Glycoprotein Hormone Subunits Research Articles

Immunoperoxidase staining of primate pituitaries with antibodies against the beta subunits of human pituitary glycoprotein hormones.

The immunocytochemical specificity of antibodies against the beta subunits of human pituitary glycoprotein hormones was tested on human and monkey hypophyses. Anti-luteinizing hormone (LH) beta stained only gonadotrophic cells in both species, and anti-thyroid-stimulating hormone (TSH) beta only thyrotrophic cells. In monkeys, anti-follicle-stimulating hormone (FSH) beta demonstrated only gonadotrophs, but in man the antibody stained thyrotrophs equally well. Staining of the two cell types was diminished to a similar degree by dilution of the anti-FSH beta antibody. Human gonadotroph heterogeneity was revealed by absorption of anti-FSH beta with LH beta or TSH beta, which abolished immunostaining of a subset of gonadotrophs. Limitations in the immunocytochemical specificity of antibodies against beta subunits of pituitary glycoprotein hormones may be a consequence of incomplete purity of the generating antigens, structural overlap among the beta subunit of the three hormones, or a combination of these.

Cell-free translation of the rat pituitary messenger RNA coding for the precursors of α and β subunits of lutropin

Lutropin is a glycoprotein belonging to a group of pituitary glycoprotein hormones which includes also follitropin and thyrotropin. It is composed of two dissimilar, noncovalently linked subunits, (Y and /3. The amino acid sequences of (Y and fl subunits of glycoprotein hormones from many species have been established and are well known. Within a species, the sequence is identical or nearly identical for (Y subunits of all pituitary hormones. In the human being, it is also identical to that of a placental glycoprotein hormone, chorionic gonadotropin. The amino acid sequence of the hormone-specific fl subunits differs, although considerable homology exists in some portions of their primary structure (reviewed in [ 1 I). Each subunit of LH and hCG contains two N-asparaginelinked oligosaccharides. Astructure has been proposed for asparagine-linked oligosaccharides of human LH and CC; these might be identical [2.3]. Messenger RNA has been extracted from placental tissue and has been translated in vitro by systems derived from wheat-germ embryo, ascites tumors or reticulocyte lysates [4-71. The first evidence for the cell-free synthesis of LHa and LHp from bovine origin by separate mRNAs appeared in [8,9].

Human growth hormone DNA sequence and mRNA structure: possible alternative splicing.

We have determined the complete sequence of the human growth hormone (hGH) gene and the position of the mature 5' end of the hGH mRNA within the sequence. Comparison of this sequence with that of a cloned hGH cDNA shows that the gene is interrupted by four intervening sequences. S1 mapping shows that one of these intervening sequences has two different 3' splice sites. These alternate splicing pathways generate hGH peptides of different sizes which are found in normal pituitaries. Comparison of sequences near the 5' end of the hGH mRNA with a similar region of the alpha subunit of the human glycoprotein hormones reveals an unexpected region of homology between these otherwise unrelated peptide hormones.

Identification of luteinizing hormone-releasing factor and alpha subunit of glycoprotein hormones in ductal carcinoma of the mammary gland.

The immunoperoxidase technique was used to identify luteinizing hormone-releasing factor (LRF) and the alpha subunit of glycoprotein hormones in ductal carcinoma of the mammary gland. Eleven tumors were studied, using antisera against LRF and alpha subunit, and against the beta subunits of hCG, LH, FSH and TSH. Six of the tumors were both LRF- and alpha-positive and three were negative. One tumour was LRF-positive/alpha-negative, and one was LRF-negative/alpha-positive. None of the anti-beta subunit antisera gave positive staining, and normal breast tissue as well as chronic cystic mastopathy (five cases) were negative with all antisera tested. The results with the anti-alpha and anti-beta subunit antisera suggest that the material stained is free alpha subunit rather than any of the native glycoprotein hormones which share the common alpha subunit.

Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit.

The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.

Heterogeneity of the Human Chorionic Gonadotropin α-Subunit Secreted by Cultured Choriocarcinoma (JEG) Cells*

The cultured human choriocarcinoma cell line, JEG-clone 3, secretes substantial quantities of both biologically active hCG and an immunoreactive alpha-subunit (JEG-alpha). This study is concerned with a comparative characterization, using RIA, of the chromatographic properties (via gel exclusion and isoelectric focusing) of JEG-alpha and standard urinary hCG-alpha. Most of the molecules comprising the JEG-alpha fraction have an apparent molecular weight greater than that of hCG-alpha. Both hCG-alpha and JEG-alpha exhibit heterogeneity on electrofocusing. However, JEG-alpha contains a major component with an isoelectric pH (pI) of 4.8; this is a minor component, if present at all, in hCG-alpha. The JEG-alpha pI 4.8 component chromatographs with an apparent molecular weight greater than hCG-alpha, while a minor JEG-alpha pI 7.0 component chromatographs with an apparent molecular weight similar to that of the standard. Heterogeneity is expected in the carbohydrate moieties of the glycoprotein hormone subunits. Results of studies on the incorporation of 14C- and 3H labeled amino acids into JEG-alpha suggest that heterogeneity also exists in the protein moiety of JEG-alpha. An interesting possibility is that the form(s) of JEG-alpha with larger apparent molecular weight represents a precursor of the alpha-subunit used to form hCG.

The circulating alpha subunit of human chorionic gonadotrophin in gynaecologic tumours.

AbstractThe circulating levels of the immunoreactive alpha subunit of glycoprotein hormones were measured by the hCG alpha subunit radioimmunoassay in 101 patients with non‐trophoblastic gynaecological cancer and in 164 serum samples obtained from 10 patients with choriocarcinoma and five patients with hydatidiform mole. Serum samples from six patients with choriocarcinoma and four with hydatidiform mole were further examined for the existence of free alpha by chromatography on a calibrated Sephadex G‐100 column. In patients with non‐trophoblastic cancer the immunoreactive alpha subunit levels were not different from those of controls in the corresponding age groups. In trophoblastic disease all hCG‐positive samples showed also alpha subunit immunoreactivity when tested by radioimmunoassay, but no isolated alpha elevation was found. Chromatographic analyses revealed free alpha subunits in three out of six patients with choriocarcinoma and two out of four patients with hydatidiform mole. A patient who eventually died of choriocarcinoma was carefully followed for the free alpha secretion, and no alpha elevation was found in any of the 16 serum samples taken at various phases of disease. The present study shows that, unlike pancreatic islet cell tumours in vivo and HeLa cells in vitro, non‐trophoblastic gynaecologic cancer in vivo does not appear to have any significant alpha secretion. In trophoblastic disease free alpha subunits may coexist with native hCG more frequently than it was originally assumed, but no isolated alpha secretion could be demonstrated. The intriguing disparity between the alpha secretion by normal and abnormal trophoblasts was confirmed in this study, but the biological basis and significance of this phenomenon remain obscure.

Excess production of free alpha subunits by mouse pituitary thyrotropic tumor cells in vitro.

Primary cultures from three serially transplanted mouse pituitary thyrotropic tumors, Furth 97A, 97B, and 97C, and one newly generated tumor, NIH 101A, were shown to secrete free alpha subunits and a beta subunit (TSH-beta) in addition to intact TSH. The three glycopeptides were measured in specific, heterologous radioimmunoassays. Basal secretion of alpha subunit was found to be in excess in all four thyrotropic tumor cultures, ranging from 5.7- to 8.9-fold higher than that of intact TSH and 30- to 35-fold higher than that of free TSH-beta. In cultures from 97A, TRH (10(-7) M) stimulated the secretion of TSH, alpha subunit, and TSH-beta by 175-185% and (T4, 10(-5) M) inhibited TSH to 19%, alpha subunit to 68%, and TSH-beta to 58% of control. Secretion of TSH and its subunits in cultures from 97B was not consistently affected by TRH or T4. In cultures from 97C, TSH and alpha secretion was inhibited in a dose-dependent fashion by T3 from 2.5 x10(-9) to 1.6x10(-7) M. Gel chromatography of incubation media from tumor 97A revealed elution volumes of TSH and alpha similar to those of purified rat glycopeptides and confirmed the excess secretion of free alpha subunit. In contrast, the alpha to TSH ratios in media from cultures of pituitary cells from normal and thyroidectomized rats were 1.6 and 0.8, respectively. The total production of TSH and its subunits was measured in cultures from tumor 97A and confirmed that alpha subunit was synthesized excessively. The ratio of the plasma concentrations of alpha to TSH in tumor-bearing mice was 1.6 compared to 1.3 in thyroidectomized mice, and 0.36 in thyroidectomized rats. The alpha to TSH ratios in extracts of the corresponding thyrotropic tissues (tumor or pituitary) were 2.7, 1.7, and 0.40, respectively. These data are analogous to those reported in humans with thyrotropic and certain other pituitary tumors, and support hypothesis that the subunits of glycoprotein hormones are independently synthesized.

Studies on the subunits of the human glycoprotein hormones in relation to reproduction

Studies on the subunits of the human glycoprotein hormones in relation to reproduction

Effect of conformation of hCG-beta on generation of hCG-specific antibody.

Most antisera generated to isolated highly purified beta subunits of human glycoprotein hormones are not sufficiently sensitive to detect physiologic blood levels of the native hormone. In the dissociated state, beta subunits assume a conformation different from that in the native hormone. Since antisera to alpha subunits have essentially no cross-reactivity between species, highly purified hCG-beta was combined with bTSH-alpha. That hybrid served as immunogen to assess whether sensitive, specific hCG antisera would more likely result than using hCG-beta alone. Of five animals immunized, three developed sufficiently sensitive and specific antisera. The results of these studies strongly suggests that human glycoprotein beta subunits combined with non-human alpha subunit are more likely to yield specific, sensitive antisera than when either isolated beta subunit or the native human glycoprotein hormone, containing common alpha determinants, serves as immunogen.

The effects of denaturant and temperature on the circular dichroic spectrum of ovine lutropin-alpha.

Circular dichroic (CD) spectra of the glycoprotein hormone subunit, lutropin-alpha, have been obtained under denaturing conditions (6 M guanidinium chloride, GdmCl) with the disulfides intact, reduced, and reduced and S-carboxymethylated. Above 260 nm, these spectra are similar (although not identical), showing that the disulfide chromophores do not contribute appreciably to the near ultraviolet CD spectrum under denaturing conditions. However, these spectra differ considerably from that of native lutropin-alpha over this spectral region demonstrating that the tertiary structure of lutropin-alpha has a major influence on the near ultraviolet CD spectrum. The magnitude of the 232.5 nm positive CD extremum of lutropin-alpha was found to increase with increasing GdmCl concentration and with decreasing temperature. Between 5 degrees C and 45 degrees C, the ellipticity [theta] at 232.5 nm changed in a linear fashion with temperature; at other wavelengths the thermal changes in [theta] were quite small. [theta] at 232.5 nm increased by about 80% over a 3 M GdmCl range; in contrast, [theta] at 216 nm (indicative of beta-structure) exhibited maximal changes over a 0.5 M GdmCl range. These results indicate some conformational flexibility in lutropin-alpha. The positive 232.5 nm CD extremum has been assigned to one or more tyrosyls which are influenced by an intact disulfide. The latter was demonstrated by showing that reduced and S-carboxymethylated lutropin-alpha failed to exhibit the positive extremum.

Alpha Subunit Contamination of Human Albumin Preparations: Interference in Radioimmunoassay1

Certain preparations of human serum albumin have been found to decrease the apparent titer of antisera to alpha subunit of human TSH (hTSH-alpha) and the sensitivity of the resultant radioimmunoassay for the immunologically common alpha subunit of the human glycoprotein hormones. Utilizing bovine serum albumin as the carrier protein, antisera to hTSH-alpha had 20-fold higher titers, and the resultant radioimmunoassay demonstrated 20-fold greater sensitivity for alpha subunit. Interference in the alpha immunoassay was not caused by protease since protease inhibitors did not eliminate it. Gel chromatography of human serum albumin revealed alph immunoactivity in an elution position identical to that of standard alpha subunit. Only certain human serum albumin preparations demonstrated interference in the radioimmunoassay because of the species specificity of the alpha subunit.A possible explanation for the alpha subunit contamination of human albumin preparations may be contamination of the serum source with placental blood, which contains large quantities of the alpha subunit of human chorionic gonadotropin.

Antibodies to reduced S-carboxymethylated alpha subunit of bovine luteinizing hormone and their application to study of the purification of gonadotropin from salmon ( Oncorhynchus tshawytscha) pituitary glands

Although antisera raised against either the β (hormone specific) subunits or the α (common) subunit of bovine thyrotropin (TSH) or luteinizing hormone (LH) show no cross reaction with partially purified salmon gonadotropin, cross reactivity is found between reduced S-carboxymethylated (RCM) salmon preparations and antisera against RCM bovine LH-α. Corresponding antisera against derivatized bovine β subunits do not cross react significantly. Further purification of salmon gonadotropin preparations by chromatography was evaluated, after their derivatization, by immunodiffusion against the anti-RCM bovine LH-α. Based on absorption to concanavalin A-Sepharose columns and subsequent elution with methyl mannoside, both the gonadotropic activity and the immunoreactivity are associated with glycoprotein(s). Successive chromatography on CM-cellulose and DEAE-cellulose resulted in three distinct fractions, two of which exhibited very weak gonadotropic activity when tested in chicks but which, after derivatization, had identical cross reactivity against anti-RCM LH-α. Both these fractions show two components on gel electrophoresis in the presence of sodium dodecyl sulfate. Their reduced S-carboxymethylated derivatives yield quite similar peptide maps in which the areas occupied by glycopeptides show staining properties very similar to those of bovine LH-α. The results demonstrate the usefulness of antisera against unfolded mammalian α subunits in the investigation of the glycoprotein hormones of lower vertebrates and strongly indicate that a peptide chain with a sequence related to the common subunit of mammalian glycoprotein hormones is also present in fish gonadotropin(s).

Secretion of alpha subunit of glycoprotein hormones by pituitary adenomas.

In 60 patients with pituitary adenomas, the serum concentration of the alpha subunit of the glycoprotein hormones (serum alpha) was measured by a sensitive and specific radioimmunoassay. Five patients had markedly elevated serum alpha prior to therapy (range 14.5-23.0 ng/ml). These 5 patients included 2 hyperthyroid men with inappropriately high serum thyrotropin, one of whom also had acromegaly, a man with hyperprolactinemia and elevated cerebrospinal fluid alpha, a postmenopausal woman with low serum gonadotropins and hyperprolactinemia, and a man with central hypothyroidism and hypogonadism. Three of the 5 were restudied after therapy; serum alpha in these three decreased from19.5 to 10.6, 23.0 to 2.0, and 17.0 to 12.0 ng/ml. Alpha in these 3 patinets' serum eluted similarly to normal pituitary alpha by gel chromatography. The other 55 patinets, including twenty with acromegaly, fifteen with galactorrhea, and two with Nelson's syndrome, had serum alpha.less than 0.5-5.0 ng/ml. In addition, 22 patients with "empty sella" syndrome (no pituitary tumor) had alpha less than 0.5-5.0 ng/ml. Normal men and premenopausal women had serum alpha concentrations of less than 0.5-2.5 ng/ml; normal postmenopausal women, 1.0-7.0 ng/ml; and patients with primary hypothyroidism, 0.7-9.0 ng/ml. The decreased alpha response to thyrotropin and luteinizing hormone-releasing hormones (TRH and LHRH) implied a relative autonomy of pituitary tumor alpha secretion; the mean alpha increment in the 5 patients with elevated serum alpha was 15% after TRH administration and 10% after LHRH. Normal individuals and patients with primary hypothyroidism demonstrated greater mean per cent alpha increments after TRH or LHRH. In certain patients with an enlarged sella turcica, an elevated serum alpha with little or no increase in secretion after TRH and LHRH may suggest the presence of pituitary tumor.

Alpha and beta glycoprotein hormone subunits (hLH, hFSH, hCG) in the serum and pituitary of the human fetus.

Utilizing specific radioimmunoassay and gel chromatographic separation methods, the concentration of a glycoprotein hormone subunit and the beta subunit of hCG, hLH, and hFSH was determined. The alpha glycoprotein hormone subunit is present throughout gestation in the serum and pituitary of the human fetus; little or no subunit is apparent in contrast to that of intact LH and FSH.

An acrylamide gel recombination assay for glycoprotein hormone subunits.

A specific and sensitive assay for recombination of the subunits of hCG and other glycoprotein hormones (LH, FSH, and TSH) is presented. The assay depends on the ability of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate clearly the subunits from native hormone. Iodinated subunit is incubated with its identical unlabeled subunit and complementary subunit. Quantitative displacement of the radioactivity to the native hormone position as followed by gel slicing is used as a direct index of recombination. The assay is reproducible, sensitive to less than one picomole of hormone formed, and offers a simple means of evaluating recombination of glycoprotein chains as distinct from biological activity.

Partial reduction of disulfide bonds in the hormone-specific subunits of TSH and LH

Partial reduction at pH 7.0 of the hormone specific (β) subunit of either bovine thyrotropin or luteinizing hormone with dithioerythritol results primarily in the opening of a single disulfide bridge. The partially reduced subunits were alkylated with [1- 14C] iodoacetic acid, followed by complete reduction and alkylation with non-radioactive iodoacetic acid. Isolation and degradation of the radioactive tryptic peptides shows that the bond primarily reduced in each β subunit links analogous half-cystine residues in the two sequences (88–95 in TSH-β and 93–100 in LH-β). These results are the first direct evidence of similar disulfide structures in hormone specific subunits of glycoprotein hormones.

Differences between purified ectopic and normal alpha subnits of human glycoprotein hormones.

"Ectopic" proteins, not distinguished immunologically from the common alpha subunit of the human glycoprotein hormones, were purified approximately 10,000-fold from a gastric carcinoid tumor (A.L.-alpha) and from tissue culture medium of bronchogenic carcinoma cell lines (ChaGo-alpha). The purified A.L.-alpha was homogeneous by sodium dodecyl sulfate (SDS) gel electrophoresis while the purified ChaGo-alpha showed multiple components, some of which represented aggregated species. In SDS gel electrophoresis, the apparent molecular weights of A.L.-alpha (15,000) and dithioerythritol-reduced ChaGo-alpha (13,000) were significantly lower than those of the alpha subunits of human chorionic gonadotropin (hCG-alpha), luteinizing hormone, follicle-stimulating hormone, or thyroid-stimulating hormone (22,000-23,000). Binding experiments with [35S]-SDS suggested that these apparent differences in molecular weight resulted, at least in part, from diminished binding of the SDS by the normal compared to the ectopic alpha subunits. In gel chromatography, the apparent molecular weights of A.L.-alpha (27,000) and ChaGo-alpha (30,000) were slightly higher than those of normal alpha subunits (23,000-24,000). Both A.L.-alpha and ChaGo-alpha were not distinguished from hCG-alpha in ion-exchange chromatography. The composition of A.L.-alpha was similar to that of hCG-alpha in 13 amino acids but showed decreased phenylalanine and increased valine; glucosamine was identified in both A.L.-alpha and hCG-alpha. Under conditions in which hCG-alpha combined with the hCG beta subunit (hCG-beta) to produce 95% of the expected gonadotropin-binding activity in a rat testis radioreceptor-assay, A.L.-alpha incubation with hCG-beta resulted in only 2% of the expected activity, and ChaGo-alpha incubation with hCG-beta produced no detectable activity. These characteristics of ectopic alpha subunits may reflect abnormalities of neoplastic protein synthesis or carbohydrate attachment which result in polypeptides with chemical and immunologic similarity to normal subunits but with differences in physical and combining properties; alternatively, the ectopic subunits may represent as yet unrecognized alpha precursor forms.

Neutralization of human thyrotropin by antisera to subunits of glycoprotein hormones.

The effects of antisera directed against various human glycoprotein hormone subunits on the biologic activity of human pituitary thyrotropin (hTSH), human chorionic thyrotropin (hCT), bovine pituitary thyrotropin (bTSH), and long-acting thyroid stimulator (LATS) were studied. Relative thyroid-stimulating potencies were determined using a McKenzie mouse assay. Antiserum to the human chorionic gonadotropin α-subunit (hCGα) completely neutralized hTSH but not hCT, bTSH, or LATS. Antisera to the hCG β-subunit (hCGβ) only partially neutralized hTSH and had no inhibiting effect on the biologic activity of hCT, bTSH, and LATS. In contrast, antisera to the human luteinizing hormone β-subunit had no significant effect on hTSH biologic activity. These results confirm previous reports indicating common antigenic determinants on hCGα subunit and hTSH and suggest similarity of the α subunits of hCG and hTSH. Placental thyroid-stimulating activity was not neutralized by either anti-hCGα or anti-hCGβ sera, indicating that hCT is immunologically distinct from hTSH and hCG, and suggesting that hCT does not have an α-subunit structure immunologically similar to that in the other human glycoprotein hormones.

Absence of intrinsic biological activity in LH and hCG subunits.

All glycoprotein hormone subunit preparations possess low biological activity when subjected to conventional bioassays. To determine whether the subunits of hCG and LH possess intrinsic biological activity, a variety of hCG and bovine or ovine LH subunits of known biological activity in vivo were assayed in vitro for target cell binding and steroidogenic activity in the rat testis. Binding-inhibition activity in the rat testis homogenate-125I-hCG radioligand-receptor assay system, and steroidogenic activity in the isolated rat testis, were found in all cases to be of similar magnitude to the biological activity of subunits measured in vivo by conventional bioassays. Because enhanced binding-inhibition or steroidogenic properties of isolated subunits were not demonstrable during the appropriate in vitro studies, it was concluded that the individual subunits do not possess preferential binding or activation functions in the dissociated state. In addition, the close agreement between the results of bioassay ...