Heterogeneity of free alpha-subunit in term placenta

Free alpha-subunit in normal term placenta was examined for molecular weight, electric charge and ability to combine with standard hCG-beta in comparison with extracellular free alpha-subunit and standard hCG-alpha dissociated from urinary hCG in vitro. The gel chromatography on Sephadex G-100 of the placental extract revealed three major immunoreactive hCG-alpha peaks, designated as P alpha-A (Kav = 0.32-0.46), P alpha-B (0.47-0.58) and P alpha-C (0.59-0.70), near the position of standard hCG-alpha. In the isoelectric focusing, while P alpha-A was mainly distributed over the acidic region, the major components of P alpha-B and P alpha-C were distributed over the basic region. Furthermore, in the combination study with standard hCG-beta, such a alpha-subunit with acidic pI scarcely showed any combining activity whereas alpha-subunit with basic pI revealed significant combining activity. These results suggest the following possibilities: that 1) the various size species of placental alpha-subunit may be responsible for the progressive glycosylation; 2) the small alpha-subunit with basic pI may combine with beta-subunit to form immunoreactive hCG; 3) the alpha-subunit, which has not associated with beta-subunit, may be converted to a large and incombinative form with acidic pI by further glycosylation, followed by secretion as a free alpha-subunit.     

Purification and characterization of the glycoprotein hormone alpha-subunit-like material secreted by HeLa cells

The protein secreted by HeLa cells that cross-reacts with antiserum developed against the alpha-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10(5) ng of alpha/mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-alpha had a composition very similar to that of the urinary hCG alpha-subunit. Peptide fingerprints of the HeLa protein and hCG-alpha revealed that several of the Tyr-, Met-, and Cys-containing tryptic peptides were held in common, thus identifying the tumor protein as a glycoprotein hormone alpha-subunit with a primary structure similar to that of hCG-alpha. However, comparison of hCG-alpha and HeLa-alpha demonstrated that the tumor-associated subunit was not identical with its normal counterpart. Only two of the three Tyr-containing tryptic peptides present in hCG-alpha could be detected in HeLa-alpha after iodination with 125I. HeLa-alpha eluted prior to hCG-alpha during Sephadex G-75 chromatography, but the subunits coeluted when the tumor protein was first subjected to mild acid hydrolysis. The purified tumor protein had an apparent molecular weight greater than that of the urinary alpha-subunit when analyzed by SDS-PAGE (Coomassie blue staining), and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI (4.7-5.5 compared to 6.5-7.8), and removal of sialic acid by mild acid hydrolysis did not entirely eliminate this difference. Immunoprecipitation and electrophoresis of alpha-subunit from HeLa cultures labeled with [3H]fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-alpha hydrolysates by HPLC confirmed previous reports that the placental subunit does not contain fucose. HeLa alpha-subunit was unable to combine with hCG beta-subunit to form holo-hCG under conditions where the hCG alpha-subunit was able to do so. The results indicate that, regardless of whether or not a single alpha-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors     

Origin and occurrence of human chorionic gonadotropin beta-subunit core fragment

Human chorionic gonadotropin (hCG) beta-subunit core fragment (beta-fragment) is present in the urine of pregnant individuals as well as those with trophoblast disease and certain other cancers at concentrations 0.8 (early pregnancy) to 7 (second trimester pregnancy)-fold greater than that of hCG. The core fragment may be directly secreted by trophoblast tissue into the circulation or possibly originates from peripheral degradation of circulating hormone by the kidney. We examined the former hypothesis. We examined 24-h organ cultures of trophoblast tissue from first, second, and third trimester pregnancy. The media from this tissue contained hCG, free beta-subunit, and beta-fragment. The amount of beta-fragment present exceeded that of hCG, as was observed in second and third trimester pregnancy urine. The beta-fragment immunoreactive material produced by trophoblast tissue was compared to a standard preparation of urinary beta-fragment. The material in medium was identical to the standard beta-fragment in its elution pattern from a gel filtration column, from a reverse-phase HPLC column, from an ion-exchange gel, and from an immobilized lectin affinity column, and also by electrophoresis and immunoblotting with fragment-reactive monoclonal antibodies. We conclude that beta-fragment can also originate directly from trophoblast tissue, and could be the principal hCG beta-immunoreactive molecule secreted.     

Characterization of glycoprotein hormone free alpha-subunit from human pituitary and placenta extracts

Standard alpha-subunit dissociated from glycoprotein hormones differs from individual (free) alpha-subunit found in sera or in cell culture media; secreted free alpha-subunit is larger, more acidic and lacks the ability of recombining in vitro with standard hCG-beta. It is unclear whether the large free alpha-subunit is only a secretory product or whether it is also present in tissue. Herein were studied the molecular size, the isoelectric pH, and the recombining activity of free alpha-subunit obtained from pituitary and placenta extracts. Sephadex exclusion chromatography showed the presence of both a large and a small form, and a changing large/small free alpha-subunit ratio in the various extracts. Most of the large form obtained from placenta extracts electrofocused into two peaks of pI 4.8 and 5.1. The large form showed no recombining activity with standard hCG-beta while the small free alpha-subunit recombined as well as did standard hCG-alpha. The observation of three common characteristics (a larger size, a pI 4.8, and a lack of recombining activity) suggests a similarity between the large secreted form and a fraction of the free alpha-subunit in tissue.     

Antigenic determinants on human choriogonadotropin alpha-subunit. I. Characterization of topographic sites recognized by monoclonal antibodies

Immunochemical studies were designed to localize antigenic regions recognized by two monoclonal antibodies directed against the alpha-subunit of human choriogonadotropin (hCG-alpha) and to provide information on the three-dimensional structure of hCG and its alpha-subunit. Monoclonal antibody HT13 bound to a region accessible on both hCG and the free alpha-subunit, whereas monoclonal antibody AHT20 recognized a site localized only on the free alpha-subunit. By studying the cross-reactivity of these antibodies to homologous proteins, we found that antibody HT13 did not bind to equine or ovine lutropin, whereas AHT20 was capable of binding to both subunits. This observation suggests that AHT20 recognized a structurally related antigenic determinant on alpha-subunits of different species. To delineate the portions of hCG-alpha contributing to the antigenic determinants of AHT20 and HT13, we performed competitive inhibition assays using reduced and carboxymethylated hCG-alpha, deglycosylated hCG-alpha, hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core 1), or disulfide-bridged peptides comprising residues 1-35 and 52-91 of hCG-alpha (hCG-alpha core 2). Reduced and carboxymethylated hCG-alpha did not inhibit the binding of 125I-labeled hCG-alpha to both antibodies, whereas deglycosylated hCG-alpha was as active as hCG-alpha, suggesting that antigenic determinants of both antibodies are mainly discontinuous and do not reside on the oligosacharide part of the alpha-subunit. hCG-alpha core 1 had the same capacity as intact hCG-alpha to inhibit the binding of 125I-hCG-alpha to both antibodies, indicating that the 5 COOH-terminal residues of hCG-alpha do not participate in the antigenic determinants. hCG-alpha core 1 was as potent as hCG-alpha in inhibition experiments performed with HT13, whereas, in striking contrast, hCG-alpha core 2 did not compete with 125I-hCG-alpha for binding to AHT20, suggesting that the peptides released after proteolysis of the alpha-subunit by trypsin participate in the epitope of AHT20 and are not included in the antigenic determinant of HT13. In an attempt to elucidate the amino acid residues constituting the antigenic sites of HT13 and AHT20, hapten inhibition experiments were carried out using as competitive inhibitors five different synthetic peptides spanning the primary structure of hCG-alpha. None of these peptides inhibited the binding of 125I-hCG-alpha to HT13. In contrast, two peptides analogous to regions 23-43 and 33-59 of hCG-alpha exhibited significant potency in competing with 125I-hCG-alpha for binding to AHT20.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antigenic determinants on human choriogonadotropin alpha-subunit. II. Immunochemical mapping by a monoclonal antipeptide antibody

In order to study antigenic site(s) present in the carboxyl-terminal part of the alpha-subunit of human choriogonadotropin (hCG-alpha), we attempted to produce site-specific antibodies directed against a 34-residue synthetic peptide analogous to region 59-92 of hCG-alpha. From a fusion experiment performed with a mouse injected with hCG-alpha-(59-92)-peptide conjugated to tetanus toxoid as immunogen, we selected a monoclonal antipeptide antibody (designated FA36) which has high binding activity for 125I-hCG-alpha but not for 125I-hCG in a radioimmunoassay. This antibody is of the IgG1 subclass and displays an affinity constant for 125I-hCG-alpha of 3.1 x 10(8) M-1. Hapten inhibition experiments performed by either radioimmunoassay or enzyme-linked immunosorbent assay with synthetic peptides spanning different portions of the region (59-92) demonstrated that the binding site of FA36 resides on (minimally) the six COOH-terminal amino acids of hCG-alpha, namely Cys-Tyr-Tyr-His-Lys-Ser, and that FA36 binds preferentially to peptides containing a carboxyl group on the COOH-terminal residue. Monoclonal immunoradiometric assays were established to determine the location of antigenic regions recognized by FA36, by antibody AHT20 (which binds only to hCG-alpha), and by antibody HT13 (which binds to both hCG and hCG-alpha). FA36 has the capacity to bind to hCG-alpha bound to either AHT20 or HT13, demonstrating that both AHT20 and HT13 antibodies are directed against antigenic regions distinct from the epitope of FA36. Monoclonal immunoradiometric assays were also carried out to study the binding of FA36 to hCG, the ovine and equine lutropin alpha-subunit, or hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core). Whereas significant binding of 125I-FA36 was observed with the ovine lutropin alpha-subunit, no binding was found with the equine lutropin alpha-subunit. As expected, FA36 did not bind to hCG-alpha core. Binding was also not detected with hCG, confirming that FA36 is specific for free hCG-alpha and that the COOH-terminal part of hCG-alpha is either weakly or (more likely) not at all accessible in the alpha/beta-dimer for antibody binding. Finally, immunoblots performed on hCG-alpha-(59-62)-peptide and various denatured alpha-subunits indicated that, with the exception of the equine lutropin alpha-subunit, FA36 detected various denatured alpha-subunits and particularly the alpha-subunit of carp gonadotropin-thyrotropin. This latter observation suggests a high degree of homology between the COOH-terminal regions of the alpha-subunits of fish gonadotropin and analogous mammalian hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.     

Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein.

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.     

Beta-subunit of bovine rod photoreceptor cGMP phosphodiesterase. Comparison with the phosphodiesterase family

A group of cDNA clones encoding the beta-subunit of bovine rod photoreceptor cGMP phosphodiesterase were isolated for structural analysis. The encoded polypeptide has 853 residues with a calculated molecular mass of 98 kDa. The beta-subunit is 72% identical to the rod cGMP phosphodiesterase alpha-subunit. Like the alpha-subunit and the cone alpha'-subunit, the beta-subunit belongs to the family of phosphodiesterase genes. The beta- and alpha-subunits are more similar to each other than either is to the cone alpha'-subunit, suggesting either that the beta- and alpha-subunits diverged more recently or that their divergence was restrained by the rod functional environment.     

Release of human chorionic gonadotropin (hCG) and its alpha-subunit (hCG-alpha) from perifused human placenta

The release of human chorionic gonadotropin (hCG) and its alpha-subunit (hCG-alpha) from the normal human placenta and the effect of some stimulatory agents on their release were studied in vitro using a perfusion system. Each perfusate was assayed for hCG and hCG-alpha in its own homologous radioimmunoassay systems. Both hCG and hCG-alpha were released from the placenta at any stage of gestation in our perfusion system. Much more hCG than hCG-alpha was released from the placenta in early gestation. By comparison, however, hCG-alpha increased gradually with the gestational age. The amount of hCG-alpha released was almost equal to that of hCG in the placenta in the 17th gestational week. After the 22nd gestational week, hCG-alpha was released in larger quantities than hCG, and about 10 times more hCG-alpha than hCG was released from the term placenta. These results were also confirmed by gel filtration of perfusates on a Sephadex G-100 column. hCG-alpha, compared with hCG, was present in excess in gel filtrated perfusates in the last two trimesters. By adding 1 mM dibutyryl cyclic AMP to the perifusion medium, the release of both hCG and hCG-alpha was stimulated significantly. Synthetic luteinizing hormone releasing hormone (LH-RH) at concentrations of 10 ng/ml and 100 ng/ml had no effect, but at a high concentration (1 microgram/ml), LH-RH stimulated the release of them. Moreover, mouse epidermal growth factor (EGF) stimulated not only the release of hCG and hCG-alpha but also their production, because both hCG and hCG-alpha levels rose progressively with the time course in the presence of EGF. The present studies demonstrate that the perifusion system of chorionic tissues is a useful method for investigating the release of hCG and its subunits in vitro.     

Disruption of N-linked glycosylation of bovine luteinizing hormone beta-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer

The single site for N-linked glycosylation of the beta-subunit of bovine LH (LH beta) was disrupted by oligonucleotide-directed mutagenesis to assess its potential roles in the biosynthesis, transport, and hormonal activity of the LH alpha/beta heterodimer. Pulsechase studies performed with stably transfected Chinese hamster ovary cells that expressed both alpha-subunit (fully glycosylated) and nonglycosylated LH beta revealed that turnover, transport, and secretion of newly synthesized, nonglycosylated LH beta were effectively blocked over a 22-h span. Free nonglycosylated LH beta, like free wild-type LH beta, was sequestered inside the cell; therefore, the intracellular retention of uncombined LH beta-subunit is not due to a signal located within the N-glycan moiety. Nevertheless, an older pool of unlabeled, nonglycosylated LH beta-subunit was available for combination with newly synthesized alpha-subunit, as verified by immunoprecipitation of radiolabeled alpha-subunit from cell lysates and culture medium with anti-LH beta-antiserum. This heterodimer displayed normal kinetics of secretion (t 1/2 = 2.4 h) as compared to fully glycosylated LH (t 1/2 = 2.1 h). The wild-type and mutant forms of LH were also purified from culture supernatants of the two cell lines, and were compared for their relative abilities to stimulate progesterone secretion in cultured rat Leydig cells. Both proteins displayed similar potency (ED50 = 32 vs. 41 ng/ml, respectively) and maximal stimulation of progesterone release Pmax = 2.7 vs 2.5 micrograms/ml), indicating that N-linked glycosylation of the LH beta-subunit does not play a significant role in LH signal transduction. Collectively, these results indicate that N-linked glycosylation is important for intracellular degradation of free LH beta, but is not essential for either its assembly with alpha-subunit or the transport and secretion of biologically active heterodimer.     

Translation of beta-tubulin mRNA in vitro generates multiple molecular forms

We describe the in vitro expression and characterization of the isolated beta-tubulin subunit in rabbit reticulocyte lysates and compare its assembly and chromatographic properties with that of the isolated alpha-subunit and the tubulin heterodimer. The beta-tubulin polypeptides, derived from a single chicken beta-tubulin cDNA, were found in three distinct molecular forms: a multimeric or lysate-associated form, beta I (Mr approximately 180,000); the free beta-subunit beta II (Mr approximately 55,000); and the hybrid heterodimer alpha(rabbit) beta(chick), beta III (Mr approximately 80,000-100,000). The hybrid heterodimers were 100% assembly competent, whereas beta-tubulin in the "associated" beta I and the monomeric beta II forms displayed only approximately 70 +/- 15 and 25 +/- 10% competence, respectively, in coassembly assays with bovine brain tubulin. This reduced functionality was not a consequence of diminished beta-subunit stability or protein denaturation. By comparing the elution positions of the three beta forms, the monomeric alpha-subunit, and tubulin dimer purified from bovine brain, we demonstrate that anion-exchange columns (Mono-Q) interact preferentially with the alpha-subunit and chromatograph tubulin dimer on the basis of alpha-subunit isotype. The rate of exchange of the free beta-subunit into bovine tubulin dimer was followed chromatographically. The exchange was slow at 4 degrees C and rapid at 37 degrees C where it is essentially complete in 40 min in the presence of 2.5 mg/ml bovine microtubule protein. Exogenous GTP, a potent effector of microtubule assembly, binds exchangeably to beta II and enhances the recovery of this form from the Mono-Q column, suggesting that GTP binding may occur at identical sites in the isolated beta-subunit and in the tubulin heterodimer.     

The glycoprotein alpha-subunit is critical for secretion and stability of the human thyrotropin beta-subunit

TSH is a member of a family of heterodimeric glycoprotein hormones which have a common alpha-subunit but differ in their hormone-specific beta-subunit. To study the posttranslational processing and assembly of human TSH, eukaryotic expression vectors were constructed that contained either the human TSH beta gene only or both the TSH beta and alpha-genes. These vectors were transfected into Chinese hamster ovary cells and stable cell lines synthesizing TSH beta or TSH dimer were isolated. The kinetics of secretion of TSH beta and the rate of assembly of TSH dimer were compared to the known secretion and assembly of human LH and human CG. In the absence of the alpha-subunit, CG beta is secreted efficiently, but TSH and LH beta-subunits are slowly degraded intracellularly (t1/2 approximately equal to 6 h) and less than 10% is secreted into the medium. In the presence of the alpha-subunit CG beta was also secreted efficiently as dimer but only 50% of the LH beta appeared in the medium as LH dimer. However, unlike LH beta, the alpha-subunit efficiently combines with TSH beta since greater than 95% was secreted as TSH dimer. Thus, the determinants for human TSH beta secretion and assembly are unique from the other human glycoprotein hormone beta-subunits.     

Influence of cyclic nucleotides on the processing of the carbohydrate part of the alpha-subunit of human choriogonadotropin by first trimester human placenta tissue

In pulse-chase experiments ([35S]Met as radioactive label) 4 intracellular forms of the alpha-subunit (apparent molecular weights of 11, 16.5, 19.5, and 23.4 kDa) were observed whereas almost no label was incorporated into the beta-subunit. The 23.4 kDa form was secreted as free alpha-subunit, the others were precursors of the alpha-subunit contained in secreted human choriogonadotropin. The rate-limiting step seemed to be the processing of the 19.5 kDa precursor by alpha-mannosidase II. 8-bromo-cAMP increased the total amount of intracellular forms of the alpha-subunit and accelerated significantly the velocity of all glycosylation steps. It seemed to cause a higher efficacy of the alpha-mannosidase II reaction. In the presence of 8-bromo-cAMP intracellular as well as extracellular alpha-subunits showed a higher sialic acid content.     

Biosynthesis, assembly and secretion of fibrinogen in cultured rat hepatocytes.

The biosynthesis, assembly and secretion of fibrinogen were investigated in cultured rat hepatocytes which were incubated with [35S]methionine. When initial rates of the synthesis of three fibrinogen subunits were compared, the A alpha-subunit was found to be synthesized significantly slower than the B beta- and gamma-subunits. Pulse-chase experiments revealed that the secreted fibrinogen contained different proportions of the newly synthesized subunits, depending upon the chase times. Radioactivity in the A alpha subunit, which initially had the highest level of the three, was rapidly decreased in parallel with the chase time. The gamma-subunit had an increasing amount of the radioactivity in the secreted molecule during the chase periods, whereas that in the B beta-subunit was gradually decreased at the later stages of chase. Analysis of intracellular components of fibrinogen confirmed that the nascent A alpha-subunit was most rapidly exhausted, and the gamma-subunit occupied the largest proportion among the non-assembled subunits at later stages of chase. Taken together, these results suggest that the synthesis of A alpha-subunit, which has the lowest rate, could be the rate-limiting step in the production and secretion of fibrinogen in cultured rat hepatocytes, in contrast with what has been proposed for human and rabbit fibrinogen, namely that the synthesis of B beta-subunit is the rate-limiting step. The results also indicate that there is a large intracellular pool of gamma-subunit.     

The clinical evaluation of the simultaneous measurements of human chorionic gonadotropin (hCG) and its alpha-subunit in sera of patients with trophoblastic diseases

The concentrations of human chorionic gonadotropin (hCG) and its free immunoreactive alpha-subunit (hCG-alpha) in the sera of patients with trophoblastic diseases were measured by hCG and hCG-alpha radioimmunoassay (RIA), respectively. In the sera of 12 women with hydatidiform mole large amounts of hCG and considerably high level of hCG-alpha were detected in all cases. After the evacuation of mole the serum level of these glycoproteins decreased, the leve of hCG-alpha declined more rapidly than hcg. in the sera of patients with destructive mole the concentration of hCG-alpha was usually lower than that of hCG. After hysterectomy and chemotherapy the levels of hCG-alpha declined practically paralleling that of hCG. However, when hCG had decreased to undetectable level, hCG-alpha could no longer be detected in all cases. Although in the serum of patient with choriocarcinoma involving the uterus and lungs the concentration of hCG-alpha was almost as high as that of hCG, the secretory pattern of hCG and hCG-alpha might not be closely related. The changes in the serum level of free hCG-alpha as well as that of hCG parelled the clinical course of the patients examined in this study. The present results suggest that measurements of the serum free hCG-alpha may be a useful parameter to follow the clinical course and to evaluate the efficacy of treatments of trophoblastic diseases.     

Phosphorylation of the glycoprotein hormone alpha-subunit secreted by human tumor cell lines

The synthesis of ectopic proteins by tumors is thought to result from derepression of normally silent genes. One approach to a better understanding of this phenomenon is to characterize the physicochemical properties of the ectopic products, comparing them to their normal counterparts. In the following communication, evidence will be presented to indicate that the glycoprotein hormone alpha-subunits secreted by a number of human tumor cell lines are phosphorylated. This novel covalent modification occurs in cell lines derived from both trophoblastic (JAR, JEG) and nontrophoblastic (HeLa, ChaGo) tumors. A choriocarcinoma cell line (JAR), which secretes both hCG-alpha and hCG-beta, phosphorylates only the alpha-subunit.     

Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.     

Homodimers of mutant tryptophan synthase alpha-subunits in Escherichia coli.

Tryptophan synthase alpha-subunit from Escherichia coli functionally exists as a heterotetramer of alpha(2)beta(2) with beta-subunit. While wild-type and mutant (F139W, T24M/F139W, and T24L/F139W) alpha-subunits were expressed as a monomer from recombinant plasmids in Escherichia coli, T24A/F139W, T24S/F139W, and T24K/F139W mutant alpha-subunits were abnormally expressed as soluble homodimers in addition to monomers. Monomers of dimer-forming mutant alpha-subunits retain high affinity to beta-subunit, high activity in stimulating catalytic activities of beta-subunit, and nearly intact content of secondary structure, indicating that the global structures of these monomers are identical to that of F139W alpha-subunit. However, fluorescence spectra of Trp139 and ANS binding indicate that significant perturbations occur in the mutant proteins. Interestingly, these defective properties of monomers caused by residue replacement were partially repaired by the dimer formation. As a result, it is suggested that dimers may be formed by domain or loop swapping, and that residue 24 may play important role in maintaining on-pathway of alpha-subunit folding.