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.     

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.     

Human chorionic gonadotropin beta-subunit affects the folding and glycosylation of alpha-cys mutants

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit but differ in their hormone-specific beta-subunits. Both subunits have five and six disulfide bonds, respectively, which consist of cystine knot structure. It is evident from numerous studies that the structure of beta-subunits is rigid, whereas that of alpha-subunit is flexible and can be molded by a beta-subunit. Previously, we reported that secreted forms of a mutants where either cysteine residue in the disulfide bond 7-31 or 59-87 was converted to alanine contained a disulfide-linked homodimer in addition to a monomer. To study whether the hCGbeta-subunit affects the conformations of alpha mutants, alpha-subunits lacking either the 7-31 or 59-87 disulfide bond were expressed with wild-type (WT) hCGbeta in Chinese hamster ovary cells, and homodimer formation and glycosylation of dimerized alpha-subunit were assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-alpha or -hCGbeta serum, digestion with endoglycosidase-H or -F, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. Our data showed that a homodimer was not observed in the half-Cys mutants except one, where cysteine at position 7 was converted to alanine, in the presence of beta-subunit. This finding indicated that hCGbeta-subunit rescued the a half-Cys mutants from the formation of intermolecular disulfide-linked homodimer by preferentially combining with the alpha mutants. In both free WT and all mutants treated with endoglycosidase-H, no or faint bands were recognized as the same migration as seen in endoglycosidase-F treatment. Even in the endoglycosidase-H sensitive cases, the amount of sensitive alpha-subunits was less than 5% of total alpha-subunits. In contrast to free alpha-subunits, distinct endoglycosidase-H sensitive bands were seen in both WT and mutants, although the ratio was various. We concluded that hCGbeta-subunit affects the folding and glycosylation of the alpha-subunit mutants.     

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.     

Unmasking a new recognition signal for O-linked glycosylation in the chorionic gonadotropin beta subunit

hCGbeta subunit is distinguished among the other members of the family of the glycoprotein hormones by the presence of four serine O-linked oligosaccharide units in the last 25 amino acids. This carboxy terminal peptide (CTP) influences the intracellular behavior of the subunit and is important for maintaining the biological half-life of hCG. To examine how the O-linked oligosaccharides affect the metabolic behavior of hCG, we generated a CGbeta mutant devoid of the native O-linked acceptor sites. An alternative site not used in the native subunit was glycosylated and the structure of this oligosaccharide differed from the wild-type O-linked carbohydrates. This glycosylation occurred at serine 130 in the CTP and in contrast to the wild type O-linked oligosaccharides, sialic acid is a major component of the alternatively linked carbohydrate. The data show that deleting the native acceptor sites exposes a new site for O-glycosylation and promotes a differential intracellular processing of the beta subunit. These results support the hypothesis that the CTP participates in the folding of the newly synthesized subunit, which is manifested by the post-translational changes reported here.     

Characterization of inhibin forms and their measurement by an inhibin alpha-subunit ELISA in serum from postmenopausal women with ovarian cancer

The aim of this study was to characterize the molecular wt forms of inhibins A and B and its free alpha-subunit present in serum from women with ovarian cancer as a basis for developing improved monoclonal antibody-based inhibin assays for monitoring ovarian cancer. Three new inhibin alpha-subunit (alphaC) ELISAs were developed using monoclonal antibodies directed to three nonoverlapping peptide regions of the alphaC region of the inhibin alpha-subunit. To characterize serum inhibin molecular wt forms present in women with ovarian cancer, existing inhibin immunoassays (inhibin A, inhibin B, and pro-alphaC) and the new alphaC ELISAs were applied to sera from women with granulosa cell tumors and mucinous carcinomas previously fractionated using a combined immunoaffinity chromatography, preparative SDS-PAGE, and electroelution procedure. The distribution and molecular size of dimeric inhibins and alpha-subunit detected were consistent with known mol wt forms of inhibins A and B and inhibin alpha-subunit and their precursor forms present in serum and follicular fluid from healthy women. The alphaC ELISAs recognized all known forms of inhibin and the free inhibin alpha-subunit, although differences between alphaC ELISAs were observed in their ability to detect high mol wt forms. To assess which of the alphaC ELISAs was preferred in application to ovarian cancer, the alphaC ELISAs were applied to serum from a range of normal postmenopausal women (n = 61) and postmenopausal women (n = 152) with ovarian (serous, mucinous, endometrioid, clear cell carcinomas, and granulosa cell tumors) and nonovarian (breast and colon) cancers. Despite differences in their ability to detect high mol wt forms of inhibin, the alphaC ELISAs showed similar sensitivity (i.e. proportion of cancer patients correctly detected) and specificity (proportion of controls correctly detected) indexes in the detection of mucinous carcinomas (84% and 95%) and granulosa cell tumors (100% and 95%) compared with earlier inhibin RIA or polyclonal antibody-based immunofluorometric assays. A combination of the alphaC ELISAs with the CA125 assay, an ovarian tumor marker that has a high sensitivity and specificity for other ovarian cancers (serous, clear cell, and endometrioid), resulted in an increase in sensitivity/specificity indexes (95% and 95%) for the all ovarian cancer group. These new monoclonal antibody-based inhibin alphaC ELISAs now provide practical and sensitive assays suitable for evaluation as diagnostic tests for monitoring ovarian cancers.     

Assembly and expression of a synthetic gene encoding the bovine glycoprotein hormone alpha-subunit.

The glycoprotein hormones are a family of alpha beta heterodimeric proteins which are responsible for gonadal and thyroid function. In previous studies we employed chimeric glycoprotein hormone beta-subunits to identify amino acid residues critical for binding to receptors and antibodies. To facilitate similar studies of the alpha-subunit of these hormones, we assembled a 406 bp synthetic gene which encodes the human alpha-subunit leader sequence and the secreted portion of the bovine alpha-subunit. It contains unique restriction sites that can be used for cassette mutagenesis or for making human/bovine alpha-subunit chimeras. The gene was assembled from eight long oligodeoxynucleotides in a single ligation and its structure verified by DNA sequencing. Co-transfection of COS-7 cells with the synthetic gene and the cDNA for human chorionic gonadotropin (hCG) beta-subunit resulted in the secretion of a functional alpha beta heterodimer which bound to luteinizing hormone receptors. The protein was recognized by several monoclonal antibodies including B109, an antibody to a conformational epitope which binds hCG but not the free bovine alpha-, human alpha-, or hCG beta-subunits. This suggests that the binding site for B109 may be formed by residues located primarily within the hCG beta-subunit and that formation of this epitope requires a change in conformation of the beta-subunit when it combines with the alpha-subunit.     

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.     

Effect of additional N-glycosylation signal in the N-terminal region on intracellular function of the human gonadotropin alpha-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. The alpha-subunit has two N-glycosylation sites at Asn52 and Asn78. To obtain more information on the relationship between the structure and function of the alpha-subunit, we introduced a novel N-glycosylation site in the N-terminal region by mutating Asp3 and Gln5 into Asn and Thr, respectively. Glycosylation mutants were expressed alone or with hCGbeta-subunit in Chinese hamster ovary cells. New N-linked oligosaccharides were efficiently added to the wild-type and mutant alpha-subunits lacking N-glycan at Asn52 (alpha deltaAsn1), Asn78 (alpha deltaAsn2), and both (alpha deltaAsn(1 + 2)). The new sugar chain did not affect secretion and assembly except that 1) it increased the intracellular degradation of alpha deltaAsn(1 + 2), and 2) it augmented the assembly of alpha deltaAsn1 with hCGbeta-subunit. Amino acid changes generated the attachment of O-glycosylation in free alpha-subunit but not in assembled form. These data indicate that the newly introduced N-glycosylation consensus sequence is functional, and that the N-terminal region of the alpha-subunit is flexible and can be modified without affecting the intracellular function. Furthermore, amino acid sequences in the N-terminus are involved in the O-glycosylation in free alpha-subunit.     

Use of a mutant cell line to study the kinetics and function of O-linked glycosylation of low density lipoprotein receptors.

A rapidly reversible defect in protein O-glycosylation exhibited by a line of mutant Chinese hamster ovary (CHO) cells was used to study the kinetics and function of O-glycosylation of the low density lipoprotein (LDL) receptor. The mutant line, genotype LDLD, cannot synthesize UDP-N-acetylgalactosamine under normal culture conditions and, therefore, cannot add mucin-type O-linked oligosaccharides to proteins. The UDP-N-acetylgalactosamine pools in LDLD cells can be filled rapidly when N-acetylgalactosamine is added to the culture medium, thus restoring normal synthesis of O-linked carbohydrates. Pulse-chase metabolic labeling experiments were used to show that (i) the first step in the O-glycosylation of LDL receptors can occur posttranslationally; (ii) after O-linked sugar-deficient LDL receptors reach the cell surface, they are not subject to subsequent O-linked sugar addition, suggesting that they do not return to compartments in which O-glycosylation takes place; (iii) O-linked carbohydrate chains on the LDL receptor itself are required for normal stability and function; and (iv) the instability of the O-linked sugar-deficient LDL receptor is due to proteolytic cleavage and the release into the medium of the bulk of the NH2-terminal extracellular domain of the receptor. It appears that O-glycosylation of the LDL receptor and several other cell surface glycoproteins permits stable cell-surface expression by preventing proteolytic cleavage of the extracellular domains of these proteins.     

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common alpha-subunit and distinct beta-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGbeta have been separately fused to the N-terminus of the alpha-subunit to give N-des[1-91]hCGbeta-alpha-C and N-des[1-91,110-114]hCGbeta-alpha-C, i.e. fusion proteins of the hCGbeta determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the alpha-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-beta-alpha cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-beta-alpha was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-beta-alpha fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGbeta in addition to the determinant loop/seat belt.     

Fragmentation of the beta-subunit of human chorionic gonadotropin produced by choriocarcinoma

When human chorionic gonadotropin (hCG) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions with dithiothreitol (DTT), a smaller weight material (CTP'), in addition to the beta-subunit, could be detected by Western blot analysis using antiserum for hCG beta-carboxy-terminal peptide (CTP). The CTP' band was much more apparent with urinary hCG from a patient with choriocarcinoma than with that from normal pregnant women. Second-dimensional electrophoresis of the choriocarcinoma hCG (c-hCG) after reduction with DTT indicated that the CTP', Mr 25,000, was released from the beta-subunit. The carbohydrate structure of the CTP' was analyzed by affinity with lectin-peroxidase on a nitrocellulose membrane. The CTP' did not interact with Concanavalin A, but exhibited strong interaction with both RCA120 and Arachis hypogaea after removal of sialic acid, indicating that it was released as a fragment containing an O-linked sugar chain as was found in the hCG beta carboxy-terminal portion. Western blot analysis using the antisera for hCG alpha, hCG beta, and hCG beta-CTP showed that the CTP' contains not only the carboxy-terminal portion but also a part of the internal (core) portion of the beta-subunit molecule. This dissociation of the c-hCG beta was further supported by the presence of a faster moving component (FMC) which may correspond to the NH2-terminal side counterpart. The desialylated FMC could be detected by Concanavalin A and RCA120 but not by Arachis hypogaea, indicating that it contains N-linked rather than O-linked sugar chains. The FMC does not contain any of the epitopes for the antisera examined in Western blot. These results indicate that the beta-subunit of the choriocarcinoma urine hCG has an unusual site which is dissociated into two components of Mr 25,000 (CTP') and Mr 18,000 (FMC) by DTT reduction.     

Regulation of the alpha and thyrotropin beta-subunit messenger ribonucleic acids by thyroid hormones

We studied the regulation of mRNAs encoding the alpha- and beta-subunits of TSH by thyroid hormones (T4 and T3) in mouse thyrotropic tumors and pituitary glands. Hypothyroid male (LAF1) mice bearing thyrotropic tumor (TtT97) were injected daily with T4 for 0, 1, 5, 12, or 33 days. After day 33, plasma levels of TSH and free (unassociated) TSH beta-subunit were reduced to less than 1% of control levels, whereas free alpha-subunit was reduced to 6% of control levels. Steady state levels of subunit mRNAs in extracts of the thyrotropic tissues were measured by blot hybridization analyses using mouse subunit-specific cloned cDNAs. Treatment of mice with T4 caused a rapid decline in the levels of tumor mRNAs for both alpha and TSH beta; after day 1, alpha and TSH beta mRNA levels decreased to 35% and 10% of control values, respectively. Levels of TSH beta mRNA were undetectable after 5 days of T4 treatment, whereas levels of alpha-subunit mRNA remained at 30-35% of control levels even after day 33. In a separate experiment, TSH beta mRNA decreased to 42% of the control level (P less than 0.05), whereas alpha-subunit mRNA remained at 64% of the control level (P = NS) 4 h after a single injection of T4. Finally, T3 also caused a rapid decrease in the levels of both subunit mRNAs in the anterior pituitary glands of hypothyroid mice, but the effect was more complete on TSH beta mRNA levels. We conclude that thyroid hormones have rapid suppressive effects on the levels of mRNAs encoding the subunits of mouse TSH in the thyrotrope. The suppressive effects of thyroid hormones occur more rapidly and are greater for TSH beta than alpha-subunit mRNAs. The parallel changes observed in the subunit mRNA levels and the plasma subunit protein levels in animals treated with thyroid hormones suggest that the changes in the plasma levels of TSH and subunits may reflect effects of thyroid hormones on TSH gene expression in addition to effects on secretion.     

Development and in vitro characterization of human recombinant thyrotropin.

We have gained insight into the molecular mechanism of human thyrotropin (hTSH) action through cloning of the human TSHbeta subunit gene, development of recombinant TSH and novel analogues and chimeras produced by site-directed as well as cassette mutagenesis. A variety of loss of function mutations have shown several key domains in both the alpha- and beta-subunits that are important for high-affinity ligand interaction with the receptor. In contrast the specificity of receptor interaction was shown to be determined primarily by areas within the hTSH-beta "seat-belt" region. We have also designed various gain of function mutants (superagonists) using evolutionary considerations, homology modeling, and sequence comparisons within the cystine knot growth factor superfamily. Such superagonists resulted from increasing the positive charge by introduction of lysine or arginine residues or neutralization of negatively charged residues of the peripheral hairpin loops of each subunit in various combinations. Certain superagonists increased receptor binding, in vitro and in vivo bioactivity 100- to 1000-fold, more than that achieved previously for any other known protein ligand. In vivo metabolic clearance and biologic activity could be separately modulated by alteration of TSH carbohydrate structure including production of chimeras that added sites of O-glycosylation and/or covalently linked the alpha- and beta-subunits. These data suggest that electrostatic interactions resulting from net positive charge in TSH and net negative charge in its receptor play an important role in high-affinity TSH receptor binding and signal transduction. Insights gained from the design of such novel recombinant TSH analogues and chimeras should have many diagnostic and therapeutic applications. These include the design of improved in vitro assays for thyrotropic factors as well as the design of second generation recombinant TSH analogues for the detection and treatment of thyroid cancer.     

Heterogeneity of human luteinizing hormone. Effect of neuraminidase treatment on biologically active hormone and alpha- and beta-subunits

By preparative isoelectric focussing of a highly purified LH preparation in a sucrose density gradient, four biologically active LH components were isolated. The effect of neuraminidase treatment of each component on the charge heterogeneity was studied by isoelectric focussing followed by in vitro biological and immunochemical techniques. The number of biologically active components with pI-values greater than 7 containing varying amounts of sialic acid is at least six. The pI-value of the most basic (= asialo) LH component was 9.26. The two most basic components were not present in our preparation (NM14) before neuraminidase treatment. It is concluded that the difference between the pI-values of LH components is caused by a difference in sialic acid content. When an intact LH component was incubated with neuraminidase there was detectable dissociation owing to the elevated temperature (37 degrees C) and necessary acidic conditions of the incubation. Under these conditions we found the same subunits as we have described before. The most basic alpha-subunit had a pI-value of 9.29, whereas two beta-subunits with pI greater than 9 were observed at pI 9.26 and pI 9.9. On the other hand, when an LH component was forced to dissociate by incubation at 56 degrees C prior to neuraminidase digestion, two additional alpha-subunits were found. From this it is concluded that in the intact LH molecule, some sialic acid residues are poorer substrates for neuraminidase action than in the free subunits.     

The role of the oligosaccharide chains of thyrotropin alpha- and beta-subunits in hormone action.

TSH, a dimeric glycoprotein hormone, has two N-linked complex-type oligosaccharide chains on the alpha-subunit and one on the beta-subunit. The oligosaccharide chains of TSH are important for the expression of hormonal activity, but the contribution of those on each of the subunits to the activity is not clear. In the current study we have determined the relative importance of the oligosaccharide chains of TSH subunits using a recently reported method of enzymatic deglycosylation. The alpha- and beta-subunits of bovine TSH were deglycosylated with endoglycosidase-F and recombined to obtain differentially deglycosylated TSH. The derivatives showed no differences in their receptor-binding activities. The in vitro bioactivity of these derivatives was assessed by measuring adenylyl cyclase activity in bovine thyroid membranes and stimulation of cAMP production and growth in FRTL-5 cells. In the adenylyl cyclase assay, deglycosylation of the alpha-subunit alone had a more profound effect on the activity [maximal stimulatory activity (Vmax), 13% that of alpha.beta) than when the beta-subunit alone was deglycosylated (Vmax, 50% that of alpha.beta). In the FRTL-5 assays, removal of carbohydrate from TSH alpha, but not the beta-subunit, caused a 2- to 3-fold increase in the concentration required for half-maximal stimulation, with minimal change in the apparent Vmax. The adenylyl cyclase assay in bovine membranes was more sensitive to carbohydrate removal than the assays of rat FRTL-5 cells, in which the derivatives with lower activity were able to stimulate cAMP and growth to near-maximal levels, albeit at 3-fold higher concentrations. These results indicate that the carbohydrate chains in both subunits of TSH, particularly those in the alpha-subunit, are important in hormone action. In contrast to previous reports, our study shows that the beta-subunit plays a role in signal transduction.     

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.     

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.     

Serum "big insulin-like growth factor II" from patients with tumor hypoglycemia lacks normal E-domain O-linked glycosylation, a possible determinant of normal propeptide processing.

The insulin-like growth factor II (IGF-II) gene is overexpressed in many mesenchymal tumors and can lead to non-islet-cell tumor hypoglycemia (NICTH). ProIGF-II consists of the 67 aa of IGF-II with a carboxyl 89-aa extension, the E domain. A derivative of proIGF-II containing only the first 21 aa of the E domain [proIGF-II-(E1-21)] has been isolated by others from normal serum and has O-linked glycosylation. We found that the "big IGF-II" of normal serum, as detected by an RIA directed against residues 1-21 of the E domain of proIGF-II, was reduced in size by treatment with neuraminidase and O-glycosidase. The big IGF-II, which is greatly increased in NICTH sera, was unaffected by neuraminidase and O-glycosidase treatment. We have also shown that big IGF-II from normal serum is retained by jacalin lectin columns and that big IGF-II from NICTH serum was not retained, indicating that it lacked O-glycosylation. Normal O-linked glycosylation may be required for proper peptidase processing of proIGF-II. The lack of normal O-linked glycosylation by tumors may explain the predominance of big IGF-II in NICTH sera. In normal serum, most of the IGF-II is present in a 150-kDa ternary complex with IGF-II binding protein (IGFBP) 3 and alpha subunit. In NICTH serum, however, the complexes carrying big IGF-II are < 50 kDa. We investigated whether big IGF-II of NICTH was responsible for this abnormality. Tumor big IGF-II and IGF-II were equally effective in forming the 150-kDa complex with purified IGFBP-3 and 125I-labeled alpha subunit. Both 125I-labeled IGF-II and 125I-labeled proIGF-II-(E1-21), when incubated with normal serum, formed the 150-kDa complex as detected by Superose 12 exclusion chromatography. We conclude that the nonglycosylated big IGF-II of NICTH serum can form normal complexes with serum IGFBPs. The defective binding in NICTH is attributable to defective IGFBP-3 binding.     

Conformation of the alpha-subunit of glycoprotein hormones: a study using polyclonal and monoclonal antibodies.

The conformation of the common alpha-subunit of human glycoprotein hormones, luteinizing hormone (hLH), follicle-stimulating hormone (hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) was probed using a highly specific polyclonal antiserum against the alpha-subunit of hCG and several monoclonal antibodies (MAbs) produced against hCG which recognized the alpha-subunit in free and combined form. The alpha-subunit was found to be conformationally altered (compared to its conformation in the isolated state) when it was in combination with various beta-subunits as indicated by shifts in the displacement curves of binding of [125I]hCG alpha to the polyclonal antiserum. The extent of the change was dependent on the beta-subunit present with minimum change being observed with hLH beta, intermediate with hCG beta and maximum change with hFSH and TSH beta-subunits. However, the affinity constants of this antiserum for all four hormones were nearly similar. Further, it was also found that binding of any one of the glycoprotein hormones to this antibody could be completely inhibited by any other hormone suggesting that the conformation of the alpha-subunit in all the four hormones is probably very similar. This was further investigated using five hCG MAbs capable of recognizing the alpha-subunit, but with different epitope specificities. All these MAbs could recognize all the four hormones suggesting the presence of the epitopes in these proteins. These epitopes were conformation specific since the MAbs did not bind reduced and carboxymethylated alpha-subunit. Displacement analysis using [125I]hCG as the tracer showed that two epitopes have nearly the same conformation in all the four hormones, while two were partially modified depending on the beta-subunit present. Based on these results, it is concluded that the alpha-subunit of glycoprotein hormones has nearly the same conformation, though subtle differences do exist.