Selenomethionyl analog of recombinant human choriogonadotropin

Selenomethionyl and high mannose type analog of recombinant human choriogonadotropin (hCG) to solve the crystallization and phase problems has been obtained by gene transfer methodology. SF9 insect cells were infected with the recombinant viruses containing hCG alpha and hCG beta cDNAs in selenomethionine containing methionine-free Grace's medium. The selenomethionyl hCG (SehCG) was purified from the culture medium by one step immunoaffinity chromatography using an immobilized monoclonal antibody against hCG beta. The presence of selenomethionine was demonstrated by amino acid analysis of SehCG. The amino acid composition indicated that more than 84% of methionine residues were substituted by selenomethionine. Its sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis yielded a single 38-kDa protein band under nonreducing conditions. The carbohydrate analysis of SehCG was consistent with the presence of four N-linked high mannose type carbohydrates and four O-linked simple disaccharide chains. The in vitro immunological and biological studies of SehCG indicated that selenomethionine substitution had no effect on the immunopotency, receptor binding, and steroidogenic activities of the hormone.     

Human choriogonadotropin binds to a lutropin receptor with essentially no N-terminal extension and stimulates cAMP synthesis.

The lutropin (LH) receptor, which belongs to the family of G-protein coupled receptors, consists of an extracellular hydrophilic N-terminal extension of 341 amino acids and a membrane-embedded C-terminal region of 333 amino acids. This C-terminal region comprises a short N terminus, seven transmembrane domains, three cytoplasmic loops, three exoplasmic loops, and a C terminus. Recently, it was reported that the N-terminal extension of the LH receptor alone or a naturally occurring variant LH receptor similar to the N-terminal extension is capable of binding the hormone with an affinity slightly higher than that of the native receptor. This finding raises a question as to whether the N-terminal extension represents the entire hormone binding site and, if so, how is hormone binding transduced to the activation of a G-protein? In an attempt to answer this important question, we have prepared truncated receptors containing an N-terminal extension as short as 10 amino acids. Surprisingly, the truncated receptors were not only capable of binding the hormone, albeit with low affinities, but also capable of stimulating cAMP synthesis. These results suggest a possibility that the hormone, at least in part, interacts with the membrane-embedded C-terminal region and modulates it to activate adenylate cyclase. The low hormone binding affinities of the truncated receptors taken together with high affinity hormone binding to the N-terminal extension of the LH receptor indicate the existence of two or more contact points between the receptor and the hormone.     

Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity

The lutropin-choriogonadotropin (LH/CG) receptor is a cell surface receptor comprised of two domains of roughly equivalent size. The amino-terminal half of the receptor is relatively hydrophilic and is located extracellularly, whereas the carboxyl-terminal half of the receptor shares amino acid homology with other receptors that couple to G proteins and is similarly thought to span the plasma membrane seven times, ending with a relatively short carboxyl-terminal tail. In order to test the role of the extracellular domain in binding hormone, we constructed a mutated rat luteal LH/CG receptor cDNA (termed pCLHR-D2), which encodes for only the extracellular domain, and used it to transiently transfect human kidney 293 cells. Here we report that the expressed extracellular domain of the LH/CG receptor is capable of binding human CG with a high affinity, comparable with that of the full-length receptor. Thus, not only is the extracellular domain of the glycoprotein hormone receptors involved in binding hormone, but it alone is capable of conferring high affinity binding. Unexpectedly, it was also found that this truncated receptor is not secreted into the culture media but remains trapped within the cells.     

Effects of antibodies to choriogonadotropin in malignant growth

Summary We have studied the effects of preimmunization with a conjugate of the -subunit of choriogonadotropin and tetanus toxoid (CG-tt) on the growth of the implanted R 3230 AC mammary adenocarcinoma (in Fischer 344 rats) and the implanted 5123 1-1 hepatoma (in Buffalo rats) after 20 days, in order to determine if the in vivo production of antibodies against CG could modify the relationship between host and malignant growth. The results obtained demonstrated that active immunization against CG retarded significantly (P<0.01) the growth of the two transplantable tumors. Anti-CG antibodies were also determined and constantly found in the sera of all the preimmunized rats of both strains while no antibodies to CG were found in the control animals.     

Enhanced thermal stability of chemically deglycosylated human choriogonadotropin

Exposure of aqueous solutions of native human choriogonadotropin (hCG), asialo-hCG (A-hCG), and chemically deglycosylated hCG (DG-hCG) to heat treatment revealed significant differences in their stability. Solutions of hCG and A-hCG were rapidly inactivated above 50 degrees C. On the other hand, solutions of DG-hCG were comparatively more stable under similar conditions as shown by the retention of significant receptor binding, immunological, and hormonal antagonistic activities. Heated solutions (100 degrees C) of hCG and A-hCG quickly lost their ability to enhance the fluorescence of the probe 1-anilino-8-naphthalenesulfonate (1,8-ANS) indicating dissociation into subunits. DG-hCG solutions were more stable in this respect suggesting significant preservation of conformational features required for the interaction with 1,8-ANS. Solutions of hCG and A-hCG which had been thermally denatured (100 degrees C, 10 min) required almost 48 h at 37 degrees C to regain complete ANS binding ability as well as receptor binding activity. Under the same conditions, heated solutions of DG-hCG completely regained these abilities in less than 2 h. A similar pattern was observed with acid (pH 2.0)-dissociated hCG, A-hCG, and DG-hCG. While heated solutions of hCG had no effect on the action of native hCG in vitro, heated DG-hCG solutions still retained their ability to antagonize the cyclic AMP accumulation or steroidogenesis induced by native hCG in rat interstitial cells. Thus, removal of carbohydrate residues (approximately 75% loss) from hCG renders the hormone more resistant to thermal denaturation.     

Properties of the phosphorylated beta subunit of human choriogonadotropin

The beta subunit of human choriogonadotropin (hCG) was previously shown to be phosphorylated by beef skeletal muscle cAMP-dependent protein kinase (Keutmann, H. T., Ratanabanangkoon, K., Pierce, M. W., Kitzmann, K., and Ryan, R. J. (1983) J. Biol. Chem. 258, 14522-14527). The phosphorylation site was primarily at Thr 97, located within the "determinant loop" region proposed by Ward and Moore (Ward, D. N., and Moore, W. T. (1979) Animal Models for Research in Fertility and Contraception (Alexander, N. J., ed) pp. 151-164, Harper and Row, Baltimore, MD) to be important for hormonal activity and specificity. Biological and immunological studies were carried out to determine the effect of this modification on the activity of hCG. The phosphorylated hCG beta recombined with hCG alpha to form phosphorylated hCG (*hCG). The recombined *hCG retained full immunological activity in a radioimmunoassay using anti-hCG serum and 125I-hCG. The biological activities of the *hCG on appropriate rat gonadal cells, as studied by hCG radioreceptor assay, follitropin radioreceptor assay, and adenylate cyclase assay, were 0.29 +/- 0.04, 0.29 +/- 0.07, and 0.69 +/- 0.13 times as potent as hCG, respectively. The reduced receptor binding activity of *hCG was not due to dissociation of the hormone into its subunits. The far ultraviolet CD spectrum of the *hCG showed no gross conformational change induced by phosphorylation. We conclude the following: 1) Thr 97 is not involved in subunit-subunit interaction and is not part of the hCG antigenic site recognized by this particular antiserum; 2) Thr 97 does appear to participate in the hCG-receptor interactions; 3) modification of Thr 97 does not result in an enhancement of follitropin-like activity.     

Structural requirements for mutational lutropin/choriogonadotropin receptor activation

Different activation mechanisms of glycoprotein hormone receptors, which are members of the G protein-coupled receptor superfamily, have been proposed. For example, the large ectodomain of glycoprotein hormone receptors may function as an inverse agonist keeping the transmembrane domain in an inactive conformation. To provide support for this hypothesis, we have generated different lutropin/choriogonadotropin receptor (LHR) constructs lacking the ectodomain. Although some ectodomain-deficient LHR constructs were targeted to the cell surface, cAMP levels remained unchanged under basal conditions and agonist application but could be increased by a mutation within the transmembrane domain 6 (D578H). Taking advantage of a constitutive activating mutation (S277N) located in the extracellular domain, we showed that the intact leucine-rich repeat-containing ectodomain is essential for constitutive activation of the LHR by mutation of the hinge region. Our findings support an activation scenario in which agonist binding or mutational alterations expose a structure within the ectodomain, which then activates the transmembrane core.     

Tryptic digestion of the alpha subunit of human choriogonadotropin

In order to further characterize chemical, physicochemical, and immunochemical properties, as well as structure-function relationships, of the common alpha subunit of human glycoprotein hormones, a tryptic core was prepared from the alpha subunit of human choriogonadotropin. The core was purified in greater than 80% yield using gel permeation and anion-exchange chromatography, and, following reduction and S-carboxymethylation, the constituent peptides were purified by gel permeation and high performance liquid chromatography. The disulfide-bridged peptides comprising the alpha core were identified as residues 1-35 and residues 52-91 by amino acid composition and amino acid carboxyl sequence analyses of the reduced, S-carboxymethylated peptides. The alpha tryptic core contained both N-asparagine carbohydrate moieties, but was devoid of residues 36-51 and the carboxyl-terminal serine at position 92. The small peptides cleaved from residues 36-51, a known potential O-glycosylation region of the alpha subunit, were purified and identified. The tryptic core retained full immunopotency relative to the intact subunit in the binding to polyclonal and monoclonal antibodies directed against the alpha subunit. The region consisting of residues 36-51 is not part of the epitope recognized by these antibodies. With antisera generated to the reduced, S-carboxymethylated subunit, peptide 1-35, but not 52-91, was immunoreactive. This finding is consistent with the known dominant antigenicity of the amino-terminal region in the reduced, S-carboxymethylated molecule. The core exhibited no appreciable interaction with the complementary beta subunit, and, not surprisingly, was unable to compete with intact hormone binding in a radioreceptor assay using rat testicular homogenates. Circular dichroic spectroscopy was used to probe gross features of tertiary structure (240-300 nm) and secondary structure (190-240 nm). The tryptic core and each of the two constituent peptides exhibited spectra above 240 nm that resembled that of the reduced, S-carboxymethylated subunit more than that of the native material, thus suggesting a significant loss of tertiary structure in the core and isolated peptides. This finding is unexpected in consideration of the full retention of immunopotency by the alpha core although consistent with failure of the core to combine with intact complementary beta subunit. The intact subunit as well as the isolated constituent peptides exhibit little if any helicity in aqueous solution. Interestingly, the reduced, S-carboxymethylated chain and peptide 52-91 displayed helicity in 80% trifluoroethanol, a helicogenic solvent.     

Recombinant carbohydrate and selenomethionyl variants of human choriogonadotropin.

Recombinant human choriogonadotropin and selenomethionyl human choriogonadotropin (rhCG and SehCG) were expressed in baculovirus expression system by coinfection of SF9 insect cells by recombinant viruses, AcMNPV-hCG alpha and AcMNPV-hCG beta containing hCG alpha and hCG beta cDNAs. The expression efficiency of both rhCG and SehCG was quite high. The association of the alpha and beta subunits into a dimer was apparently complete since no detectable amount of rhCG beta was found in the rhCG eluate from the monoclonal hCG beta antibody immunoaffinity column. Both rhCG and SehCG preparations were homogeneous as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography. The apparent molecular mass of rhCG and SehCG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions was about 38 kDa while under reducing conditions the heterodimer dissociated to yield beta and alpha subunits with molecular masses of 22.5 and 18 kDa, respectively. The carbohydrate analysis of rhCG showing the presence of 2.1, 3.3, 7.38, 4.2, and 27.8 residues of Fuc, GalNAC, GlcNAC, Gal, and Man, respectively, per mole of the hormone was consistent with the presence of 4 N-linked high mannose type carbohydrate hydrate and 4 O-linked simple carbohydrate chains, probably made up of Gal-GalNAC. Despite the altered glycosylation, rhCG demonstrated close similarity to the native urinary hCG in amino acid composition, receptor binding, and in its ability to stimulate cAMP and steroidogenesis. This indicates that there is no specificity of carbohydrate required for biological activity. Furthermore, it implies that the alteration from the complex to high mannose type carbohydrates in rhCG does not affect its proper folding. Finally, amino acid analysis of SehCG showed that 84% of methionine residues in rhCG were replaced by selenomethionine.     

Evidence for impaired subunit interaction in chemically deglycosylated human choriogonadotropin

Using three different methods evidence was obtained that native and deglycosylated choriogonadotropin show differences in their conformations which might account for the antagonistic properties of deglycosylated choriogonadotropin: 1. In the deglycosylated hormone additional peptide bonds were susceptible to chymotrypsin. 2. In the far ultraviolet circular dichroism only small differences existed between native and deglycosylated choriogonadotropin. However, in 80% hexafluoropropanol the deglycosylated hormone adopted a higher degree of ordered structure. 3. At 37 degrees C the deglycosylated hormone showed a 13 fold increase of the dissociation rate into subunits at pH 3 in comparison to native choriogonadotropin. The results provide evidence that in chemically deglycosylated choriogonadotropin the subunit interactions are disturbed due to conformational changes.     

Characterization of a cleavage product in the human choriogonadotropin beta-subunit

The various molecular forms of human chorionic gonadotropin present in a crude preparation of urine from pregnant women were analyzed by two-dimensional gel electrophoresis and immunoblotting with monoclonal antibodies directed to synthetic peptides corresponding to the carboxyl-terminal part of either the alpha or beta-subunit. Under reducing conditions, immunoblotting with antibodies directed to the beta-subunit revealed the presence of a low-molecular-weight material of 22 kDa. This molecular form had large heterogeneity, as analyzed by isoelectrofocusing; it was immunoreactive with antibodies directed to the 111-145 region. Using microsequencing techniques, we found that the fragment had a NH2 terminal portion corresponding to the sequence of the beta-subunit appearing from residue 48. Thus, the 22-kDa fragment comprises the 48-145 portion of the beta-subunit and is probably a cleavage product of the native protein with intrachain nicking.     

The beta-subunit of human choriogonadotropin interacts with the exodomain of the luteinizing hormone/choriogonadotropin receptor and changes its interaction with the alpha-subunit.

Human CG (hCG) consists of a common alpha-subunit and a hormone-specific beta-subunit. Similarly, its receptor is also composed of two domains, an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain). hCG initially binds the exodomain of the receptor after which the resulting hCG/exodomain complex is thought to interact with the endodomain. This secondary interaction is considered responsible for signal generation. Despite the importance, it is unclear which hormone subunit interacts with the exodomain or the endodomain. As a step to determine the mechanisms of the initial and secondary interactions and signal generation, we investigated the interaction of the hormone-specific beta-subunit in hCG with the receptor's exodomain. A photoactivable hCG derivative consisting of the wild-type alpha-subunit and a photoactivable beta-subunit derivative was prepared and used to label the exodomain. The analysis and immunoprecipitation of photoaffinity labeled exodomain demonstrate that the beta-subunit in hCG makes the direct contact with the exodomain.     

The differential binding affinities of the luteinizing hormone (LH)/choriogonadotropin receptor for LH and choriogonadotropin are dictated by different extracellular domain residues

The high degree of amino acid sequence homology and the divergent ligand binding affinities of the rat (r) and human (h) LH receptors (LHRs) allowed us to identify amino acid residues of their extracellular domain that are responsible for the different binding affinities of bovine (b) and hLH, and human choriogonadotropin (hCG) to the hLHR and rLHR. Because of the proposed importance of the beta-sheets of the leucine-rich repeats (LRRs) of the extracellular domain of the LHR on hormone binding, we examined 10 divergent residues present in these regions by analyzing two complementary sets of mutants in which hLHR residues were substituted with the corresponding rLHR residues and vice versa. These experiments resulted in the identification of a single residue (a Ile or Ser in the C-terminal end of LRR2 of the hLHR or rLHR, respectively) that is important for hLH binding affinity. Surprisingly, however, this residue does not affect hCG or for bLH binding affinity. In fact, the results obtained with bLH and hCG show that several of the divergent residues in the beta-sheets of LRR1-9 affect bLH binding affinity, but none of them affect hCG binding affinity. Importantly, our results also emphasize the involvement of residues outside of the beta-sheets of the LRRs of the LHR in ligand binding affinity. This finding has to be considered in future models of the interaction of LH/CG with the LHR.     

Sulfation of the choriogonadotropin alpha subunit in human placental explants

Incubation of first trimester placental explants with [35S]O4 resulted in incorporation of radioactive sulfate into free and dimer forms of alpha subunit of human chorionic gonadotropin. Sulfate was not attached to N-linked oligosaccharides since it was not released by endoglycosidase F. Analysis of pronase digest revealed the presence of tyrosine-O-[35S]O4. Comparison of tryptic peptides of alpha subunit labeled with several amino acids identified the penultimate carboxyterminal peptide as the sulfation site. Since the C-terminal region of the hCG alpha plays a critical role in receptor binding of the hormone, modification in this region may regulate hormonal activity.     

Variant forms of the pig lutropin/choriogonadotropin receptor.

The cloning and sequencing of porcine lutropin/choriogonadotropin (LH/hCG) receptor messenger RNAs have shown the presence of a full-length receptor (pLHR-A) and of shorter variants lacking either the transmembrane and the intracellular domains (pLHR-B and pLHR-C) or only the transmembrane domain (pLHR-D). Moreover, immunoblotting of testicular membrane extracts has detected 85-, 68-, and 45-48-kDa proteins reacting with antireceptor antibodies. Transfection experiments were performed to assign the protein species to the various messenger RNAs and to study the function of the various receptor species. COS-7 and L-cells transfected with an expression vector encoding full-length receptor pLHR-A yielded a protein of apparent molecular mass of 105 kDa. This corresponded to the complete receptor which had undergone a different glycosylation pattern to that found in testis, since after digestion with peptide N-glycosidase F both the 105-kDa COS-7 protein and the 85-kDa testicular glycoprotein yielded a holoprotein of approximately 63 kDa. Transfection with pLHR-A also yielded a high proportion of the 68-kDa glycoprotein which was shown by digestion with endoglycosidase H to be a high-mannose precursor of the full-length receptor. The existence of a large pool of precursor species in both transfected cells and Leydig cells evokes possible physiological regulations at the level of receptor maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of recombinant human choriogonadotropin in Chinese hamster ovary glycosylation mutants

Human CG, a member of the glycoprotein hormone family that includes LH, FSH, and TSH, is composed of two nonidentical subunits each containing two asparagine linked (N-linked) oligosaccharides. The role of the oligosaccharides in the action of these hormones is unclear. To examine the structure-activity relationships of the glycoprotein hormone oligosaccharides using nonenzymatic and nonchemical methods, we transfected CG subunit genes into mutant cell lines derived from Chinese hamster ovary cells. Two mutant cell lines that synthesize truncated oligosaccharides were used. Cell line 15B, lacking N-acetylglucosaminyltransferase I, synthesizes N-linked carbohydrates containing Man5 oligomannosyl structures, and 1021, defective in transporting CMP-sialic acid into the Golgi, results in sialic-acid deficient glycoproteins. The binding of these derivatives to the LH/CG receptor did not differ significantly from purified CG (CR119), but the ability of the mutant hormones to stimulate cAMP biosynthesis in vitro is reduced compared to wild-type CG or CR119. Since the amino acid sequence of CG from the mutant and wild-type cells is identical, these data indicate that oligosaccharide structures, while not influencing receptor binding, directly affect signal transduction.     

Effects of adenine nucleotides on choriogonadotropin alpha and beta subunit synthesis

The alpha subunit of human chorionic gonadotropin (CG) contains a discrete cAMP response element in the 5' flanking region of the gene. Since cAMP also stimulates the synthesis of the CG beta subunit the presence of a cAMP cis element in the CG beta gene was examined. Deletion mutants bearing various lengths of CG beta 5' region in front of the chloramphenicol acetyl transferase (CAT) gene were transfected in placental tumor cells. No discrete cAMP response element could be identified. Unexpectedly we also observed that AMP and adenosine not only stimulated CAT activity driven by CG beta promoter sequences but also enhanced synthesis of CG alpha and beta subunits in cultured choriocarcinoma cells. GMP, CMP, guanosine, and cytosine were inactive at comparable concentrations. These data suggest that the response of the CG alpha and beta genes to the non-cyclic adenine derivatives occurs by a mechanism that differs from cAMP.     

Characterization of the expression products of recombinant human choriogonadotropin and subunits

Human choriogonadotropin (hCG) is a placental glycoprotein hormone composed of a 92-amino acid alpha subunit noncovalently linked to a 145-amino acid beta subunit. We report here the expression of biologically active hCG in mouse C127 cells transfected with expression vectors containing the DNA coding for both subunits. In addition, the same cell line was used to express the alpha subunit alone. The expression products were purified by affinity chromatography using specific monoclonal antibodies to hCG or its subunits. The system secreting biologically active hCG also produced a 10-fold or greater molar excess of free beta subunit. The dimeric hormone, as well as the excess beta subunit, resembles the standard urinary hCG and beta subunit by chemical and biological criteria. In contrast, when the vector encoding for the alpha subunit was expressed alone, the alpha subunit had a higher molecular weight than both standard alpha and the alpha found in the expressed dimeric hormone. The molecular weight difference between expressed alpha subunit and standard alpha was found to reside in the alpha peptide consisting of residues 52-91 which contained all of the carbohydrate of the alpha subunit. The N-asparagine-linked carbohydrate moieties in the recombinant alpha were found to be triantennary in contrast to biantennary in urinary alpha, and this hyperglycosylation was responsible for the higher molecular weight of the alpha subunit when it was expressed alone. We found no evidence of O-threonine glycosylation at position alpha 39 reported to be present in free forms of the alpha subunit; however, the companion paper (Corless, C.L., Bielinska, M., Ramabhadran, T. V., Daniels-McQueen, S. Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. (1987) J. Biol Chem. 262, 14197-14203) finds a small quantity of O-glycosylation. Since the excess beta subunit appears to be of normal size and contains the expected complement of sugars, only free alpha subunit seems to be a potential substrate for addition of extra sugar moieties. No large beta subunit forms have been found by others, while large alpha subunits have been described both clinically and in tissue culture systems. These observations imply that the conformation of the free alpha subunit, in the regions of the glycosylation recognition sites, allows easier access for glycosyltransferases than those same sites in the beta subunit. When alpha is combined with beta, the local structures around the alpha glycosylation sites are apparently altered so as to make the synthesis of triantennary chains less favorable.