also differs from the beta-subunit of hCG in its carbohydrate structure, lacking sialic acid and having a low but variable amount of galactose. A beta-fragment containing the same two NH2-terminal sequences was also isolated from a single pregnant woman's urine. The two major polypeptides comprising the beta-fragment contain a total of nine half-cystine residues, raising the possibility that a free thiol may exist or that a third undetected disulfide-bridged peptide is present in the intact fragment. However, tests for the presence of a free thiol have been negative. Another intrinsic characteristic of the beta-fragment is the formation of a variable amount of dimer in solutions of neutral pH. beta-fragment will not combine with intact alpha-subunit. Despite the absence of regions beta-(1-5), beta-(41-54), and beta-(93-145), the beta fragment is recognized by the SB-6 antibody and most monoclonal antibodies elicited to the beta-subunit, thus excluding half of the amino acids of the beta-subunit from the epitope(s) where these antibodies bind.     

Carbohydrate moieties of small placental hCG: requirement of mannose structure for biological activity

The 43 kDa human chorionic gonadotropin (hCG) (SP-hCG) was purified from human placenta and analyzed for sugar moieties. The low hexosamine content suggests that SP-hCG probably lacks O-linked sugar chains in the beta-subunit and incompletely formed N-linked sugar chains in the alpha- and beta-subunits. In the present study SP-hCG was hydrolyzed with various glycosidases. Treatment of hCG or SP-hCG with O-glycan peptide hydrolase increased the mobility of asialo-hCG beta in reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) while that of SP-hCG beta was unaffected, indicating that SP-hCG beta does not contain NeuNAc-Gal-GalNAc unit. Alpha-Mannosidase and endoglycosidase H hydrolyzed mannose and the high mannose-GlcNAc moieties, respectively, from alpha- and beta-subunits of SP-hCG, but not from the subunits of authentic hCG. Glycopeptidase F hydrolyzed completely the N-linked sugar chains from SP-hCG subunits, producing alpha- and beta-subunits with estimated Mr of 15,000 and 18,500, respectively. The biological activity of purified SP-hCG is about 50-80% of highly purified authentic hCG. In an in vitro system SP-hCG increased cAMP accumulation and testosterone production by rat Leydig cells to the same levels as that induced by hCG. However, the biological activity of SP-hCG was markedly reduced, following treatment with endoglycosidase H or alpha-mannosidase. To attain the level of testosterone production equivalent to that induced with untreated SP-hCG, 10-20 times higher dose of treated SP-hCG was required. On the other hand, cAMP accumulation induced with treated SP-hCG even at a very high concentration was substantially lower than that attained with untreated SP-hCG. In conclusion, the mannose moieties are essential structural components of the hormone in stimulating cAMP accumulation and steroidogenesis by rat Leydig cells.     

Detection of the free beta subunit of human chorionic gonadotropin (HCG) in cultures of normal and malignant trophoblast cells, pregnancy sera, and sera of patients with choriocarcinoma

Immunoaffinity adsorption techniques, utilizing specific antisera for hCG and its subunits bound to Sepharose 4B, have been employed to separate hCG alpha beta dimer and free subunits of hCG. As previously reported by this and a number of other laboratories, trophoblast cells (in vivo and in vitro) produce free alpha subunit in addition to hCG dimer. We have now shown that cultured JAr choriocarcinoma cells also secrete free beta subunit: 37% of the total beta subunit (combined and free) secreted by JAr cells is in the free form. Moreover, in pooled sera from choriocarcinoma patients 15% of total beta subunit is free, and in media from placental explant cultures and in pooled first trimester pregnancy sera 11% and 6.5%, respectively, of total beta subunit are in the free form. The free beta s are all of similar mol wt to the combined forms, and associate with urinary hCG alpha to form hCG. Free alpha s, which are larger than the combined forms, are unable to associate with urinary hCG beta to form hCG. We propose that the supply of combinable alpha subunit, rather than beta, limits dimer formation.     

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.     

HUMAN CHORIONIC GONADOTROPHIN AND SUBUNITS—HETEROGENEITY IN SERUM OF MALE PATIENTS WITH TUMOURS OF THE GENITAL TRACT

The pattern of secretion of human chorionic gonadotrophin (hCG) and its subunits in male subjects with tumours of the genital tract was examined by gel filtration, radioimmunoassay, immunoradiometric assay, bioassay and binding to Concanavalin A. The predominant form of hCG was the intact molecule but all patients had increased levels of free beta subunit. The intact hCG was active in a mouse Leydig cell bioassay and was normally glycosylated. High concentrations of free alpha subunit were not found and the ratio of alpha subunit: beta subunit was less than that in normal pregnancy. It is concluded that hCG-secreting tumours of the male genital tract are similar to choriocarcinoma in the female in that large quantities of intact hormone are produced with a disproportionate increase in free beta sub-unit.     

Purification and properties of a monoclonal antibody specific to the free beta-subunit of human chorionic gonadotropin (hCG beta) and its use in the isolation of free hCG beta produced by choriocarcinoma cells

The free beta-subunit of hCG is secreted by several tumors and is reported to be different from native hCG beta. We have developed a monoclonal antibody, B158, specific for free hCG beta to facilitate the detection and isolation of tumor-derived free hCG beta in the presence of intact hCG and hCG alpha. B158 belongs to the immunoglobulin G1 subclass, has high affinity for hCG beta (Ka, 1.05 X 10(9) M-1), and can be obtained in large quantities. The sensitivity of this antibody to detect free hCG beta in a RIA is less than 1 ng. B158 has negligible cross-reactivity with hCG, human LH, and other intact glycoprotein hormones and reacts with the free beta-subunits of deglycosylated hCG, human LH, and ovine LH. The antibody completely inhibits the recombination of hCG beta with hCG alpha indicating the antigenic site to be in the subunit interaction region or its vicinity. Comparison of the amino acid sequences of the various cross-reacting and noncross-reacting hormones indicates that the antigenic site may be discontinuous and conformational. B158 was purified to homogeneity from ascites fluid by DEAE-Affi-Gel blue and hCG beta affinity chromatography. Immobilized pure B158 antibody was used to isolate free hCG beta in a homogeneous form and high yield from BeWo choriocarcinoma cell culture supernatants. This free hCG beta has a slightly higher mol wt than standard hCG beta and recombines normally with hCG alpha. BeWo cells appear to produce only one species of free hCG beta.     

Serum HCG beta-core fragment is masked by associated macromolecules.

To determine if beta-core fragment is present in serum from individuals with pregnancy and choriocarcinoma, samples were analyzed by gel filtration and fractions assessed by immunoassays (Mr = 15,000). In agreement with published reports, only minute amounts of beta-core fragment were detected (less than 0.1% of hCG level). A small amount of beta-core fragment activity was identified near the void volume of the column (Mr greater than 60,000). This high molecular weight material was pooled and dissociated with 3M ammonium thiocyanate, and gel filtration repeated. A much more significant beta-core fragment peak was then detected (Mr = 15,000). After dissociation, beta-core fragment was 18% (mol/mol) of the hCG level in early pregnancy serum, 91% in mid-pregnancy serum, 50% in term pregnancy serum, but only 1.3% in choriocarcinoma serum, pre-therapy. We conclude that the major form of beta-core fragment found in serum is associated with other macromolecules, which mask the beta-core fragment epitope to existing hCG beta antibodies. Measurements made on the dissociated beta-core fragment complex are much more in keeping with pregnancy levels found in urine.