Identification and characterization of subpopulations of the free alpha-subunit that vary in their ability to combine with chorionic gonadotropin-beta

The free (uncombined) alpha-subunit of hCG is secreted in excess over alpha beta dimer from both malignant and nonmalignant trophoblast cells and is secreted ectopically from a variety of other malignant cell types. The free alpha-subunits from various sources are distinguishable from those that combine because they migrate more heterogeneously and more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than dimer alpha. We have previously identified three posttranslational modifications that may contribute to the altered mobility of the free alpha-subunit and to its inability to combine with the beta-subunit: 1) preferential phosphorylation of the free alpha-subunit, 2) O-glycosylation of free alpha, and 3) differences in the processing of the asparagine-linked oligosaccharides between the free and combinable forms. We have purified three populations of the alpha-subunit from the JAR choriocarcinoma cell line and from ChaGo, a bronchogenic carcinoma cell line that ectopically synthesizes only the alpha-subunit, in order to identify the posttranslational modifications that contribute to the altered mobility on SDS-PAGE. Fractionation of the oligosaccharides released from the alpha forms with peptide N-glycosidase has shown that the faster migrating alpha forms on SDS-PAGE have less completely processed oligosaccharide chains. Twenty-two to 25% of the JAR free alpha and 35-41% of the ChaGo alpha forms that migrate the fastest on SDS-PAGE recombine with beta in an in vitro recombination assay under conditions where 62% of the dimer alpha form recombines. In contrast, only 5-12% and 16-21% of the JAR free alpha and ChaGo alpha forms, respectively, that migrate the slowest on SDS-PAGE recombine with beta. The form of JAR free alpha least capable of combining with beta contains on O-linked glycan on Thr-39. This same site is a substrate for phosphorylation by JAR cells. However, most of ChaGo alpha fails to recombine with beta even though ChaGo alpha contains little O-linked carbohydrate. These results suggest that the larger asparagine-linked complex glycans on the slower migrating alpha forms are the major limiting factor for subunit combination. Although these modifications may not be rate limiting for combination in the rough endoplasmic reticulum, they may prevent dimerization of the free subunits later in the secretory pathway.     

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.     

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.     

Differential secretion of O-glycosylated gonadotropin alpha-subunit and luteinizing hormone (LH) in the presence of LH-releasing hormone

The pituitary hormones LH, FSH, and TSH are secreted as dimers of two subunits, alpha and beta. The alpha-subunit, identical in all three hormones, is produced by the pituitary in excess of beta and secreted as free subunit. Bovine free alpha does not combine with purified beta-subunit and contains an extra O-linked oligosaccharide not found on dimer alpha. We have developed an assay to quantitate this modified form of alpha in the medium of incubated steer pituitary slices. The assay, based on reverse phase HPLC analysis of radiolabeled alpha-subunit tryptic peptides, shows that under basal conditions, 75% of secreted free alpha is O-glycosylated. When the secretagogue LHRH is added to the slices, a 14-fold increase in LH dimer release is observed, but secretion of the modified alpha is increased by only 2-fold. Our results indicate that the majority of free alpha-subunit secreted by the pituitary contains O-linked oligosaccharide, and that secretion of this form of alpha differs from that of LH dimer.     

Combination of the chorionic gonadotropin free beta-subunit with alpha.

The rate-limiting event for combination of hCG alpha- and beta-subunits in JAR choriocarcinoma cells is the rate of disulfide bond formation in the beta-subunit. This is accompanied by a conformational change that produces a combination-competent form of the beta-subunit. The combination reaction, however, is incomplete, and 50% of the synthesized beta molecules remain uncombined (free). In addition, 70% of biosynthetically labeled free beta is degraded in the cell. Possible explanations for incomplete dimer formation include 1) biochemical differences between the free and combined beta-subunits that limit combination of free beta, and 2) an inefficient combination reaction due to low intersubunit affinities or limiting concentrations of combination-competent subunits within the cell. To examine whether the biochemical differences between free and combined beta-subunits that we have previously observed affect the combined beta-subunits that we have previously observed affect the combination competence of free beta, free and dimer beta-subunits were purified from the culture medium and lysates of JAR cells and examined for their ability to combine with alpha purified from pregnancy urine in an in vitro combination assay. Secreted free and dimer beta obtained from culture medium combined to the same extent with urinary alpha. Although the combination efficiencies were lower for the intracellular forms, the free and dimer beta-subunits purified from cell lysates also combined to the same extent with urinary alpha. Thus, biochemical differences that exist between the beta forms do not prevent combination of free beta with alpha in an in vitro combination assay. To examine the second possibility, we speculated that if high concentrations of hCG subunits remained in the rough endoplasmic reticulum (ER) for extended periods of time, the extent of dimer formation would increase in the cell. To increase the residence time of hCG subunits in the ER, JAR cells were treated with carbonyl cyanide trifluoromethoxyphenylhydrazone, an agent that inhibits the translocation of hCG subunits from the ER to the Golgi. Treatment of cells with trifluoromethoxyphenylhydrazone in long and short term pulse-chase labeling studies did not result in an increase in the extent of dimer formation. Thus, the subunit combination reaction in JAR cells may be incomplete due to subtle conformational differences in the free beta-subunit; however, these differences do not inhibit the combination of the free beta-subunit in vitro.     

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.     

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.     

The effect of carbonyl cyanide trifluoromethoxyphenylhydrazone and methylamine on the processing and secretion of the glycoprotein hormone chorionic gonadotropin by human choriocarcinoma cells

Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), a protonophore, and methylamine, a weak base, agents that dissipate hydrogen gradients across cellular membranes, were used to probe the coupling of hydrogen gradients to the processing and secretion of the glycoprotein hormone hCG by human choriocarcinoma cells (JAR) in culture. Both drugs disrupted the processing of asparagine-linked oligosaccharides such that the secreted hCG forms contained mostly high mannose rather than complex oligosaccharide chains. As the concentrations of FCCP were increased above 1 microgram/ml and those of methylamine above 12.5 mg/ml, the secretion of the labeled hCG dimer and free alpha-subunit was progressively inhibited. Both FCCP and methylamine also inhibited the incorporation of [35S] methionine and [3H]mannose into hCG subunits. Nevertheless, the inhibition of secretion was clearly apparent as an intracellular accumulation of the hCG subunit precursors in spite of the diminished incorporation of radioactive substrates. The intracellular hCG precursors that accumulated in the drug-treated cells contained predominantly Man8-9GlcNAc2 units, structures characteristic of glycoproteins localized in the endoplasmic reticulum. Both FCCP and methylamine inhibited hCG secretion at concentrations that did not lower the cellular content of ATP. We postulate on the basis of these results that a hydrogen gradient across the membrane either of the rough endoplasmic reticulum or the transitional vesicle is coupled to the rough endoplasmic reticulum to Golgi translocation step such that dissipation of the proton gradient blocks the secretion of hCG.     

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.     

Discordant and variable production of human chorionic gonadotropin and its free alpha- and beta-subunits in early pregnancy

Monoclonal antibodies specific to dimer hCG (alpha beta), free beta-subunit (beta hCG), free alpha-subunit (alpha hCG), and total beta hCG (dimer + free beta hCG) were used to monitor discordant production of hCG and its subunits during the early stages of embryo implantation. Sera collected 3 to 22 days postovum retrieval were assayed from patients participating in an in vitro fertilization program. In the majority of patients with ongoing pregnancies (n = 16), rising levels using the dimer hCG assay were first detected at an average time of 9.2 days post retrieval compared to 8.3 days for the total beta hCG assay and 6.5 days for the free beta hCG assays. Between days 5 to 9 after retrieval, the rises in the total beta hCG assay were due to predominantly free beta hCG subunit production. The proportion of total beta hCG levels due to free beta hCG subunits declined progressively from day 9 to less than 5% by day 22. Free alpha hCG levels remained low with rising levels first detected from day 18. A second group of patients (n = 12) had delayed, slowly rising levels in the total beta hCG assay which were first detected at an average of 12.4 days and associated with mainly biochemical pregnancies (n = 10). In these patients, rising total beta hCG levels were due to predominantly free beta hCG production. The subsequent loss of their pregnancy may be due to poor luteal support associated with delayed rises in dimer hCG levels. In nonconceptual cycles (n = 47) no significant rises were detected in dimer or free hCG subunits. Although the major form of hCG in circulation is dimer hCG, the origin of free beta hCG production in the early stages of implantation may be due to poorly differentiated trophoblastic tissue. Thus falling levels of free beta hCG subunits associated with increasing dimer hCG production may reflect increasing alpha hCG production by the proliferating layer of cytotrophoblastic cells.     

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.     

Coordinate regulation of the messenger ribonucleic acids encoding the alpha- and beta-subunits of human chorionic gonadotropin in HeLa cells and butyrate-resistant variants

Short term exposure of HeLa S3 cells to sodium butyrate induces accumulation of the mRNAs encoding both the alpha- and beta-subunits of hCG. Both mRNAs accumulate with the same kinetics and reach maximal levels with the same concentration of butyrate, suggesting that the levels of alpha and hCG beta mRNAs are coordinately regulated. In addition, induction of both mRNAs is tightly coupled with a similar increase in secreted levels of hCG subunit protein, further suggesting that regulation of CG biosynthesis in HeLa cells occurs before translation. To determine whether HeLa cells which have overcome the growth inhibitory properties of butyrate show uniformly high levels of alpha and hCG beta mRNAs, we isolated clonal variants by stepwise selection with increasing concentrations of butyrate. Of 69 isolated variants, at least 2 (AO and B2) display different patterns of CG gene expression. In AO cells, alpha-subunit mRNA was not detectable, while in B2 cells, the level of alpha-subunit mRNA was similar to that of wild-type HeLa S3 cells. Conversely, hCG beta mRNA levels in both variant cell lines approximated levels found in butyrate-treated HeLa S3 cultures, suggesting that the mRNAs for alpha and hCG beta are regulated independently. Analysis of genomic DNA by blot hybridization indicated that the alpha-subunit gene was present as a single unrearranged copy in HeLa S3 cells and in both variants, indicating that differences in alpha-subunit gene expression are not associated with major genomic alterations, but probably reflect more subtle changes.     

Synthesis and secretion of human chorionic gonadotropin and its subunits in choriocarcinoma cells: a comparative study with normal placental cells.

The human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.     

Synthesis of chorionic gonadotropin subunits in human choriocarcinoma clonal cell line JEG-3: carbohydrate differences in glycopeptides from free and combined alpha-subunits

The glycoprotein hormone hCG and its free alpha-subunit are secreted by the clonal choriocarcinoma cell line JEG-3. Free hCG alpha has a larger apparent mol wt (22,000-24,000) than the combined hCG alpha (18,000-19,000) obtained by dissociation of the hCG secreted by these cells. Techniques developed for the specific isolation and purification of the free and combined hCG alpha forms and for the preparation of glycopeptides from these subunits have permitted detection of the incorporation of D-[3H]glucosamine [( 3H]GlcN) and L-[3H]fucose into both alpha-subunit forms. Relative to their [35S]methionine content, 2.3-fold more [3H]GlcN and 6-fold more L-[3H]fucose were incorporated into free hCG alpha than into combined hCG alpha. Analyses of [3H]GlcN glycopeptides prepared from free and combined hCG alpha indicate that the 22,000- to 24,000-dalton subunit form contained more [3H]GlcN and 27% more of the GlcN metabolite N-acetylneuraminic acid than the 18,000- to 19,000-dalton hCG alpha-subunit, than both hCG alpha forms contained two major N-linked oligosaccharide chains differing primarily in their NeuAc content, and that most of the [3H]GlcN was incorporated as GlcN or metabolites of GlcN other than N-acetylneuraminic acid. These studies provide direct chemical evidence of a higher content of carbohydrate in the larger free alpha-subunit form.     

The antigenic structure of the human glycoprotein hormone alpha-subunit. I. Characterization of anti-alpha monoclonal antibodies.

The glycoprotein hormones CG, LH, FSH, and TSH are composed of two noncovalently linked subunits, alpha and beta. The beta-subunit confers hormone specificity, while the alpha-subunit is homologous within a species. To help in determining the antigenic structure of the common alpha-subunit, six monoclonal antibodies (mAbs) to the free or heterodimeric alpha-subunit of human (h) gonadotropic hormones have been prepared and, along with two previously isolated mAbs, have been characterized for binding specificity to alpha- and beta-subunits and the human glycoprotein hormones, CG, LH, FSH, and TSH. Each mAb was derived from hybidomas of FO myeloma cells fused with spleen cells from mice immunized with free alpha-subunit, hCG or hFSH. mAbs A101, A102, and E512 were specific for the alpha-subunit but showed the highest affinity for the intact hormone; K2.18, K94.6, E501, E502, and E511 were specific for free alpha. All of the antibodies inhibited binding of 125I-hCG to luteal membrane receptor, and 125I-labeled mAbs did not recognize hCG/receptor complex. Characterization by two-site binding assays using alpha, hCG, or hFSH as antigen revealed that all the mAbs bind to unique sites on alpha which may be overlapping, and which are modified in the intact hormone. The antigenic sites for mAbs E502, E511, and K2.18 are at least partially linear because they bind to reduced, carboxymethylated alpha.     

Processing of O-linked glycosylation in the chimera consisting of alpha-subunit and carboxyl-terminal peptide of the human chorionic gonadotropin beta-subunit is affected by dimer formation with follicle-stimulating hormone beta-subunit

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunits. hCGbeta is unique among beta-subunits due to a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharides. We previously reported that there were differences in O-glycosylation between two chimeras consisting of alpha-subunit and CTP, i.e. a variant with CTP at the N-terminal region (Calpha) and another analog with CTP at the C-terminus (alphaC) of the alpha-subunit. To address whether O-glycosylation is influenced by the heterodimer formation, Calpha and alphaC were expressed alone or with FSHbeta-subunit in Chinese hamster ovary cells. The O-linked glycosylation was assessed by continuous labeling with [(35)S]methionine/cysteine, immunoprecipitation with anti-alpha or anti-FSH serum, serial digestion with endoglycosidase-F and neuraminidase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The decrease in molecular weight of dimeric chimeras digested with endoglycosidase-F was greater in Calpha than that in alphaC after treatment with neuraminidase, revealing that both chimeras have different numbers of sialic acids on O-linked carbohydrates. By treating with endoglycosidase-F, the dimeric alphaC migrated faster than its free form, whereas the mobility difference between assembled and unassembled forms of Calpha was very little. These data indicate that processing of O-glycosylation is affected by the backbone polypeptide chain(s).     

Similarity of the clearance rates of free alpha-subunit and alpha-subunit dissociated from intact human chorionic gonadotropin, despite differences in sialic acid contents

It is well known that the MCR of proteins can be influenced by their carbohydrate structure; i.e. the presence of terminal galactose on proteins results in uptake by hepatic receptors for galactose-terminated glycoproteins. Thus, a protein with its galactose residues covered or removed exhibits a far longer half life in plasma than one with its galactose residues exposed. The free alpha-subunit of human CG (hCG) has been shown to have a different carbohydrate composition than does the alpha-subunit dissociated from the intact hormone. In our laboratory, analysis of alpha-subunits isolated from pregnancy urine indicated that the alpha-subunit dissociated from hCG (hCG alpha) contains primarily monosialylated oligosaccharides, whereas the free alpha-subunit contains more than one sialic acid per oligosaccharide. This difference in the degree of sialylation prompted us to examine the clearance rates of these two subunits. Accordingly, free alpha and hCG alpha were purified by affinity chromatography and labeled with 125I. The labeled subunits were injected iv into rats and serum samples were removed at various time intervals over a 2-h period. The amount of [125I]alpha-subunit remaining in the serum was determined by immunoprecipitation using an antiserum to hCG alpha. The disappearance curves for the two subunits were indistinguishable and could be analyzed by a biexponential model. The t 1/2 of the faster component was 5 min, while the t 1/2 of the slower component was 74 min. In order to determine whether or not terminal galactose was present on either the hCG alpha or the free alpha-subunit, the labeled molecules were subjected to lectin column chromatography using Ricin or peanut lectin linked to agarose. Both of these lectins bind galactose but have different specificities with respect to the penultimate sugar. Both subunit preparations contained only minor amounts of material which could bind or either lectin. However, after desialylation, both hCG alpha and free alpha bound extensively to Ricin, indicating the presence of penultimate galactose residues in both. We conclude that terminal galactose residues are not present on the oligosaccharides of either hCG alpha or free alpha-subunits, and that the difference observed in the sialic acid contents of the two subunits does not affect their rates of clearance.     

Effects of retinoic acid on differentiation of choriocarcinoma cells in vitro

Choriocarcinoma cells maintain multiple hormonal functions in culture. We have found that these cells secreted no immunoreactive pregnancy-specific beta 1-glycoprotein (PS beta G), a placental protein. Choriocarcinoma cells can be induced to synthesize low levels of PS beta G by retinoic acid, 8-bromo-cAMP (8BrcAMP), cholera toxin, methyl-isobutylxanthine (MIX), and 5-bromo-2'-deoxyuridine (BrdUrd). The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave synergistic induction of PS beta G. The simultaneous addition of retinoic acid and BrdUrd failed to give even additive induction. In addition to stimulating PS beta G production, retinoic acid increased the production of hCG and its alpha-subunit (hCG alpha) by choriocarcinoma cells. The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave additive induction for hCG and hCG alpha. Passage of choriocarcinoma cells in medium containing retinoic acid induced a stable altered phenotype characterized by elevated levels of PS beta G, hCG, and hCG alpha. These retinoid-treated choriocarcinoma cells remained responsive to 8BrcAMP or compounds that increase intracellular cAMP concentrations and to BrdUrd; the production to PS beta G and hCG was greatly stimulated by 8BrcAMP, cholera toxin, or MIX, and the production of hCG and hCG alpha was greatly inhibited by BrdUrd. However, the production of hCG alpha was only slightly induced by these cAMP modulators, and the production of PS beta G was not increased by BrdUrd.