Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem.

One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common alpha subunit and a hormone-specific beta subunit. Subunit assembly is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormone-specific posttranslational modifications, and signal transduction. To address whether alpha and beta subunits can be synthesized as one chain and also maintain biological activity, a chimera composed of the human chorionic gonadotropin (hCG) beta subunit genetically fused to the alpha subunit was constructed. The resulting polypeptide hCG molecule not only was efficiently secreted but also displayed an increased biological activity in vitro and in vivo. These data show that the alpha and hCG beta subunits encoded as a single chain retain a biologically active conformation similar to that seen in the heterodimer. This approach can be used to investigate structure-function relationships of the glycoprotein hormone family that were previously not tractable because of the absolute dependence on assembly for the biological response. Moreover, other bioactive multisubunit ligands can be engineered where the combination efficiency and specificity of heterodimers and homodimers are otherwise difficult to control.     

Synthesis and secretion of gonadotropins including structure-function correlates

The synthesis and secretion of the gonadotropic hormones involves coordination of signal transduction, gene expression, protein translation, post-translational folding and modification and finally secretion. The production of biologically active gonadotropin thus requires appropriately folded and glycosylated subunits that assemble to form the heterodimeric hormone. Here we overview recent literature on regulation of gonadotropin subunit gene expression and current understanding of the assembly and secretion of biologically active gonadotropic hormones. Finally, we discuss the therapeutic potential of understanding glycosylation function towards designing new forms of gonadotropins based on observations of physiologically relevant parameters such as age related glycosylation changes.     

Biological activity of single chain chorionic gonadotropin, hCGalphabeta, is decreased upon deletion of five carboxyl terminal amino acids of the alpha subunit without affecting its receptor binding

The strategy of translationally fusing the two subunits of human chorionic gonadotropin (hCG) has been used to produce recombinant single chain hCG in which the C-terminus of the alpha subunit is fused to the N-terminus beta without any linker using Pichia pastoris expression system. The Pichia-expressed hCGalphabeta (phCGalphabeta) attained an overall conformation similar to that of hCG, and could bind to the receptor and elicit biological response, suggesting that receptor binding and signal transduction can take place even with a molecule having blocked the C-terminus of the alpha subunit. The carboxyl terminal of the alpha subunit has been shown to be involved in hormone binding and signal transduction of all the heterodimeric glycoprotein hormones. However, deletion of five amino acids from the C-terminus of the alpha subunit in the single chain hCG did not alter the overall conformation of the fusion molecule and its receptor binding ability, but led to a significant reduction in its ability to elicit biological response. These data show that these five amino acids at the C-terminus of the alpha subunit in the single chain hCG are not absolutely essential for attaining a conformation required for receptor binding, but are essential for obtaining a full biological response.     

Comparison of the biological and immunological properties of glycosylation deficient human chorionic gonadotropin variants produced by site directed mutagenesis and chemical deglycosylation.

Appropriate glycosylation of gonadotropins is essential for the full expression of their biological activity. In this investigation we have compared the properties of glycosylation deficient human chorionic gonadotropin (hCG) produced by chemical deglycosylation (HF treatment--DG-hCG) or recombinant techniques (site directed mutagenesis). Among the recombinant hCG molecules secreted into the culture medium, the following variants containing selective N-glycosylation deletions at delta alpha 1 or delta alpha 1,2 or in both subunits could not stimulate steroidogenesis in mouse Leydig tumor cells (MA-10 cell line) and in this respect were very similar to DG-hCG. The other variants were fully active like native hCG, but the alpha + delta beta 1,2 recombinant hCG was a partial agonist. In radioimmunoassay with antibodies against native hCG, the DG-hCG as well as all recombinant hCG variants, including the wild type (WT), were similar. However, with antisera against DG-hCG or affinity purified antibodies specific to DG-hCG the alpha/delta 1,2 beta mutant, the WT hormone and native hCG were less active. In this assay mutants containing N-glycosylation deletions in the alpha subunit as well as the delta alpha 1,2/delta beta 1,2, variant showed higher activity. A similar pattern was evident in reaction with a selected monoclonal antibody showing preferential binding of 125I-labeled DG-hCG (antagonist). Affinity purified antibody directed against native hCG conformation was successful in converting DG-hCG and some inactive glycosylation deficient variants into an active form by stimulating progesterone in MA-10 cells. These data suggest that there are similarities as well as subtle differences in conformation of DG-hCG and various recombinant hCG molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Converting heterodimeric gonadotropins to genetically linked single chains New approaches to structure activity relationships and analogue design.

One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure; the subunits combine early in the secretory pathway and only the dimers are capable of binding to receptors. Therefore, assembly is rate limiting in the production of functional heterodimers, a problem encountered when removing the carbohydrates from one or both subunits as discussed in this review. If the heterodimers can be expressed as single chains, this might avoid mutagenesis-induced defects in secretion and combination of individual subunits for structure-function studies and analogue design. Here we discuss the feasibility of this approach for such problems.     

Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris

The pituitary-derived glycoprotein hormone FSH plays a central role in controlling vertebrate gonadal function. In female mammals the maturation of ovarian follicles is critically dependent upon stimulation by FSH. Moreover, injection of exogenous FSH is used extensively to stimulate increased numbers of follicles to ovulate. Structurally FSH is a heterodimeric glycoprotein composed of two non-covalently associated polypeptide subunits. The tertiary structures of both the alpha- and beta-subunits are constrained by intramolecular disulphide bonds and are post-translationally modified with two N-linked carbohydrate moieties, the structure of which appears to modulate in vivo biological activity. Here we report the expression of ovine FSH (oFSH) as a biologically active single-chain polypeptide using the methylotrophic yeast Pichia pastoris. Sequences encoding the mature oFSH alpha- and beta-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the beta-chain joined to the N-terminus of the alpha-chain, with the chains separated by a two amino acid linker sequence. This fusion gene was itself fused to two alternative Pichia leader sequences (mating factor alpha and acid phosphatase) and transformed into the Pichia strains GS115 and SMD1168. The recombinant fusion protein (oFSHbetaalpha) was expressed at approximately 0.1 microg/ml in 'shake-flask' cultures. The Pichia-expressed tethered protein was biologically active in an in vitro bioassay, had a molecular mass of 28 kDa, as determined by SDS-PAGE, and bound the bovine FSH receptor with a binding profile similar to that of native oFSH.     

Production of biologically active tethered tilapia LHbetaalpha by the methylotrophic yeast Pichia pastoris

In fish, luteinizing hormone (LH) stimulates processes leading to final oocyte maturation and ovulation in females, and spermiation in males. The hormone is a heterodimeric glycoprotein composed of two non-covalently associated subunits. In this study, we describe the expression of tilapia LH (tLH) as a biologically active, single-chain polypeptide using the methylotrophic yeast Pichia pastoris. The tLHbeta and alpha mature protein-coding sequences were joined to form a fusion gene that encodes a "tethered" polypeptide in which the tLHbeta-chain forms the N-terminal part and the alpha-chain forms the C-terminal part. A "linker" sequence of six amino acids (three Gly-Ser pairs) was placed between the beta- and alpha-chains to assist in the chimerization of the subunits, and a six-His tail was placed at the end of the beta-subunit, to enable purification of the recombinant protein. Western blot analysis of the pituitary LH resolved by SDS-PAGE yielded a band of 35 kDa, while the recombinant tLHbetaalpha had a molecular mass of 45 kDa, and was found to possess only N-linked carbohydrates. Recombinant tLHbetaalpha stimulated the release of 11-ketotestosterone from mature testes, whereas its release from immature testes was less pronounced.     

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.     

Conversion of human choriogonadotropin into a follitropin by protein engineering.

Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the alpha subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their beta subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG beta-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH beta and hFSH beta in receptor recognition and activation. Since the beta subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH beta subunits, coexpressed them with the human alpha subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH beta residues 33-52 for hCG beta residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG beta residues 108-145 or substitution of residues in the "determinant loop" located between hCG beta residues 93 and 100.     

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.     

Replacement of the invariant tyrosine in the CAGY region of the human chorionic gonadotropin β subunit

The mammalian glycoprotein hormone β subunits contain a highly conserved amino acid sequence, Cys-Ala-Gly-Tyr-Cys (residues 34–38 of human chorionic gonadotropin β), that is denoted as the ‘CAGY region’. Using site-directed mutagenesis we have replaced Tyr-37 in hCGβ, i.e., the invariant Tyr in all known mammalian CG, LH, FSH, and TSH β subunits, with two hydrophobic amino acid residues, Phe and Leu. The resultant mutant forms were characterized for α subunit binding and the resulting heterodimers were analyzed for biological activity using two in vitro assays with transformed murine Leydig cells (MA-10). Chinese hamster ovary cells containing a stably integrated gene for bovine α were transiently transfected with a eukaryotic expression vector containing a Rous sarcoma viral promoter and the wild-type and mutant cDNAs. The hCGβ(Phe-37) mutant bound to α essentially to the same extent as hCGβ wild-type, while the hCGβ(Leu-37) mutant formed somewhat less heterodimer. The heterologous heterodimeric mutant and wild-type gonadotropins were equipotent in a competitive binding assay with [¹²⁵I]hCG. In a steroidogenic assay, the mutant hormones were active, but they appeared slightly less potent than the wild-type form. Thus, this invariant Tyr can be replaced with another aromatic amino acid residue or with a hydrophobic, but not aromatic amino acid residue in hCGβ without any dramatic effect on function. These results indicate that Tyr-37 in hCGβ, while not obligatory, may participate, either directly or indirectly, in subunit assembly and that the hydroxyl group may function in a modulatory role in signaling.     

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.     

Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor

LH and FSH are heterodimeric glycoprotein hormones, composed of a common alpha-subunit non-covalently associated with a hormone-specific beta-subunit. Repeated efforts to isolate catfish FSH (cfFSH) have not been successful and only catfish LH (cfLH) has been purified from catfish pituitaries. Recently, however, we succeeded in cloning the cDNA encoding the putative cfFSHbeta; the cDNAs for the alpha- and beta-subunit of cfLH have been cloned before. Here we report the expression of biologically active cfLH and cfFSH in the soil amoeba, Dictyostelium discoideum. The biological activity of the recombinant hormones was analyzed using cell lines transiently expressing either the cfLH receptor or the cfFSH receptor. Moreover, a primary testis tIssue culture system served to study the steroidogenic potency of the recombinant hormones. Our results demonstrated that Dictyostelium produced biologically active, recombinant catfish gonadotropins, with recombinant cfLH being almost indistinguishable from its native counterpart, purified from pituitaries. Although recombinant cfFSH has significant effects in the bioassays used in this study, the specific function of native cfFSH in the control of reproduction and its expression patterns are not yet understood.     

Translational fusion of two beta-subunits of human chorionic gonadotropin results in production of a novel antagonist of the hormone

The strategy of translationally fusing the alpha- and beta-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGalphabeta expressed using Pichia expression system. Using the same expression system, another analog, in which the alpha-subunit was replaced with the second beta-subunit, was expressed (hCGbetabeta) and purified. hCGbetabeta could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGalphabeta and hCGbetabeta using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGbetabeta interacts with two LH receptor molecules. These studies demonstrate that the presence of the second beta-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of alpha-subunit, the molecule is unable to elicit response. The strategy of fusing two beta-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.     

Properties of human chorionic gonadotropin produced in vitro by ovarian carcinoma cells.

The present study was designed to compare the immunological, physical, and biological properties of native hCG with an hCG molecule secreted ectopically in vitro by an ovarian adenocarcinoma cell line maintained in long term tissue culture. The hCG produced by the cell line was concentrated by ultrafiltration of the tissue culture medium. The inhibition curves generated by serial dilutions of the culture medium concentrates were parallel to those obtained with purified urinary hCG in the beta-hCG RIA and the rat Leydig cell radioreceptor assay (RRA). The ectopic hCG also reacted with an antibody generated against the carboxyl-terminal peptide (109-145) of beta-hCG. The immunoreactive material cochromatographed with urinary hCG on a Sephadex G-100 column, as determined by the beta-hCG RIA and RRA. Neither free alpha nor free beta subunits were found in the tissue culture medium. The tissue culture gonadotropin was adsorbed onto a Concanavalin A-Sepharose column and could be eluted with alpha-D-methylglucoside. The biological activity of the ectopic hCG was 9289 IU/mg, as determined by the ventral prostate weight (VPW) method in hypophysectomized immature male rats. The biological to immunological ratios by the ventral prostate weight method and RRA were 1.79 and 2.17, respectively. The in vivo disappearance rate of ectopic hCG after injection into immature female rats was significantly faster than that of placental or urinary hCG, but was considerably slower than the disappearance rate of human LH. These studies demonstrate that the immunoreactive and biologically active portions of the hCG produced by the ovarian adenocarcinoma cell line and native hCG are similar or identical. The faster disappearance rate of the ectopic hCG in the rat model may be due to incomplete sialylation of the oligosaccharide moiety of the hCG molecule.     

Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells.

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunits. The beta subunit of hCG (hCG beta) is unique among the beta subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either the hCG beta gene alone or together with the hCG alpha gene were transfected into a mutant Chinese hamster ovary cell line, IdID, which exhibits a reversible defect in O-glycosylation. Our results reveal that hCG beta can be secreted normally in the absence of its O-linked oligosaccharides. hCG beta devoid of O-linked carbohydrate can also combine efficiently with hCG alpha and be secreted as an intact dimer. We conclude that in Chinese hamster ovary cells, the hCG beta O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.     

Expression of active secreted forms of human amyloid beta-protein precursor by recombinant baculovirus-infected insect cells.

Three alternatively spliced forms of the amyloid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vector system. The recombinant proteins were secreted into the culture medium by infected insect cells, and APP molecules were detected in insect cells and medium 2 days after infection with the recombinant APP-baculoviruses. A partial sequence of the NH2 terminus of the secreted protein revealed identity with the native secreted protein and showed that the signal peptide was recognized and properly cleaved in insect cells. Purified secreted recombinant APP-751 comigrated with protease nexin 2 purified from platelets and fibroblasts. A 15-kDa COOH-terminal fragment of APP was also detected in cells infected with recombinant baculoviruses, suggesting that recombinant APP proteins were cleaved at the COOH-terminal end like native APP protein. Recombinant APP-751 and APP-770 formed complexes with epidermal growth factor-binding protein, whereas APP-695 did not. In addition, recombinant APP-751 and APP-770 inhibited trypsin and chymotrypsin activity, whereas APP-695 did not. Growth of a human fibroblast cell line, A-1, that required APP for complete growth, was restored upon addition of secreted recombinant APP-695 or APP-751. Thus, the appropriately sized, secreted recombinant APP proteins produced in this expression system are biologically active.     

Cellular localization of chorionic gonadotropin in human fetal kidney and liver

Earlier, we reported that second trimester human fetal kidney and, to a much lesser extent, human fetal liver were capable of synthesizing and secreting the beta-subunit of hCG. Recently, we also have shown that these tissues, likewise, synthesize and secrete the alpha-subunit of hCG. The hCG produced is biologically active. To determine the cellular localization of these peptides, immunocytochemical studies were performed on human fetal tissues using antibodies against beta hCG, alpha hCG, and the intact hormone. Placental syncytiotrophoblast served as an immunopositive control. In the human fetal kidney, the ascending (thick) limb of the loop of Henle, distal convoluted tubule, and occasional cells in the collecting ducts were distinctly immunopositive for both beta hCG and the alpha-subunit. Small amounts of light positive staining occurred in only a few hepatocytes. Placental syncytiotrophoblast was routinely positive for both subunits, but fetal lung and striated muscle were negative. These immunocytochemical results indicate that immunoreactive beta hCG as well as the alpha-subunit are present in placental syncytiotrophoblast, in the distal renal nephron, and in a limited population of hepatocytes. The qualitative number and intensity of immunopositive cells closely correlate with the quantitative amounts of their hCG subunit synthesis. Taken together with our previous biosynthetic data, the immunocytochemical localization reported here indicates the probable cellular sites of alpha- and beta hCG synthesis in these tissues. The presence of comparable alpha- and beta-subunit staining in identical cell populations suggests that both hCG subunits and, therefore, perhaps intact hCG are produced at these same cellular sites during fetal life.