Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin β-subunit by disruption of disulfide bonds

The human chorionic gonadotropin β-subunit (hCGβ) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGβ produced in Chinese hamster ovary (CHO) cells: wild-type hCGβ (βWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of βWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled βWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGβ molecule. Abbreviations: hCGβ, human chorionic gonadotropin β-subunit; βWT, wild type hCGβ; CHO, Chinese hamster ovary; Endo-H, endoglycosidase H; Endo-F, endoglycosidase F This revised version was published online in November 2006 with corrections to the Cover Date.     

Is deglycosylated human chorionic gonadotropin an antagonist to human chorionic gonadotropin? Characterization of deglycosylated human chorionic gonadotropin action in two testicular interstitial cell fractions

In order to determine the significance of carbohydrate residues of human chorionic gonadotropin (hCG) in receptor interaction and signal transduction leading to steroidogenesis, the effect of deglycosylated hCG (DG-hCG) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fraction I light cells, previously found to bind 125I-labeled hCG with high affinity without producing testosterone, also bound 125I-labeled DG-hCG with high affinity (Kd 7.2.10(-10) M) without stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studies in which DG-hCG was shown to be an antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as result of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological response in the same cell; and (d) DG-hCG partially antagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.     

The carbohydrate structures ofβ-galactosidase from human liver

Acid -galactosidase (EC3.2.1.23) was obtained from human liver in a pure monomeric state (M_r63 000). The carbohydrate content of the enzyme was established to be, 9% by weight; mannose,N-acetylglucosamine, galactose andN-acetylneuraminic acid were found to be the constituent monosaccharides. The carbohydrate structures of the enzyme were studied at the glycopeptide level by employing 500 MHz¹H-NMR spectroscopy, carbohydrate composition analysis and methylation analysis involving GLCMS. Based upon the intensities of relevant signals in the¹H-NMR spectrum, approximately 60% of the chains were found to be of theN-acetyllactosamine type, having the structure The rest appeared to be of the oligomannoside type (Man₅-₆GlcNAc₂Asn). The carbohydrate composition and methylation analysis results sustained these findings, although the calculation of the distribution based upon these techniques indicated a somewhat lower percentage ofN-acetyllactosamine type chains. There are approximately three oligosaccharide chains per molecule. These findings offer an explanation for the abnormal distribution of -galactosidase in tissues and cultured fibroblasts of patients with I-cell disease.     

Production of the β-subunit of human chorionic gonadotropin in Escherichia coli and its export mediated by the heat-labile enterotoxin chain-B signal sequence

The PCR-amplified β-subunit of the human chorionic gonadotropin structural gene (fihCG) was cloned under the control of the tac promoter and the heat-labile enterotoxin chain B (LTB) signal sequence (LTBss). βhCG was successfully produced, processed and exported to the periplasmic space in Escherichia coli. Expression of βhCG was confirmed by immunoblot analysis using an anti-βhCG polyclonal antibody. The processing of the protein was very efficient, as only the processed band could be detected at all time points during the course of induction. Expression was evident soon after the addition of the lactose analogue, IPTG. These results demonstrate that E. coli cells can synthesize, process and export βhCG using the LTBss.     

Purification and characterization of monoclonal antibodies against the free α-subunit of human chorionic gonadotrophin

Monoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free alpha-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-alpha hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free alpha-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free alpha-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary hormones either as free subunits or intact molecules as follows: alpha hCG 100%; intact hCG 1.8%; beta hCG 0.14%; alpha FSH 24.5%; intact FSH 0.8%; beta FSH 0.09%; alpha LH 20.5%; intact LH 0.9%; beta LH 0.08%; alpha TSH 50.5%; intact TSH 3.7%; beta TSH 0.07%; The affinity constant (K) of this Mab with respect to free alpha-subunit of hCG was found to be 1.5 x 10(7) I/mol as determined by the simple antibody dilution analysis method.     

Effect of cyclic 3′:5′-AMP derivatives,prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture

Summary The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N⁶, O^2′-dibutyryl cyclic 3′:5′-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process. malignant trophoblast cultures were incubated with 3′:5′-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmuno-assayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretio of hCG, the N⁶- and O^2′-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3′:5′-GMP (cGMP), dbcBMP, 5′-AMP, adenosine, L-epinephrine and prostaglandins E₁, E₂, F_1α and F_2α were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast. This investigation was supported by Grant Nos. CA14232 and CA16539 awarded by the National Cancer Institute, DHEW.     

Amino acid and carbohydrate compositions of human serum dopamine-β-hydroxylase

Dopamine--hydroxylase has been purified from human serum. The amino acid composition has been determined and found to be similar to that of the enzyme purified from human pheochromocytoma. Human serum dopamine--hydroxylase is a glycoprotein, containing 13.11 g carbohydrates/100 g protein. Individual sugar determinations showed the presence of fucose, galactose, glucose, mannose,N-acetylglucosamine,N-acetylgalactosamine andN-acetylneuraminic acid.     

Determination of the structure of the carbohydrate chains of acid α-glucosidase from human placenta

Acid α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz ¹H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man₅GlcNAcGlcNAc-ol and Man₇GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man_2–3GlcNAc[Fuc]_0–1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.     

Regulation of low density lipoprotein receptor mRNA levels by estradiol 17β and chorionic gonadotropin in human placenta

Inhibition of synthesis of estradiol 17 by the addition of inhibitors of aromatase, a key enzyme in the biosynthesis of estradiol 17, or addition of tamoxifen - an estrogen receptor antagonist, to human placental minces resulted in an increase in the level of LDL-receptor mRNA. This increase could be blocked by the simultaneous addition of estradiol 17. A concentration dependent effect of estradiol 17 on the level of LDL-receptor mRNA was seen both in first trimester, and term placenta. Addition of human chorionic gonadotropin (hCG) to term placental minces also increased the LDL-receptor mRNA levels. When hCG and cycloheximide were added together, an additive effect was observed. The results obtained in this study suggest that the LDL-receptor mRNA levels in the human placenta are regulated by estradiol 17 and hCG.     

Infertility in mice induced by the rhesus monkey chorionic gonadotropin β-subunit glycoprotein (rmCGβ) using DNA immunization

The recombinant eukaryotic expression vector pCMV4-rmCG, inserted full-length cDNA of the -subunit of rhesus monkey chorionic gonadotropin (rmCG), as DNA immuno-contraceptive against CG glycoprotein, has previously demonstrated the biological expression of rmCG in vitro and in vivo. The plasmid DNA of pCMV4-rmCG was inoculated into BALB/c mice at different doses and routes as DNA immuno-contraceptive to understand its antifertility effect. The results of immune responses indicated that the intradermal inoculation is the optimal pCMV4-rmCG DNA delivery method for BALB/c mice, and the dose of 10 g should be enough to elicit immune response. With different doses from 10–50 g, marked reductions in the fertility of the female mice after two intramuscular inoculations of pCMV4-rmCG DNA were seen, while the similar level of humoral immune responses were induced. With the dose of 20 g of pCMV4-rmCG DNA, the mice showed reduction in fertility from intraperitoneal, and intradermal to intramuscular inoculating method. The antifertility effect of antiserum from immunized mice confirmed that the antibodies elicited by pCMV4-rmCG DNA could prevent pregnancy in female mice. At the same time, the full-length cDNA of -subunit of mouse chorionic gonadotropin (muCG) was cloned from placenta and sequenced for the first time (GenBank Accession No. AF333067). Sequence analysis showed that muCG shares 99.6% homology with rmCG and 90.6% with hCG respectively. The results indicated that the infertility of BALB/c mice induced by pCMV4-rmCG contraceptive should be further studied as a CG DNA contraceptive.     

The application of single beta-human chorionic gonadotropin (β-hCG) level measurement in women undergoing single blastocyst transfer

We previously explored the associations between β-hCG on the 14th day post–embryo transfer (ET) and reproductive outcomes and established a series of cutoff values to predict different outcomes. The aim of this study was to explore the parameters associated with β-hCG levels and establish β-hCG cutoff values in women undergoing single blastocyst transfer. The patients were transferred with either fresh or frozen-thawed blastocysts. Serum β-hCG levels were compared among different groups. Cutoff values of β-hCG were established and applied to divide the patients into different groups, among which the β-hCG groups were compared. Develop day negatively affected β-HCG levels in those who were pregnant or gave live birth (P < 0.001, 0.008). Inner cell mass significantly affected β-hCG levels in women who were pregnant or gave live birth (P = 0.013, 0.044). Trophectoderm significantly affected β-hCG levels in women with most reproductive outcomes, except biochemical pregnancy (BP) (P = 0.184). The cutoff values of β-hCG for predicting positive outcomes were 194.1, 503.0, 1048.0, and 2590.5 mIU/L. BP rates and adverse pregnancy outcome rates were significantly lower in the higher β-hCG groups (P < 0.001). Shorter gestational age and lower birth weight and length (P = 0.005, 0.041, 0.003) were observed in the lowest-concentration β-hCG group. The application of a single β-hCG measurement was sufficient to predict reproductive outcome in women undergoing blastocyst transfer, under the full consideration of blastocyst parameters. However, the association between β-hCG and obstetric outcomes remains to be investigated and fully explained.     

Single chain human chorionic gonadotropin,hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the α and β subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGαβ) was used to investigate roles of critical residues of the α subunit in hormone receptor interaction and biological activity. The α subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type β subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGα P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGα T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the α subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions.     

The porcine ovarian luteinizing hormone/human chorionic gonadotropin receptor II. Is the purified receptor an oligomer of identical subunits?

Purified porcine luteinizing hormone/human chorionic gonadotropin receptors were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis following reduction and thermal denaturation and stained with Coomassie Brilliant Blue. A major protein of Mr = 77 +/- 4 X 10(3) and a minor protein of Mr = 66 +/- 4 X 10(3) were observed. Iodoreceptor proteins were resolved into a major component of Mr = 77 +/- 3 X 10(3) and a minor component of Mr = 62 +/- 5 X 10(3) after reduction and thermal denaturation. In the absence of reduction, the iodoreceptor had a major component of Mr 63 +/- 3 X 10(3). Purified human chorionic gonadotropin specifically transferred part of the iodoreceptor from the Mr = 63 X 10(3) species to an Mr = 110-120 X 10(3) species. Purified receptors were analyzed by nondenaturing polyacrylamide gel electrophoresis and identified by specific binding of iodo-human chorionic gonadotropin. Three binding species with approximate Mr = 60 X 10(3), 130 X 10(3), and 260 X 10(3) were identified. Iodoreceptors co-migrated with the Mr = 60 X 10(3) species under the same conditions. Similar results were obtained following renaturation of receptors separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reduction and thermal denaturation. These results suggest for the first time that the porcine corpus luteum luteinizing hormone/human chorionic gonadotropin receptor may be a hormone binding monomer of Mr = 60-65 X 10(3), and that the monomer may associate to form hormone binding polymeric receptor complexes.     

Purification of β-glucuronidase and structural assessment of the carbohydrate chains by lectin affinity immunoelectrophoresis

The purification of rat liver -glucuronidase from a lysosomal fraction by methods including affinity chromatography, chromatofocusing and preparative PAGE steps is described. Molecular weights of 300 000 and 150 000 were estimated by two dimensional gradient PAGE/immunoelectrophoresis of the lysosomal extract. Isoelectrofocusing in agarose gel followed by immunoelectrophoresis in the second dimension revealed the presence of at least five maxima in the range pH 4.3–7.4.The structural assessment of the carbohydrate chains of lysosomal and microsomal -glucuronidase was performed by lectin affinity immunoelectrophoresis. Reaction with Concanavalin A indicated the presence of bi-antennary complex, oligomannosidic and hybrid type structures, whereas the absence of tri- and tetra-antennary complex type structures was deduced from the lack of interaction with phytohemagglutinin-L. The reaction withLens culinaris agglutinin, Pisum sativum agglutinin andLotus tetragonolobus lectin revealed that part of the glycans contained a fucose (1-6)-linked to theN-acetylglucosamine attached to asparagine. The presence of terminal (1-4)-galactose residues was detected withRicinus communis agglutinin I.     

Mapping of assembled epitopic regions of human chorionic gonadotropin reveals proximity of CTPα to the determinant loop β93–100

Three overlapping assembled epitopes of βhCG have been mapped using MAb probes and a single step solid phase radioimmunoassay. These epitopes have been shown to be at receptor binding region comprising of the loop region μ Cys93-Cys100. Importance of disulphide bonds in maintaining integrity of these epitopes is assessed. Two MAbs (INN 58 and INN 22) interact with the μ region as well as the β C-terminal peptide, while the other MAb INN 24 interacts with only the μ region. Cross-reactivity pattern with μhCG and hLH as well as the reported crystal structure of hCG substantiates the epitope identification. The results demonstrate utility of MAbs as probes in investigations on three-dimensional structure of gonadotropins     

The amino acid sequences of the Fd fragments of two human γ heavy chains

The amino acid sequences of the Fd fragments of two human pathological immunoglobulins of the immunoglobulin G1 class are reported. Comparison of the two sequences shows that the heavy-chain variable regions are similar in length to those of the light chains. The existence of heavy chain variable region subgroups is also deduced, from a comparison of these two sequences with those of another gamma 1 chain, Eu, a mu chain, Ou, and the partial sequence of a fourth gamma 1 chain, Ste. Carbohydrate has been found to be linked to an aspartic acid residue in the variable region of one of the gamma 1 chains, Cor.     

Structural assessment of β-glucuronidase carbohydrate chains by lectin affinity chromatography

Rat liver -glucuronidase was studied by sequential lectin affinity chromatography. -Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed byN-[¹⁴C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography.Ulex europaeus agglutinin-agarose chromatography revealed the presence of (1-3) linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin,Ricinus communis agglutinin andPhaseolus vulgaris erythroagglutinin.     

Immobilization of cellulase through its carbohydrate side chains — A rationale for its recovery and reuse

The major types of components of cellulase [see 1,4-(1, 3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] have been adsorbed onto concanavalin A immobilized on Sepharose 4B, suggesting that they are glycoproteins. These components were covalently coupled to cyanogen bromide-activated Sepharose after aminoalkylation of their periodate-oxidized carbohydrate side chains to provide additional points of attachment of the enzyme to the support. Although there was only a 9% recovery of starting avicelase activity, the immobilized enzyme catalysed the hydrolysis of insoluble cellulose to glucose with greater efficiency than did free cellulase.     

Immunolocalization of inhibin α-subunit in the human testis

Summary The localization of inhibin -subunit in the human testis was studied at the light- and electron-microscope level with immunostaining techniques. Antibodies against specific fragments of porcine and human inhibin -subunits were utilized. At light microscopy, inhibin -subunit immunoreactivity was detected in Sertoli cells, spermatocytes and in some Leydig cells. At electron microscopy, gold labeling was found in the cisternae of the Golgi apparatus and in the endoplasmic reticulum of Sertoli and Leydig cells. Gold labeling for inhibin was also found in coated vesicles in the cytoplasm of Sertoli cells as well as in coated pits and coated vesicles in the cytoplasm of some spermatocytes. The results of the present study suggest that, in the human testis, inhibin is produced by Sertoli and Leydig cells and is taken up by spermatocytes, on which it might act in a paracrine manner.