Identification and characterization of protein glycosylation using specific endo- and exoglycosidases.

Enzymatic deglycosylation followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples. Specific glycosidases were used to remove sugars from glycoproteins in a controlled fashion leaving the protein core intact; the resulting change in molecular weight could be detected as shifts in gel mobility. Alternatively, glycan-sensitive reagents were used to visualize the intensity of glycoprotein bands before and after enzyme treatment. The ease of use of these techniques, which require only basic laboratory instrumentation and reagents, makes them the methodology of choice for initial glycobiology studies. These protocols are also well suited to screen for optimal expression conditions, since multiple glycoprotein samples can be processed at once.

Decreased levels of genuine large free hCG alpha in men presenting with abnormal semen analysis.

BACKGROUND: The pregnancy hormone human chorionic gonadotropin (hCG) and its free subunits (hCG alpha, hCG beta) are produced in the male reproductive tract and found in high concentrations in seminal fluid, in particular hCG alpha. This study aimed to elucidate changes in peptide hormone profiles in patients showing abnormal semen analyses and to determine the genuineness of the highly abundant hCG alpha. METHODS: Seminal plasma was obtained from 45 male patients undergoing semen analysis during infertility workups. Comprehensive peptide hormone profiles were established by a panel of immunofluorometric assays for hCG, hCG alpha, hCG beta and its metabolite hCG beta core fragment, placental lactogen, growth hormone and prolactin in seminal plasma of patients with abnormal semen analysis results (n = 29) versus normozoospermic men (n = 16). The molecular identity of large hyperglycosylated hCG alpha was analyzed by mass-spectrometry and selective deglycosylation. RESULTS: hCG alpha levels were found to be significantly lower in men with impaired semen quality (1346 +/- 191 vs. 2753 +/- 533 ng/ml, P = 0.022). Moreover, patients with reduced sperm count had reduced intact hCG levels compared with normozoospermic men (0.097 +/- 0.022 vs. 0.203 +/- 0.040 ng/ml, P = 0.028). Using mass-spectrometry, the biochemical identity of hCG alpha purified from seminal plasma was verified. Under non-reducing conditions in SDS-PAGE, hCG alpha isolated from seminal plasma migrated in a manner comparable with large free hCG alpha with an apparent molecular mass (Mr, app) of 24 kDa, while hCG alpha dissociated from pregnancy-derived holo-hCG migrated at approximately 22 kDa. After deglycosylation with PNGase F under denaturing conditions, all hCG alpha variants showed an Mr, app of 15 kDa, indicating identical amino acid backbones. CONCLUSIONS: The findings indicate a pathophysiological relevance of hCG, particularly its free alpha subunit, in spermatogenesis. The alternative glycosylation pattern on the free large hCG alpha in seminal plasma might reflect a modified function of this subunit in the male reproductive tract.

Efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones.

Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37 degrees C in acetic acid. The alpha-and beta-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of > 98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C4 column (80-90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.

Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions.

Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common alpha-subunit (alpha-glycoprotein hormone (alpha-GP)), beta-FSH, and beta-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the alpha-subunit without the His-tag with each of the His-tagged beta-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and alpha-GP in abundance; however, expression of the individual beta-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to ~15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose-response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of species-specific GtHs so that FSH- and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.

Isolation of alpha- and beta-subunits of peak-I hCG and generation of highly specific polyclonal antisera.

Development of polyclonal antisera is still a choice in some hard-pressed budget laboratories. In the present study, an attempt was made to isolate alpha- and beta-subunits from peak-I hCG, generation of polyclonal antisera and their characterization. The anti-hCG-a antisera showed titres of 1: 8000 and anti-hCG-beta antisera 1: 16,000 at 50% binding to radiolabelled hCG in RIA. Studies on specificity using anti hCG-beta antiserum demonstrated no cross-reaction with several hormones tested in the present study, except for hCG-beta and hCG, thus eliciting a highly specific hCG-beta antiserum.

La transferencia de embriones. Una técnica de mejoramiento animal en ganado de lidia / Embryo transfer. A technique of animal improvement in fighting livestock

Dos explotaciones del estado de Tlaxcala (uno productor de ganado de lidia y otro productor de leche), se utilizaron para evaluar dos tipos de hormonas, la gonadotropina de suero de yegua gestante (PMSG) y la hormona folículo estimulante (FSH), en relación con la cantidad y calidad de embriones que se obtuvieron por superovulación en el programa de transferencia de embriones. Para la superovulación, se formaron dos grupos de cinco hembras de lidia; administrando al primero, FSH en dosis descendentes de 100 U.I. a 37.5 U.I. durante cuatro días consecutivos, y al segundo grupo con la administración de PMSG, en una sola dosis de 2,000 U.I. Se obtuvieron con FSH, 25 embriones transferibles, 20 degenerados o inmaduros y 15 ovocitos no fertilizados, sin embargo con PMSG, se obtuvieron cinco embriones transferibles, cinco degenerados o inmaduros y cinco ovocitos no fertilizados, lo que indica que el tratamiento con FSH, es más efectivo que el de PMSG para superovular a hembras de lidia con una probabilidad estadísticamente significativa (P < 0.05).Los 30 embriones que se obtuvieron para ser transferidos, fueron transportados a la explotación de leche, donde se llevó a cabo la transferencia de los mismos a 30 vaquillas respectivamente, mismas que fueron consideradas como receptoras. Del total de las transferencias, sólo cinco vaquillas se diagnosticaron como gestantes (16.6 por ciento), coincidiendo con embriones obtenidos de hembras superovuladas con FSH; cinco (16.6 por ciento) repitieron el estro a los 21 días de ciclo; otras cinco vaquillas (16.6 por ciento) repitieron el estro hasta los 37 días de ciclo; diez (33.3 por ciento) sólo presentaron un cuerpo lúteo en el ovario derecho y las cinco restantes (16.6 por ciento) además de presentar también un cuerpo lúteo en el ovario derecho, presentaban infección uterina. Esto se corroboró estadísticamente con una probabilidad estadística (P < 0.05).

Carbohydrate analysis of glycoprotein hormones.

Complete carbohydrate composition analysis of glycoprotein hormones, their subunits, and oligosaccharides isolated from individual glycosylation sites can be accomplished using high-pH anion-exchange chromatography combined with pulsed amperometric detection. Neutral and amino sugars are analyzed from the same hydrolyzate by isocratic chromatography on a Dionex CarboPAC PA1 column in 16 mM NaOH. Sialic acid is quantified following mild hydrolysis conditions on the same column in 150 mM sodium acetate in 150 mM NaOH. Ion chromatography on a Dionex AS4A column in 1.8 mM Na(2)CO(3)/1.7 mM NaHCO(3); postcolumn, in-line anion micromembrane suppression; and conductivity detection can be used to quantify sulfate, a common component of pituitary glycoprotein hormone oligosaccharides. Mass spectrometric analysis before and after elimination of oligosaccharides from a single glycosylation site can provide an estimate of the average oligosaccharide mass, which facilitates interpretation of oligosaccharide composition data. Following release by peptide N-glycanase (PNGase) digestion and purification by ultrafiltration, oligosaccharides can be characterized by a high-resolution oligosaccharide mapping technique using the same equipment employed for composition analysis. Oligosaccharide mapping can be applied to the entire hormone, individual subunits, or individual glycosylation sites by varying PNGase digestion conditions or substrates. Oligosaccharide release by PNGase is readily monitored by SDS-PAGE. Site-specific deglycosylation can be confirmed by amino acid sequence analysis. For routine isolation of oligosaccharides, addition of 2-aminobenzamide at the reducing terminus facilitates detection; however, the oligosaccharide retention times are altered. Composition analysis is also affected as the 2-aminobenzamide-modified GlcNAc peak overlaps the fucose peak.

Two free isoforms of ovine glycoprotein hormone alpha-subunit strongly differ in their ability to stimulate prolactin release from foetal pituitaries.

alpha-Subunit dissociated from glycoprotein hormones has been previously shown to stimulate rat pituitary lactotroph differentiation and proliferation. However, whether the free form of the alpha-subunit (free alpha) can also play such a role is not known. To test whether free alpha may act on prolactin (PRL) release from ovine foetal pituitaries, this molecule was purified and two major isoforms, alphaA and alphaB were isolated. Free alphaA was found to be more acidic and more hydrophobic than both free alphaB and ovine LH alpha-subunit (oLHalpha). Free alphaA and oLHalpha exhibited a molecular mass of 14 kDa as determined by mass spectrometry, whereas free alphaB displayed a molecular mass of only 13.5 kDa because of its truncated N-terminus. All three alpha molecules bear mature-type N-linked saccharide chains including Nacetyl galactosamine residues but none of them contains O-linked oligosaccharide. The free alphaA isoform, more than the oLHalpha, was able to stimulate PRL release from ovine foetal pituitary explants in culture, whereas the free alphaB isoform displayed no activity. Moreover, the free alphaA and alphaB isoforms were able to recombine with the ovine LH beta-subunit (oLHbeta). The free alphaB/oLHbeta, and the oLHalpha/oLHbeta dimer were 4-fold more active than the free alphaA/oLHbeta dimer in a specific LH radioreceptor assay and in the stimulation of testosterone release from rat Leydig cells. The present study demonstrates that the two free alpha isoforms of ovine glycoprotein hormones exhibit distinct efficiencies in stimulating PRL release from ovine foetal pituitaries. Moreover, despite their identical ability to recombine with the oLHbeta, the free alpha isoform, which is the most efficient on PRL release, is the least efficient in conferring LH activity on the alpha/beta dimer.

Developmental changes in the glycosylation of glycoprotein hormone free alpha subunit during pregnancy.

Glycoprotein hormone alpha subunit, in its free form (free alpha), is a major placental product. Its glycosylation was found to change dramatically during the advancement of pregnancy. In this study, we have analyzed these glycosylation changes in five normal pregnancies. Binding to Lens culinaris lectin increased dramatically in all subjects between weeks 14 and 17 from the last menstrual period, indicating more core fucosylation as well as possible changes in branching of glycans. Studies using Datura stramonium agglutinin confirmed that the type of triantennary branching changed in this period of pregnancy. The precise structural nature of these changes was determined by high-pH anion-exchange chromatography and electrospray ionization mass spectrometry. Amounts of core fucosylation and of triantennary glycans increased substantially from early to late second trimester, and a shift was observed from 1-->4/1-->3- toward predominantly 1-->6/1-->6-branched triantennary structures. The glycosylation changes occurred in all five individuals at the same time period in gestation, suggesting developmental regulation of N-acetylglucosaminyltransferases IV and V and alpha6-fucosyltransferase during normal pregnancy. These enzymatic activities also appear to be affected in malignant transformation of the trophoblast. Our findings have important implications for the proposed use of specific forms of glycosylation as markers for cancer, as the relative amounts of these glycans in normal pregnancy will be determined by gestational age.

Isolation of an Alu repetitive DNA binding protein and effect of CpG methylation on binding to its recognition sequence.

The structure, expression, and evolution of Alu repetitive DNA elements have been extensively studied, but the role of these sequences in the function of primate genomes has yet to be elucidated. The contribution of Alu repetitive sequences (ARS) to the structure, maintenance, or expression of the human genome is undoubtedly mediated by one or more DNA binding proteins. As part of a larger study in this laboratory to define the molecular mechanisms that result in de-repression of the glycoprotein hormone alpha-subunit (GPH alpha) gene in a variety of tumor cell types, it was found that the gene was hypermethylated in a variety of cell lines that produce alpha-subunit at high levels and significantly less methylated in cell lines where the gene is unexpressed or expressed at low levels. This is in sharp contrast to the majority of genes examined in this regard, which show an inverse correlation between methylation and expression. The analysis was extended to a group of clones isolated from a single cell line (HeLa) that were differentially methylated over the GPH alpha gene and exhibited a 400-fold range in its expression. These analyses demonstrated that methylation of a small number of CpG dinucleotides correlated with high level expression of the gene. Two of these sites are imbedded in oppositely oriented Alu repeats located in the 5'-flanking DNA and second intron. The upstream site was examined in some detail. DNase I footprint analysis demonstrated that the protein protects a region encompassing the sequence 5'-TTGAACCCGGGAG-3', and electrophoretic gel mobility shift analysis demonstrated specific binding of a protein to an oligonucleotide containing the DNase footprint sequence. Chromatography of nuclear extracts on Sephacryl S-200, heparin--agarose, and oligonucleotide--Sepharose produced an apparently homogeneous preparation of the 50-53 kDa DNA-binding protein as judged by silver staining of sodium dodecylsulfate polyacrylamide gels. The affinity-purified material was enriched 15- to 18,000-fold over crude nuclear extracts. Binding of this protein to an oligonucleotide containing the DNase-protected sequence was severely inhibited when CpG dinucleotide in the Msp I recognition site was methylated on either the sense or antisense strands. Based on its properties, this protein has been termed MeSABp50 for methylation-sensitive Alu binding protein of 50 kDa.

Carbohydrate and peptide structure of the alpha- and beta-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma.

Human chorionic gonadotropin (hCG), purified from the urine of 14 individuals with normal pregnancy, diabetic pregnancy, hydatidiform mole, or choriocarcinoma, plus two hCG standard preparations, was examined for concurrent peptide-sequence and asparagine (N)- and serine (O)-linked carbohydrate heterogeneity. Protein-sequence analysis was used to measure amino-terminal heterogeneity and the "nicking" of internal peptide bonds. The use of high-pH anion-exchange chromatography coupled with the increased sensitivity of pulsed amperometric detection (HPAE/PAD) revealed that distinct proportions of both hCG alpha- and beta-subunits from normal and aberrant pregnancy are hyperglycosylated, and that it is the extent of the specific subunit hyperglycosylation that significantly increases in malignant disease. Peptide-bond nicking was restricted to a single linkage (beta 47-48) in normal and diabetic pregnancy, but occurred at two sites in standard preparations, at three sites in hydatidiform mole, and at three sites in choriocarcinoma beta-subunit. In the carbohydrate moiety, alpha-subunit from normal pregnancy hCG contained nonfucosylated, mono- and biantennary N-linked structures (49.3 and 36.7%, means); fucosylated biantennary and triantennary oligosaccharides were also identified (7.3 and 6.9%). In choriocarcinoma alpha-subunit, the level of fucosylated biantennary increased, offset by a parallel decrease in the predominant biantennary structure of normal pregnancy (P < 0.0001). The beta-subunit from normal pregnancy hCG contained fucosylated and nonfucosylated biantennary N-linked structures; however, mono- and triantennary oligosaccharides were also identified (4.6 and 13.7%). For O-linked glycans, in beta-subunit from normal pregnancy, disaccharide-core structure predominated, whereas tetrasaccharide-core structure was also detected (15.6%). A trend was demonstrated in beta-subunit: the proportions of the nonpredominating N- and O-linked oligosaccharides increased stepwise from normal pregnancy to hydatidiform mole to choriocarcinoma. The increases were: for monoantennary oligosaccharide, 4.6 to 6.8 to 11.2%; for triantennary, 13.7 to 26.7 to 51.5% and, for O-linked tetrasaccharide-core structure, 15.6 to 23.0 to 74.8%. For hCG from individual diabetic pregnancy, the principal N-linked structure (34.7%) was consistent with a biantennary oligosaccharide previously reported only in carcinoma; and sialylation of both N- and O-linked antennae was significantly decreased compared to that of normal pregnancy. Taken collectively, the distinctive patterns of subunit-specific, predominant oligosaccharides appear to reflect the steric effect of local protein structure during glycosylation processes. The evidence of alternative or "hyperbranched" glycoforms on both alpha- and beta-subunits, seen at low levels in normal pregnancy and at increased or even predominant levels in malignant disease, suggests alternative substrate accessibility for Golgi processing enzymes, alpha 1,6 fucosyltransferase and N-acetylglucosaminyltransferase IV, in distinct proportions of subunit molecules.

Evidence for two folding domains in glycoprotein hormone alpha-subunits.

We reconstituted ovine (o) LH alpha from its amino- and carboxyl-terminal fragments obtained as follows. oLH alpha was nicked at Arg46-Ser47 with Arg-C protease. Nicked oLH alpha disulfide bonds were broken by sulfitolysis, and its N-terminal peptide and C-terminal glycopeptide were separated by Sephacryl S-200 chromatography. Both fragments were mixed, reduced, and reoxidized. Reoxidation products were chromatographed on Sephacryl S-200, and an alpha-monomer fraction was recovered. The putative nicked alpha-monomer fraction was reassociated with native oLH beta, and the resulting oLH derivative was isolated by S-200 chromatography with a reduced yield of 11% (intact subunits yield, 67% oLH). This preparation was 2.6% as active as oLH in a LH receptor binding assay. Two additional oLH derivatives were prepared. Cleavage at alpha Arg46-Ser47 alone, followed by reassociation with native oLH beta, produced Arg-C-nicked oLH alpha:oLH beta (14% yield) that was 3.3% as active as native oLH. Reduction-reoxidation of Arg-C-nicked oLH alpha followed by reassociation with oLH beta produced reduced reoxidized-Arg-C-nicked oLH alpha:oLH beta (11% yield) that was 1.8% as active as oLH. These results indicated that the nicked oLH alpha monomer had been reconstituted from its N- and C-terminal fragments.

Purification and characterization of the acid-labile subunit of rat serum insulin-like growth factor binding protein complex.

Circulating insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), when occupied by IGF-I or IGF-II, combines with an acid-labile glycoprotein subunit (ALS) to form a high molecular weight complex. In this study, ALS from rat serum has been purified and its properties investigated. Purification involved ion-exchange chromatography, and affinity chromatography on an IGF-I-IGFBP-3 column, yielding almost 1 mg ALS from 100 ml serum. Amino-terminal sequencing confirmed the prediction from previous complementary DNA analysis but indicated that the protein may circulate in a truncated form. Rat ALS was almost as potent as human ALS in binding to human IGF-I-IGFBP-3 complex (association constant, 2.3 nM-1). A sensitive RIA was developed, with high specificity for rodent ALS. Serum ALS rose from 3 micrograms/ml in 2-day-old rats to more than 40 micrograms/ml at 10 weeks, with no sex difference. GH-deficient rats showed 60-75% lower values than controls. This study shows rat ALS to have similar binding properties, and age and GH dependence, to human ALS. The new RIA will facilitate studies of IGF and IGFBP regulation in the rat.

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.

Expression of two forms of carp gonadotropin alpha subunit in insect cells by recombinant baculovirus.

There are two types of cDNA clones (designated alpha 1 and alpha 2) encoding the alpha subunit of carp gonadotropin. These two cDNAs are derived from different genes and encode proteins that differ by seven amino acid residues (three in the signal peptide and four in the mature polypeptide). Expression of these two cDNAs in insect cells by recombinant baculovirus revealed that the alpha 1 subunit, after noncovalent association with the beta subunit, has the same potency as the native alpha subunit purified from the pituitary. In contrast, the alpha 2 subunit can associate with the beta subunit, but only to form an inactive gonadotropin. Competition of the alpha 2 subunit with the alpha 1 subunit for association with the beta subunit decreases the gonadotropin activity of the alpha/beta complex. In addition, both alpha 1 and alpha 2 subunits are secreted into the culture medium by insect cells and have an apparent molecular mass approximately 5 kDa higher than that of the native alpha subunit. These results indicate that the insect cell-derived alpha 1 subunit is biologically active and that those four amino acid changes in the mature of alpha 2 protein affect the biological activity and thus provide valuable clues for the study of the structure-function relationship of the alpha subunit of glycoprotein hormones.

Complete and partial glycophospholipid anchors are found on a fusion protein consisting of luteinizing hormone beta subunit followed by a carboxyl-terminal domain of Thy-1.

The Thy-1 antigen is anchored to the cell surface by a carboxyl-terminal glycophospholipid moiety. To investigate the extent of anchor addition which occurs when such proteins cannot move efficiently to the cell surface, we have expressed a recombinant fusion protein composed of 107 amino-terminal amino acids of bovine luteinizing hormone beta subunit and 46 COOH-terminal amino acids of murine Thy-1 (Thy-1.2 allele). Although the limited amount of fusion protein transported to the cell surface is glycophospholipid-anchored, most of the protein accumulates in an intracellular, endoglycosidase H-sensitive form. The intracellular protein has an unusual structure that contains ethanolamine but does not bind detergent, suggesting either that anchor addition proceeds via a hydrophilic partial intermediate, or that anchor-degradative enzymes exist along the secretory path.

N-terminal amino-sequencing of camel (Camelus dromedarius) luteinizing-hormone alpha and beta-subunits.

The subunits of luteinizing hormone from the Camelus dromedarius (CamLH) have been separated by reverse phase HPLC after reduction and alkylation of their disulfide bridges. The N-terminal amino-acid sequencing of the alpha and beta subunits has been performed up to the 53rd and 67th residue respectively (i.e. more than half of each polypeptide chain). These sequences have been compared to those of LH from other mammalian species in order to estimate the phylogenetic divergence of LH in this species and in order to point out characteristic features of its primary structure that can be related to its physico-chemical properties.

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.

Isolation and characterization of the gene encoding the alpha-subunit of the rat pituitary glycoprotein hormones.

The gene encoding the common alpha subunit of the rat pituitary glycoprotein hormones was isolated from a rat genomic DNA library. The gene spans approximately 8 kb, and contains four exons and three intervening sequences of 5.4 kb, 1.1 kb and 0.6 kb. Blot hybridization of restriction enzyme digests of rat genomic DNA suggests that the alpha gene is present in a single copy. The coding region and 424 bp of the 5'-flanking region of the gene were sequenced. Primer extension and S1 nuclease analyses revealed a single transcriptional start point downstream from consensus promoter elements. The organization of the rat alpha-subunit gene is similar to that of the human and bovine genes including the sizes and locations of the four exons and three introns. In addition, a region of strong sequence similarity has been identified in the 5'-flanking region of the rat, human and bovine genes. This region includes sequences which are similar to a putative triiodothyronine regulatory element and the previously identified cAMP regulatory region; such sequences may mediate the known effects of these factors on alpha-subunit gene expression.

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