Abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line, JEG-3

Over the past two decades, sugar chain structures of human chorionic gonadotropin (hCG) produced in healthy people, three types of trophoblastic disease, and some types of cell lines have been analyzed. The abnormal biantennary structure of hCG is a good marker for the diagnosis of malignant choriocarcinoma. In spite of much research, hCG with an abnormal biantennary structure is only detected in the urine of choriocarcinoma or pregnant diabetic patients. We hypothesized that the formation mechanism of the abnormal biantennary sugar chain structure is mainly caused by high GnT-IV activity. To confirm this, we measured the N-acetylglucosaminyltransferase (GnT)-IV activity and hCG productivity in three choriocarcinoma cell lines, and selected JEG-3 cells. hCG samples were purified from medium conditioned by JEG-3 cells, and their sugar chain structures were analyzed. We detected an abnormal biantennary structure, and the proportions were different from those previously reported in the urine samples of choriocarcinoma patients. These findings proved our hypothesis and suggest the usefulness of JEG-3 cells for further analyses of abnormal biantennary structure formation. Published in 2004.     

Altered glycosylation of human chorionic gonadotropin decreases its hormonal activity as determined by cyclic-adenosine 3',5'-monophosphate production in MA-10 cells

Human chorionic gonadotropin (hCG) purified from pooled urine of normal pregnant women contains four asparagine-linked sugar chains and four mucin-type sugar chains. The structures of asparagine-linked sugar chains of this hormone are constant and site-specific. hCGs obtained from the urine of patients with invasive mole or choriocarcinoma have quite different sets of oligosaccharides although the primary structures of the polypeptides and the numbers of the sugar chains are the same as those of normal hCG. In order to examine the biological activities of these hCGs with altered glycosylation, we measured the amount of cAMP produced in a murine Leydig tumour cell line, MA-10, after incubation with the hCG samples. The extent of sialylation of oligosaccharides in the three hCG samples used in this study were 88% in normal hCG, 82% in invasive mole hCG and 63% in choriocarcinoma hCG. The hormonal activity of invasive mole hCG was slightly lower while that of choriocarcinoma hCG was significantly (P less than 0.01) lower than that of normal hCG. Complete desialylation induced remarkable loss of full activities in all the samples. However, the hormonal activities of the three samples were different even after desialylation. The full activities of the desialylated samples of invasive mole hCG and choriocarcinoma hCG were 78 and 65% of that of desialylated normal hCG. These results indicated that the structures of the neutral oligosaccharide portion are also important for the expression of full hormonal activity.     

hCG对绒癌细胞系MMP-2和MMP-8表达的影响

为了探讨人绒毛膜促性腺激素（hCG)对绒并且吕细胞侵袭性相关基因表达的影响作用。采用逆转录多聚酶链反应（RT-PCR）检测方法,观察了不同浓度hCG不同处理时间对JEG-3绒癌细胞系表达基质金属蛋白酶（MMP-2和MMP-8）的影响。结果显示,绒癌细胞系JEG-3表达MMP-2和MMP-8;分别用0、50、500、5000、25000IU/LhCG处理48h后,JEG-3细胞中MMP-2mRNA的含量无明显变化。MMP-8mRNA的表达则被诱导,并随hCG作用浓度增高而增强,进一步研究处理时间对MMP表达的影响。结果发现经25000IU/LhCG处理的JEG-3细胞,MMP-8的表达随处理时间的延长逐渐增强,而MMP-2的表达则在第6h被显著诱导后逐渐降低,以上结果提示,hCG可诱导绒癌细胞系JEG-3中MMP-2和MMP-8两种基质金属蛋白酶的表达,并因此可能对绒癌细胞系的侵袭性具有影响作用。然而hCG对这两者表达的影响规律并不完全一致。     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin produced in choriocarcinoma. Appearance of triantennary sugar chains and unique biantennary sugar chains

Human chorionic gonadotropin (hCG) highly purified from urine of the patient with choriocarcinoma contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from polypeptide portion by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The structures of these sugar chains were determined by the combination of sequential glycosidase digestion, periodate oxidation, and methylation analysis. As compared with the sugar chains of normal urinary and placental hCG reported previously, they include several prominent structural differences. More than 97% of the sugar chains of choriocarcinoma hCG was free from sialic acid, while the sugar chains of normal hCG were mostly sialylated. Choriocarcinoma hCG contains unusual biantennary complex-type sugar chains in addition to regular tri-, bi-, and monoantennary sugar chains. These sugar chains have two outer chains linked at the C-2 and C-4 positions of the same alpha-mannosyl residue of the trimannosyl core. Since normal hCG does not contain any triantennary sugar chains, occurrence of Gal beta 1 leads to 4GlcNAc beta 1 leads to 4Man alpha 1 leads to group is another characteristic feature of the sugar chains of choriocarcinoma hCG. The evidence that the monoantennary sugar chain of Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc leads to Asn is not found in normal hCG and the sum total of fucosylated sugar chains is 50%, which is twice as much as normal hCG, indicated that fucosylation is also modified in choriocarcinoma tissue.     

Structures, function, and transformational changes of the sugar chains of glycohormones

Human chorionic gonadotropin (hCG), human luteinizing hormone, human thyroid-stimulating hormone, and human follicle-stimulating hormone are closely related family of proteins which share a common alpha-subunit. However, their sugar moieties are quite different. hCG contains five acidic asparagine-linked sugar chains. These five sugar chains are derived by sialylation from three neutral oligosaccharides: two biantennary (N-1 and N-2) and one monoantennary (N-3) complex-type oligosaccharides. Although hCG purified from the urine of pregnant women is more enriched in sialylated sugar chains than that purified from placenta, the molar ratio of N-1, N-2, and N-3 of these two hCGs are the same (1:2:1). Comparative study of the sugar moieties of the alpha- and beta-subunits of hCG revealed that alpha contains 1 mol each of N-2 and N-3, while beta contains 1 mol each of N-1 and N-2. This specific distribution of oligosaccharides at the four asparagine loci of the hCG molecule is now helping us to consider the functional role of the sugar moiety of glycohormones. hCG is produced not only by the trophoblast but also by various trophoblastic diseases. The hCGs purified from the urine of patients with hydatidiform mole contain the same oligosaccharides as normal hCG. However, those from the urine of choriocarcinoma patients contain five additional neutral oligosaccharides. In contrast, hCGs from invasive-mole patients contain three of the five oligosaccharides, specifically found in choriocarcinoma hCGs. The malignant transformational change of the sugar moiety of hCG can be explained by an increase of a fucosyltransferase, which forms the Fuc alpha 1----6GlcNAc group and by ectopic expression and subsequent modification of N-acetylglucosaminyltransferase IV. The appearance of tumor-specific sugar chains of hCG has been used to develop a new diagnostic method for invasive mole and choriocarcinoma.     

Structure, pathology and function of the N-linked sugar chains of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) contains five acidic N-linked sugar chains, which are derived from three neutral oligosaccharides by sialylation. Each of the two subunits (hCGalpha and hCGbeta) of hCG contain two glycosylated Asn residues. Glycopeptides, each containing a single glycosylated Asn, were obtained by digestion of hCGalpha with trypsin, and of hCGbeta with chymotrypsin and lysyl endopeptidase. Comparative study of the sugar chains of the four glycopeptides revealed the occurrence of site-directed glycosylation. Studies of the sugar chains of hCGs, purified from urine of patients with various trophoblastic diseases, revealed that choriocarcinoma hCGs contain sialylated or non-sialylated forms of eight neutral oligosaccharides. In contrast, hCGs from invasive mole patients contain sialyl derivatives of five neutral oligosaccharides. The structural characteristics of the five neutral oligosaccharides, detected in choriocarcinoma hCGs but not in normal placental hCGs, indicate that N-acetylglucosaminyltransferase IV (GnT-IV) is abnormally expressed in the malignant cells. This supposition was confirmed by molecular biological study of GnT-IV in placenta and choriocarcinoma cell lines. The appearance of tumor-specific sugar chains in hCG has been used to develop a diagnostic method of searching for malignant trophoblastic diseases. In addition, a summary of the current knowledge concerning the functional role of N-linked sugar chains in the expression of the hormonal activity of hCG has been presented.     

Identification of a soluble GM-CSF binding protein in the supernatant of a human choriocarcinoma cell line.

We identified two forms of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) made by the human choriocarcinoma cell line JEG-3 using an affinity-labeling technique. The protein was identified in the detergent-extract was 78 kDa, very similar to that of the membrane-bound GM-CSF receptor alpha chain expressed in a wide variety of hematopoietic and nonhematopoietic cells, including JEG-3. In contrast, a 62-kDa GM-CSF binding protein, or the soluble GM-CSF receptor, was identified in the supernatant of JEG-3 cells. Utilizing the same affinity labeling technique, we did not detect the soluble GM-CSF binding protein in the supernatant of several hematopoietic cell lines, such as U-937 and KG-1, which express membrane bound alpha chain as well as beta chain. The 62-kDa soluble GM-CSF receptor is produced in abundant amounts by JEG-3, but in very small amounts, if any, by hematopoietic cell lines.     

High retinoid X receptor expression in JEG-3 choriocarcinoma cells: Involvement in cell function modulation by retinoids

Retinoic acid (RA) is an important mediator of cell differentiation. It stimulates hCG secretion by JEG-3 choriocarcinoma cells in vitro after a time lag. The first aim of this study was to characterize which types of retinoid receptors (RARs and RXRs) are present in JEG-3 cells. Using Western blot analysis and immunocytochemistry with specific antibodies as well as Northern blot analysis, we found that JEG-3 cells expressed RARα and RXRα, the latter being the predominant receptor. We then analyzed the action on cell proliferation and hCG secretion of the physiological retinoids all-trans RA (RA) and 9 cis RA as well as synthetic retinoids with specific affinity for RARα and RXRα. All these retinoids were potent inhibitors of cell growth, maximal inhibition (72 ± 2%) being observed after 4 days of treatment with Ro 25, a RXRα specific ligand. Within 24 h, 9 cis RA and Ro 25 stimulated hCG secretion, and maximal stimulation (1,472 ± 10%) occurred at 48 h with the RXRα-specific ligand. The RARα-specific ligand also stimulated hCG secretion but to a lower extend and after a delay of 48 h. These results suggest a predominant role of RXRα in mediating the biological effects of retinoids on JEG-3 cells and the possible induction by RA itself of the metabolic pathway leading to 9 cis RA. J. Cell. Physiol. 176:595–601, 1998. © 1998 Wiley-Liss, Inc.     

Comparative study of the N-glycans of human monoclonal immunoglobulins M produced by hybridoma and parental cells

Cell-cell hybridization is one method of establishing cell lines capable of producing an abundance of antibodies. In order to clearly characterize antibodies produced by hybridomas, the influence of cell-cell hybridization on the glycosylation of produced antibodies should be studied. In this report, we describe structural changes of the N-glycans in immunoglobulin M (IgM) produced by a hybridoma cell line termed 3-4, which was established through hybridization of an IgM-producing Epstein-Barr virus transformed human B-cell line termed No. 12, and a human myeloma cell line termed P109. We analyzed the structures of sugar chains on the constant region of the mu-chain of IgMs produced by parental No. 12 cells and hybridoma 3-4 cells. In both parental cells and hybridoma cells, the predominant structures at Asn171, Asn332, and N395 were fully galactosylated biantennary complex types, with or without core fucose and/or bisecting GlcNAc. However, the amount of bisecting GlcNAc was markedly decreased in the hybridoma cells. Therefore, the activity of UDP-N-acetylglucosamine:beta-D-mannoside beta-1,4-N-acetylglucosaminyltransferase (GnT-III) responsible for the formation of bisecting GlcNAc was measured in parental cells and hybridoma cells. No. 12 cells showed some GnT-III activity, whereas P109 cells showed no such activity. The corresponding level of activity observed in hybridoma 3-4 cells was much lower than that in No. 12 cells. The above results demonstrated a reduction in the intracellular activity of GnT-III in the hybridoma cells, which was largely due to the influence of P109 cells. Moreover, the sugar chain structures of IgMs produced by the cells reflected the level of GnT-III activity.     

Comparative study of the mucin-type sugar chains of human chorionic gonadotropin present in the urine of patients with trophoblastic diseases and healthy pregnant women

Human chorionic gonadotropins (hCGs) highly purified from the urine of patients with trophoblastic diseases and of healthy pregnant women contain approximately four mucin-type sugar chains in one molecule. The structures of these sugar chains were studied comparatively by using a new sensitive method to obtain mucin-type sugar chains quantitatively as radioactive oligosaccharides from a small amount of glycoproteins. The mucin-type sugar chains of all hCGs include sialylated and nonsialylated Gal beta 1----3GalNAc and Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAc. In the case of normal hCG and hydatidiform mole hCG, oligosaccharides containing the tetrasaccharide core occupy approximately 10% of the total mucin-type sugar chains. The ratio of the tetrasaccharide containing oligosaccharides is increased prominently to approximately 60% in choriocarcinoma hCG. The proportion in invasive mole hCG was also increased, but less than the proportion of choriocarcinoma hCG.     

Polyamines control human chorionic gonadotropin production in the JEG-3 choriocarcinoma cell

The effect of inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) and the resultant lowering of polyamine levels upon human chorionic gonadotropin (hCG) production in JEG-3 choriocarcinoma cells was investigated. DFMO (10 mM) totally inhibited ornithine decarboxylase activity. In DFMO-treated cells, cellular spermidine concentrations fell to nondetectable levels (less than 1% of control values) within 24 h and spermine concentrations were reduced to 41.9% of controls over 6 days. DFMO caused a 70-80% inhibition of hCG production. Levels of mRNA for both the alpha and beta subunits of hCG were also inhibited relative to mRNA for tubulin. Exogenous putrescine normalized hCG production in a dose-dependent manner. Other diamines, including cadaverine, 1,3-diaminopropane, 1,6-diaminohexane, and 1,7-diaminoheptane, were ineffective in reestablishing hCG production in DFMO-treated cells. Dibutyryl cAMP (1 mM) stimulated hCG production and increased levels of mRNA for the alpha and beta subunit 5-40-fold in both DFMO-treated and control cells. Polyamines appear to have a fundamental role in hCG production in JEG-3 choriocarcinoma cells. However, dibutyryl cAMP can partially overcome or circumvent the requirement for polyamines in hCG biosynthesis.     

Rapid maturation of glycoprotein hormone free alpha-subunit (GPHalpha) and GPHalpha alpha homodimers

The dynamics of glycoprotein hormone alpha-subunit (GPHalpha) maturation and GPHalpha alpha homodimer formation were studied in presence (JEG-3 choriocarcinoma cells) and absence (HeLa cells) of hCGbeta. In both cases, the major initially occurring GPHalpha variant in [35S]Met/Cys-labeled cells carried two N-glycans (M(r app) = 22 kDa). Moreover, a mono-N-glycosylated in vivo association-incompetent GPHalpha variant (M(r app) = 18 kDa) was observed. In JEG-3 cells the early 22-kDa GPHalpha either associated with hCGbeta, or showed self-association to yield GPHalpha alpha homodimers, or was later converted into heavily glycosylated large free GPHalpha (M(r app) = 24 kDa). Micro-preparative isolation of intracellular GPHalpha alpha homodimers of JEG-3 cells and their conversion by reduction revealed that they consisted of 22-kDa GPHalpha monomers and not of large free GPHalpha. In HeLa cells, the large free GPHalpha variant was not observed, whereas GPHalpha alpha homodimers were present. Intracellularly, early GPHalpha alpha homodimers (35 kDa) and late variants (JEG-3: 44 kDa, HeLa: 39 kDa) were found. Both cell types secreted 45 kDa GPHalpha alpha homodimers. Large free GPHalpha and GPHalpha alpha homodimers were more rapidly sialylated than hCG alphabeta-heterodimers indicating a sequestration mechanism in the secretory pathway. In GPHalpha alpha homo- as well as hCG alphabeta-heterodimers the subunit interaction site, located on loop 2 of GPHalpha (amino acids 33-42), became immunologically inaccessible indicating similar spatial orientation of GPHalpha in both types of dimers. The studies demonstrate the formation, in vivo dynamics of GPHalpha alpha homodimers, and the pathways of the cellular metabolism of variants of GPHalpha, monoglycosylated GPHalpha and large free GPHalpha.     

Immunofluorescent and immunochemical evidence for the expression of cytovillin in the microvilli of a wide range of cultured human cells

We have previously purified a Mr 75,000 protein, cytovillin, from cultured human choriocarcinoma cells (JEG-3) and shown that this protein was specifically confined to the microvillus membrane of these cells. I have now studied the expression and the subcellular distribution of cytovillin in eighteen normal and transformed human cell lines and strains by using immunoblotting and indirect immunofluorescence microscopy. In all cell types, cytovillin was highly enriched in cell surface protrusions. When cell types were ranked according to their staining intensity, choriocarcinoma was highest, then amniotic epithelial cells, other choriocarcinoma cells and tumor cells, and finally fibroblastoid cells. The latter only gave faint diffuse fluorescence on the plasma membrane and, occasionally, on the microvilli. However, detergent extracts of all cell types could be shown to contain cytovillin by the use of immunoblotting techniques. Metabolic pulse-chase labelling experiments with JEG-3 cells demonstrated synthesis of cytovillin as a single-chain polypeptide. No precursor forms or specific proteolytic cleavage products could be seen either by immunoblotting or immunoprecipitation. The protein was found to be very stable with a biologic half-life of about 25 hours. The pI determined by isoelectric focusing was 6.1. These results were consistent with cytovillin being an integral component of the microvilli and other surface extensions of all human cell types examined.     

Uptake of dehydroepiandrosterone-3-sulfate by isolated trophoblasts from human term placenta, JEG-3, BeWo, Jar, BHK cells, and BHK cells transfected with human sterylsulfatase-cDNA

The human placenta lacks the enzyme 17-hydroxylase/17-20-lyase, and is thus unable to convert cholesterol into estrogens. Therefore estrogen synthesis of trophoblast cells depends on the supply of precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16-hydroxy-dehydroepiandrosterone-3-sulfate by maternal and fetal blood. To investigate the cellular internalisation of these anionic hydrophilic precursors, the uptake of [³H]-/[³⁵S]-DHEA-S and [³H]-taurocholate by isolated cytotrophoblasts, cells of choriocarcinoma cell lines (JEG-3, BeWo, Jar), BHK and BHK cells transfected with human sterylsulfatase-cDNA (BHK-STS cells) was studied. Furthermore, the activity of sterylsulfatase of these cells in suspension and in corresponding cell homogenate was measured.

During the first 5 min of incubation with [³H]-DHEA-S or [³⁵S]-DHEA-S, radioactivity of cytotrophoblasts increased significantly, while radioactivity of JEG-3, Jar, BHK and BHK-STS cells did not increase. Radioactivity of BeWo cells increased slightly. For all cell types, there was no significant difference for uptake of either substrate. During incubation with [³H]-taurocholate, radioactivity of cytotrophoblasts did not increase. Sterylsulfatase activity of cytotrophoblast homogenate was significantly lower than that of cytotrophoblast suspension. Sterylsulfatase activity of BHK-STS, JEG-3 or BeWo cell homogenate was significantly higher than that of the corresponding cell suspension. In BHK and Jar cells sterylsulfatase activity was not detectable.

Cytotrophoblasts take up DHEA-S without prior hydrolysis. BHK, BHK-STS, JEG-3, and Jar cells do not take up and BeWo cells slowly take up DHEA-S. In cytotrophoblasts extracellular DHEA-S rapidly gains access to intracellular sterylsulfatase, while in choriocarcinoma and BHK-STS cells access of DHEA-S to sterylsulfatase is limited. Our results indicate, that uptake by cytotrophoblasts is mediated by a carrier which is not expressed in choriocarcinoma or BHK cells and which is different from the known taurocholate-transporting organic anion transporting polypetides.     

Self-regulation of the endothelin receptor system in choriocarcinoma cells

The human trophoblast secretes endothelin-1 (ET-1) and expresses ET receptors. The present study tested whether the transformed BeWo, JAR and JEG-3 choriocarcinoma cells: (1) secrete endothelin-1 (ET-1); (2) express both ET-A and ET-B receptor subtypes; and (3) have the potential to allow for autologous regulation of ET-receptor proteins. The cells were cultured for 24/48 h with or without 10% FCS and, in experiments on receptor regulation, with ET-1 (5-20 nM and 10 microM). ET-1 secretion was measured by RIA and receptor levels by immunoblotting. All cell types secreted ET-1 albeit at different levels and sensitivity to FCS. All cell lines expressed both ET-A (JEG-3>BeWo=JAR) and ET-B (JEG-3=JAR>BeWo) receptor subtypes, which could be up- and downregulated depending on ET-1 concentration, culture time and FCS presence. It is concluded that BeWo, JAR and JEG-3 choriocarcinoma cells secrete ET-1 and express both ET-A and ET-B receptor subtypes. The receptor levels can be regulated by ET-1. This provides the molecular basis for an autocrine system with the potential of autologous regulation of yet unidentified ET-1-induced functions.     

Human glycoprotein hormone production in human-human and human-mouse somatic cell hybrids

The production of both protein and steroid hormones was studied utilizing somatic cell hybrids formed with human choriocarcinoma cells. The human JEG-3 cell line produced the species and organ-specific hormone human chorionic gonadotropin (hCG), the steroid hormone progesterone, and converted 19-carbon steroids to estrogens. Hybrids formed with human VA-2 cells, mouse Cl 1D cells and mouse 3T3-4EF cells had detectable hCG synthesis in 20 of 41 total clones. There was no detectable progesterone or 19-carbon aromatization to estrogens in any hybrids. These data demonstrate that the differentiated function of human protein hormone production can be retained in inter- and intra-specific somatic cell hybrids. These results also suggest that protein hormone production can occur independently of steroid production in these cells of placental origin.