Subunit interaction of human chorionic gonadotropin (hCG) with rat ovarian luteinizing hormone (LH)/CG receptor.

The subunit interaction of hCG with its rat ovarian LH/CG receptor was studied by cross-linking the solubilized receptor-hormone complex with glutaraldehyde (GA), disuccinimidyl suberate (DSS) or dithiobis(succinimidyl propionate) (DSP) and analyzing the complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The hormone was labeled either in its alpha-subunit (125I-hCG) or in its beta-subunit (3H-hCG) or the label (3H) was introduced into the receptor molecule instead of the hormone. All of the labeling procedures led to the detection of only the receptor-(alpha,beta)hCG and receptor-(alpha)hCG complexes on the autoradiograms. The sizes of these complexes were 137,000 and 106,000, respectively, under reducing conditions. These results suggest that the receptor binds one hormone molecule, and that hCG interacts with the receptor mainly through its alpha-subunit. In addition, polyclonal antibodies directed against the LH/CG receptor and the alpha- and beta-subunits of hCG were used to detect the non-reduced receptor-(alpha,beta)hCG complex in immunoblotting. As antibodies directed against both the alpha-subunit and the beta-subunit were able to detect the Mr 130,000 complex, it is conceivable that both of the subunits are at least partially exposed on the receptor-hormone complex. 125I-hCG was also cross-linked to the membrane-bound receptor. The membrane-bound complex had an Mr of 144,000 under reducing conditions, i.e. approximately 7000 higher than that of the solubilized complex (Mr 137,000). This may indicate that the membrane-bound receptor is covalently modified or differs in conformation from the solubilized receptor.     

Purification and characterization of an incompletely glycosylated form of human chorionic gonadotropin from human placenta

A small form of hCG (SP-hCG) was purified from an acetone powder preparation of human first trimester placenta by repeated gel filtration and ion exchange chromatography on a Q-Sepharose or FPLC Mono Q column. The estimated mol wt (Mr) of the small hCG by gel filtration is 43K compared to 58K for authentic hCG. The pI of SP-hCG is 10.0, suggesting deficiency of sialic acids. SP-hCG dissociates into subunits when treated with 6 M guanidine-HCl or analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two beta-subunits of SP-hCG were found with estimated Mr of 23K and 20K. Both are distinctly smaller than authentic hCG beta. A single alpha-subunit was found, with an estimated Mr of 21 K. The immunoactivity (8,900-10,000 IU/mg) of highly purified SP-hCG was comparable to that of reference hCG (CR119) determined by a RIA method using anti-hCG antibodies. The hCG/LH receptor-binding activity of SP-hCG is equivalent to that of reference hCG (CR119). Its biological activity is lower than that of reference hCG (approximately 30% or more) assayed by the in vitro stimulation of rat Leydig cells to produce testosterone and cAMP. A high dose is required to attain the same level of stimulation as reference hCG. The amino acid composition of SP-hCG is similar to that of reference hCG, whereas its hexsamine content is significantly lower. Its glucosamine content is about half that in reference hCG, while it completely lacks galactosamine. These findings suggest that SP-hCG is deficient in O-linked oligosaccharide chain in the beta-subunit, and that the N-linked oligosaccharide chains of both subunits are shortened. SP-hCG is one of the principal forms of the hormone present in first trimester placenta and may be a key intermediate in the posttranslational biosynthesis of hCG. Although it lacks O-linked sugar chains and shortened N-linked sugar chains, it possesses substantial biological activity. To have full biological activity, the hCG molecule must contain the complete complement of sugar chains.     

Phosphorylation and glycosylation of the luteinizing hormone receptor.

Purified testicular and ovarian luteinizing hormone/human chorionic gonadotropin (hCG) receptors are phosphorylated at serine and threonine residues by the catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). Occupancy of the receptors by hCG significantly increased the rate but not the extent of phosphorylation. However, prolonged preincubation of receptors with hCG reduced the subsequent rate of receptor phosphorylation. Identical phosphopeptide maps were obtained for the phosphorylated ovarian and testicular receptors. The phosphorylated receptor, like the native receptor, bound to wheat germ lectin and hCG-Sepharose and migrated as a single band of Mr 90,000 (testis) and Mr 85,000 (ovary) on NaDodSO4/PAGE. Neuraminidase treatment of receptors caused reductions of molecular weight to 82,000 (testis) and 77,000 (ovary), and further treatment with O-Glycanase had minimal effect on molecular size. However, deglycosylation with N-Glycosidase and endoglycosidase F produced a single labeled polypeptide of Mr 59,000 for both gonadal receptors. Treatment of native receptors with neuraminidase caused no apparent change in binding of gonadotropin to blotted receptors, whereas deglycosylated receptors showed a major reduction in hormone binding. These results indicate that luteinizing hormone/hCG receptors are sialoglycoproteins with predominantly N-linked glycosyl residues that account for the size difference between testicular and ovarian receptors and that may participate in the interaction with gonadotropin. Receptor occupancy by agonist leads to a conformational change that facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to hCG.     

Glycosylation characteristics of the mouse liver lactogenic receptor

The structural characteristics and glycosylation properties of the lactogenic receptor were examined in 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized plasma membranes from female mouse liver. The specific binding of the radioiodinated human growth hormone [( 125I]hGH) was displaced with an equivalent potency by both hGH and prolactin. After a mild neuraminidase treatment, this binding was increased by 40%, as a result of an increase in receptor affinity. Affinity chromatography on immobilized lectins revealed that the [125I]hGH-receptor complexes were specifically retained and eluted from ricin lectin-agarose, concanavalin A and lentil lectin, indicating the presence of N-linked glycans. Covalent cross-linking of solubilized [125I]hGH-receptor complexes with disuccinimidyl suberate, followed by analysis by sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE) under reducing conditions, and autoradiography resulted in the appearance of two bands with apparent Mr approximately 62,000 and approximately 100,000. The labelling of these bands was prevented by unlabelled hGH or ovine prolactin (oPrl) but not by bovine growth hormone (bGH). Neuraminidase treatment of the two receptor forms resulted in increased electrophoretic mobility which was inhibited by simultaneous addition of sialyl-lactose, a neuraminidase substrate. The both cross-linked forms were unaffected by endoglycosidase H, while endoglycosidase F decreased the molecular weight of each of the forms by about 8000 Da, yielding bands at Mr approximately 54,000 and approximately 92,000. In conclusion, taking into account that hGH is a Mr 22,000 polypeptide, the two forms of the receptor correspond to glycoproteins of Mr approximately 40,000 and approximately 78,000, respectively. They contain polypeptide backbones of Mr approximately 32,000 and approximately 70,000, and complex N-linked oligosaccharide chains with terminal sialic acid residues which could be involved in receptor binding affinity.     

Fragmentation of the beta-subunit of human chorionic gonadotropin produced by choriocarcinoma

When human chorionic gonadotropin (hCG) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions with dithiothreitol (DTT), a smaller weight material (CTP'), in addition to the beta-subunit, could be detected by Western blot analysis using antiserum for hCG beta-carboxy-terminal peptide (CTP). The CTP' band was much more apparent with urinary hCG from a patient with choriocarcinoma than with that from normal pregnant women. Second-dimensional electrophoresis of the choriocarcinoma hCG (c-hCG) after reduction with DTT indicated that the CTP', Mr 25,000, was released from the beta-subunit. The carbohydrate structure of the CTP' was analyzed by affinity with lectin-peroxidase on a nitrocellulose membrane. The CTP' did not interact with Concanavalin A, but exhibited strong interaction with both RCA120 and Arachis hypogaea after removal of sialic acid, indicating that it was released as a fragment containing an O-linked sugar chain as was found in the hCG beta carboxy-terminal portion. Western blot analysis using the antisera for hCG alpha, hCG beta, and hCG beta-CTP showed that the CTP' contains not only the carboxy-terminal portion but also a part of the internal (core) portion of the beta-subunit molecule. This dissociation of the c-hCG beta was further supported by the presence of a faster moving component (FMC) which may correspond to the NH2-terminal side counterpart. The desialylated FMC could be detected by Concanavalin A and RCA120 but not by Arachis hypogaea, indicating that it contains N-linked rather than O-linked sugar chains. The FMC does not contain any of the epitopes for the antisera examined in Western blot. These results indicate that the beta-subunit of the choriocarcinoma urine hCG has an unusual site which is dissociated into two components of Mr 25,000 (CTP') and Mr 18,000 (FMC) by DTT reduction.     

Similarity of the clearance rates of free alpha-subunit and alpha-subunit dissociated from intact human chorionic gonadotropin, despite differences in sialic acid contents

It is well known that the MCR of proteins can be influenced by their carbohydrate structure; i.e. the presence of terminal galactose on proteins results in uptake by hepatic receptors for galactose-terminated glycoproteins. Thus, a protein with its galactose residues covered or removed exhibits a far longer half life in plasma than one with its galactose residues exposed. The free alpha-subunit of human CG (hCG) has been shown to have a different carbohydrate composition than does the alpha-subunit dissociated from the intact hormone. In our laboratory, analysis of alpha-subunits isolated from pregnancy urine indicated that the alpha-subunit dissociated from hCG (hCG alpha) contains primarily monosialylated oligosaccharides, whereas the free alpha-subunit contains more than one sialic acid per oligosaccharide. This difference in the degree of sialylation prompted us to examine the clearance rates of these two subunits. Accordingly, free alpha and hCG alpha were purified by affinity chromatography and labeled with 125I. The labeled subunits were injected iv into rats and serum samples were removed at various time intervals over a 2-h period. The amount of [125I]alpha-subunit remaining in the serum was determined by immunoprecipitation using an antiserum to hCG alpha. The disappearance curves for the two subunits were indistinguishable and could be analyzed by a biexponential model. The t 1/2 of the faster component was 5 min, while the t 1/2 of the slower component was 74 min. In order to determine whether or not terminal galactose was present on either the hCG alpha or the free alpha-subunit, the labeled molecules were subjected to lectin column chromatography using Ricin or peanut lectin linked to agarose. Both of these lectins bind galactose but have different specificities with respect to the penultimate sugar. Both subunit preparations contained only minor amounts of material which could bind or either lectin. However, after desialylation, both hCG alpha and free alpha bound extensively to Ricin, indicating the presence of penultimate galactose residues in both. We conclude that terminal galactose residues are not present on the oligosaccharides of either hCG alpha or free alpha-subunits, and that the difference observed in the sialic acid contents of the two subunits does not affect their rates of clearance.     

Involvement of plasma membrane enzymes in the proteolytic cleavage of luteinizing hormone receptor

In vitro degradation of LH receptor after occupancy by hCG was studied. Rat ovarian membranes labeled with [125I]iodo-hCG were incubated at 37 C; as a result, 30-40% of the radioactivity initially bound was rendered soluble in the medium. The molecular complexes in the medium and in incubated membranes solubilized with 1% Triton X-100 were then cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Particulate receptor-[125I]iodo-hCG complex exhibiting an apparent mol wt of 125,000 was cleaved during incubation into two distinct components (mol wt, 96,000 and 74,000) which appeared in the medium. Using tritiated hCG (beta-subunit labeled) instead of radioiodinated hCG (alpha-subunit labeled), these same two components were also observed, indicating that they both contain intact hCG (alpha and beta) as a part of their structure. In addition to the hormone (mol wt, 48,000), these two components contain receptor fragments with mol wt of 64,000 or 38,000, demonstrated directly by labeling the particulate receptor itself with periodate-tritiated borohydride before tagging with unlabeled hCG and in vitro incubation. These receptor fragments were purified from the medium by hCG-directed immunoaffinity chromatography and detached from the hormone by pH treatment. The intact receptor extracted from the membranes with detergent and purified identically in the absence of proteolysis migrated as a 90,000 mol wt polypeptide. These results demonstrate that after hormone occupancy, proteolytic cleavage of the 90,000 mol wt receptor polypeptide occurs at two specific sites. Thiol-blocking agents selectively prevented the appearance of the larger component (hCG coupled to 64,000 mol wt receptor fragment), while metal-chelating agents markedly decreased the appearance of the smaller component (hCG coupled to 38,000 mol wt receptor fragment) in the medium. Identical observations, obtained upon incubation of plasma membranes purified by sucrose density gradient centrifugation, suggest that plasma membrane enzymes are involved.     

Synthesis and secretion of human chorionic gonadotropin and its subunits in choriocarcinoma cells: a comparative study with normal placental cells.

The human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.     

The alpha-subunit of human choriogonadotropin interacts with the exodomain of the luteinizing hormone/choriogonadotropin receptor.

The LH/CG receptor, a G protein-coupled receptor, consists of two parts, the N-terminal extracellular segment (exodomain) and the membrane-associated C-terminal segment (endodomain). hCG initially binds the exodomain of the receptor and then, the hormone/exodomain complex is thought to make the secondary contact with the endodomain of the receptor and generate a hormone signal. However, little direct evidence is available about which hormone subunits (alpha or beta) interact with which domains of the receptor. To determine whether the alpha-subunit contacts the exodomain of its receptor, hCG containing [125I]alpha and truncated exodomain lacking the endodomain were prepared. They were chemically cross-linked, and the resulting cross-linked complexes were solubilized and electrophoresed. The results indicate that the alpha-subunit of hCG was directly and specifically cross-linked to the exodomain. To verify the cross-linked exodomain by the independent method, the Flag epitope was inserted between the signal sequence and the mature exodomain. hCG containing [125I]alpha was cross-linked to the Flag exodomain, and the resulting cross-linked hCG/Flag exodomain complexes were immunoprecipitated with anti-Flag antibody. The results show that the material cross-linked to hCG containing [125I]alpha is indeed the exodomain. In conclusion, our results show the direct interaction of the alpha-subunit with the exodomain and, therefore, its crucial role in the hormone-receptor interaction in addition to its involvement in signal generation.     

Effect of individual N-glycosyl chains in the beta-subunit on the conformation of human choriogonadotropin

Human choriogonadotropin is a heterodimeric glycoprotein hormone comprised of noncovalently associated alpha- and beta-subunits. Each of the subunits has two N-glycosyl chains. In our previous communication, we investigated the role of individual carbohydrate chains in the alpha-subunit on the signal transduction function and conformation of the hormone. This paper deals with the effect of individual or both N-glycosyl chains in the beta-subunit on the function and conformation of the monomer as well as of the heterodimer. Three mutants each of hCGbeta and hCG lacking N-glycosyl chains at beta13Asn, beta30Asn and beta13,30Asn were prepared by site-directed mutagenesis by replacing Thr residues in the recognition triplet sequence at beta15 and beta32 positions with Gln. All mutant heterodimers had receptor binding and cAMP and progesterone stimulating activities comparable to wild type hCG. While the loss of carbohydrate at beta13Asn or beta13,30Asn in the case of hCGbeta monomer resulted in a 4-6%, decrease in the ordered structure, the loss of the glycosyl chain at beta 30Asn did not alter the conformation as compared with the wild type hCGbeta. Similarly, all carbohydrate deficient hCG heterodimers had a decrease of 6-8%) in the ordered structure as compared with hCG. Thus, while the individual N-glycosyl chains did not affect the function of the hormone, they did have marked effect on its conformation but the conformational changes were localized and did not perturb the receptor binding and signal transduction sites.     

The antigenic structure of the human glycoprotein hormone alpha-subunit. I. Characterization of anti-alpha monoclonal antibodies.

The glycoprotein hormones CG, LH, FSH, and TSH are composed of two noncovalently linked subunits, alpha and beta. The beta-subunit confers hormone specificity, while the alpha-subunit is homologous within a species. To help in determining the antigenic structure of the common alpha-subunit, six monoclonal antibodies (mAbs) to the free or heterodimeric alpha-subunit of human (h) gonadotropic hormones have been prepared and, along with two previously isolated mAbs, have been characterized for binding specificity to alpha- and beta-subunits and the human glycoprotein hormones, CG, LH, FSH, and TSH. Each mAb was derived from hybidomas of FO myeloma cells fused with spleen cells from mice immunized with free alpha-subunit, hCG or hFSH. mAbs A101, A102, and E512 were specific for the alpha-subunit but showed the highest affinity for the intact hormone; K2.18, K94.6, E501, E502, and E511 were specific for free alpha. All of the antibodies inhibited binding of 125I-hCG to luteal membrane receptor, and 125I-labeled mAbs did not recognize hCG/receptor complex. Characterization by two-site binding assays using alpha, hCG, or hFSH as antigen revealed that all the mAbs bind to unique sites on alpha which may be overlapping, and which are modified in the intact hormone. The antigenic sites for mAbs E502, E511, and K2.18 are at least partially linear because they bind to reduced, carboxymethylated alpha.     

Physical characteristics of the gonadotropin receptor-hormone complexes formed in vivo and in vitro.

The physical properties of detergent-solubilized gonadotropin receptor-hormone complexes, determined by density gradient centrifugation and gel filtration, were compared after in vivo and in vitro labeling of specific ovarian binding sites with radioiodinated human chorionic gonadotropin (hCG). Following intravenous administration of biologically active 125I-labeled hCG, up to 50% of the gonadotropin tracer was bound to the luteinized ovaries of immature female rats treated with pregnant mare serum/human chorionic gonadotropin. Comparable binding of 125I-labeled hCG was observed after equilibration of ovarian particles with the labeled hormone in vitro. The sedimentation properties of the solubilized receptor-hormone complexes formed in vivo were identical with those derived for the corresponding complexes formed in vitro and extracted with Triton X-100 and Lubrol PX, with sedimentation constants of 8.8 S for the Triton-solubilized complex and 7.0 S for the complex extracted with Lubrol PX. During analytical gel filtration of the Triton-solubilized receptor-hormone complex on Sepharose 6B in 0.1% Triton X-100, the partition coefficient (Kav) of the "in vivo" complex (0.32) was not significantly different from that of the complex formed in vitro (0.29). Gel filtration of the Lubrol-solubilized ovarian particles on Sepharose 6B in 0.5% Lubrol PX gave Kav values for the "in vivo" and "in vitro" labeled complexes of 0.36 and 0.32, respectively. These findings demonstrate that the physical properties of size and shape which determine the partition coefficient and sedimentation characteristics of detergent-solubilized gonadotropin receptor-hormone complexes formed in vitro are not distinguishable from those of the complexes extracted after specific interaction of the ovarian gonadotropin receptors with radioiodinated hCG in vivo.     

Characterization of a novel self-associating Mr 40,000 platelet glycoprotein

A novel platelet glycoprotein has been purified and characterized. This glycoprotein, designated Pltgp40, is an acidic sialylated 40,000-dalton protein that bears both O-linked and N-linked oligosaccharides. Treatment of Pltgp40 with neuraminidase resulted in a 5,000-dalton reduction in its Mr and a 1.5 Unit alkaline shift in the isoelectric point, indicating the presence of a large number of sialic acid residues. A similar size reduction and change in pl were observed after treatment of Pltgp40 with O-glycanase showing that sialic acids are present on O-linked oligosaccharides. Digestion of Pltgp40 with N-glycanase reduced the Mr to approximately 20,000 daltons but did not affect the isoelectric point, suggesting that Pltgp40 contains six to seven nonsialylated N-linked carbohydrate chains. High Mr proteins were observed in affinity purified Pltgp40 and were identified as detergent-stable protein oligomers consisting of multiple 40,000-dalton monomers. Immunodepletion and direct binding studies indicated that Pltgp40 was not equivalent to Ig Fc receptor type II, another 40,000-dalton glycoprotein expressed on platelets. However, Pltgp40 copurified with Fc receptor type II when platelet extracts were loaded onto human IgG affinity columns, raising the possibility that Pltgp40 may associate with Fc receptors or Fc receptor-lg complexes. Amino acid sequence analysis of the N-terminus of Pltgp40 was performed and confirmed that Pltgp40 is a novel platelet glycoprotein. Epitopes on Pltgp40 appear to be widely expressed because monoclonal antibodies against Pltgp40 also reacted with a variety of myeloid, lymphoid, and epithelial cells. Pltgp40 was detected on activated but not resting platelets, indicating that Pltgp40 is a platelet activation marker.     

Synthesis of chorionic gonadotropin subunits in human choriocarcinoma clonal cell line JEG-3: carbohydrate differences in glycopeptides from free and combined alpha-subunits

The glycoprotein hormone hCG and its free alpha-subunit are secreted by the clonal choriocarcinoma cell line JEG-3. Free hCG alpha has a larger apparent mol wt (22,000-24,000) than the combined hCG alpha (18,000-19,000) obtained by dissociation of the hCG secreted by these cells. Techniques developed for the specific isolation and purification of the free and combined hCG alpha forms and for the preparation of glycopeptides from these subunits have permitted detection of the incorporation of D-[3H]glucosamine [( 3H]GlcN) and L-[3H]fucose into both alpha-subunit forms. Relative to their [35S]methionine content, 2.3-fold more [3H]GlcN and 6-fold more L-[3H]fucose were incorporated into free hCG alpha than into combined hCG alpha. Analyses of [3H]GlcN glycopeptides prepared from free and combined hCG alpha indicate that the 22,000- to 24,000-dalton subunit form contained more [3H]GlcN and 27% more of the GlcN metabolite N-acetylneuraminic acid than the 18,000- to 19,000-dalton hCG alpha-subunit, than both hCG alpha forms contained two major N-linked oligosaccharide chains differing primarily in their NeuAc content, and that most of the [3H]GlcN was incorporated as GlcN or metabolites of GlcN other than N-acetylneuraminic acid. These studies provide direct chemical evidence of a higher content of carbohydrate in the larger free alpha-subunit form.     

Carbohydrate moieties of small placental hCG: requirement of mannose structure for biological activity

The 43 kDa human chorionic gonadotropin (hCG) (SP-hCG) was purified from human placenta and analyzed for sugar moieties. The low hexosamine content suggests that SP-hCG probably lacks O-linked sugar chains in the beta-subunit and incompletely formed N-linked sugar chains in the alpha- and beta-subunits. In the present study SP-hCG was hydrolyzed with various glycosidases. Treatment of hCG or SP-hCG with O-glycan peptide hydrolase increased the mobility of asialo-hCG beta in reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) while that of SP-hCG beta was unaffected, indicating that SP-hCG beta does not contain NeuNAc-Gal-GalNAc unit. Alpha-Mannosidase and endoglycosidase H hydrolyzed mannose and the high mannose-GlcNAc moieties, respectively, from alpha- and beta-subunits of SP-hCG, but not from the subunits of authentic hCG. Glycopeptidase F hydrolyzed completely the N-linked sugar chains from SP-hCG subunits, producing alpha- and beta-subunits with estimated Mr of 15,000 and 18,500, respectively. The biological activity of purified SP-hCG is about 50-80% of highly purified authentic hCG. In an in vitro system SP-hCG increased cAMP accumulation and testosterone production by rat Leydig cells to the same levels as that induced by hCG. However, the biological activity of SP-hCG was markedly reduced, following treatment with endoglycosidase H or alpha-mannosidase. To attain the level of testosterone production equivalent to that induced with untreated SP-hCG, 10-20 times higher dose of treated SP-hCG was required. On the other hand, cAMP accumulation induced with treated SP-hCG even at a very high concentration was substantially lower than that attained with untreated SP-hCG. In conclusion, the mannose moieties are essential structural components of the hormone in stimulating cAMP accumulation and steroidogenesis by rat Leydig cells.     

The heterogeneity of porcine 32,000 Mr inhibin alpha-subunit: a gel electrophoresis and immunoblot study

Porcine 32,000 Mr inhibin is a glycoprotein with one asparagine-linked glycosylation site on the alpha-subunit. The presence of carbohydrate on the alpha-subunit was visualized by periodate-Schiff (PAS) staining. This stain for carbohydrate also verified that the beta-subunit of 32,000 Mr porcine inhibin does not contain carbohydrate. When analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) under reducing conditions, the inhibin alpha-subunit consistently existed as a doublet, and by the PAS stain, both bands of the doublet were glycosylated. Analysis by two-dimensional (2D) PAGE further revealed the presence of charge isoforms of the alpha-subunit. The alpha-subunit of inhibin could be deglycosylated by N-glycanase, but not by endoglycosidase F, endoglycosidase D, or endoglycosidase H. When the N-glycanase-treated inhibin was analyzed by either 1D-PAGE or 2D-PAGE, the molecular size of the alpha-subunit was reduced by 3500 Mr. Each doublet band observed with reducing conditions in 1D-PAGE or 2D-PAGE for the alpha-subunit became a single band (spot) in the deglycosylated alpha-subunit. However, the charge heterogeneity detected by 2D-PAGE was retained, indicating that only a portion of this heterogeneity is attributable to the carbohydrate moiety. The in vitro biological activity of the deglycosylated inhibin was not different from the control sample. The composition of the carbohydrate in inhibin was investigated with the Dionex carbohydrate analyzer. Inhibin contains fucose, glucosamine, galactose, mannose, and glucose. Colorimetric analysis revealed the presence of sialic acid. Taken together, this implies some aspect of the peptide portion of the molecule is involved in charge heterogeneity. Inhibin may have an unusual carbohydrate component, as evidenced by the detection of glucose in inhibin samples. The absence of glucose in the carbohydrate moiety of another glycoprotein fraction that accompanied the inhibin through all the same fractionation procedures argues against the artifactual introduction of glucose in the fractionation medium per se.     

Direct demonstration of glycosylation of insulin receptor subunits by biosynthetic and external labeling: evidence for heterogeneity.

Insulin receptors of human lymphocytes (IM-9 line) were biosynthetically labeled with [3H]glucosamine, [3H]galactose, [3H]fucose, or [3H]mannose. After solubilization in Triton X-100, cell extracts were immunoprecipitated with serum from a patient containing autoantibodies to the insulin receptor. Na-DodSO4/polyacrylamide gel electrophoresis of the immunoprecipitates under reducing conditions showed the presence of major labeled subunits of apparent Mr 134,000 and 98,000 and a minor component of Mr 206,000. The ratio of activity in the 134,000 versus 98,000 Mr bands varied from 2:1 for mannose to 1.2:1 for galactose. In addition, the receptor subunits could be demonstrated when the cell surface of intact lymphocytes was labeled with NaB3H4 by using either the galactose oxidase (acts on nonreducing terminal galactose and N-acetylgalactosamine) technique or the periodate (oxidizes sialic acid) technique. With the periodate treatment, NaB3H4 labeled preferentially the Mr 98,000 band. With the galactose oxidase procedure, on the other hand, NaB3H4 labeled only the Mr 134,000 band; prior treatment with neuraminidase increased the labeling of this band and also revealed the Mr 98,000 subunit. These data demonstrate that the major subunits of the insulin receptor are complex glycoproteins that have differences in the nonreducing ends of the carbohydrate chains. In the Mr 134,000 subunit, there appear to be more exposed galactosyl or N-acetylgalactosaminyl (or both) residues, whereas the Mr 98,000 subunit appears to have a higher degree of sialylation. These labeling techniques provide new tools to examine the role of the carbohydrate moiety in insulin receptor function and turnover.     

Processing of O-linked glycosylation in the chimera consisting of alpha-subunit and carboxyl-terminal peptide of the human chorionic gonadotropin beta-subunit is affected by dimer formation with follicle-stimulating hormone beta-subunit

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunits. hCGbeta is unique among beta-subunits due to a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharides. We previously reported that there were differences in O-glycosylation between two chimeras consisting of alpha-subunit and CTP, i.e. a variant with CTP at the N-terminal region (Calpha) and another analog with CTP at the C-terminus (alphaC) of the alpha-subunit. To address whether O-glycosylation is influenced by the heterodimer formation, Calpha and alphaC were expressed alone or with FSHbeta-subunit in Chinese hamster ovary cells. The O-linked glycosylation was assessed by continuous labeling with [(35)S]methionine/cysteine, immunoprecipitation with anti-alpha or anti-FSH serum, serial digestion with endoglycosidase-F and neuraminidase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The decrease in molecular weight of dimeric chimeras digested with endoglycosidase-F was greater in Calpha than that in alphaC after treatment with neuraminidase, revealing that both chimeras have different numbers of sialic acids on O-linked carbohydrates. By treating with endoglycosidase-F, the dimeric alphaC migrated faster than its free form, whereas the mobility difference between assembled and unassembled forms of Calpha was very little. These data indicate that processing of O-glycosylation is affected by the backbone polypeptide chain(s).     

O-glycosylation of the alpha-subunit does not limit the assembly of chorionic gonadotropin alpha beta dimer in human malignant and nonmalignant trophoblast cells

Biosynthetic experiments were carried out in cultures of human malignant trophoblast cells (the JAR cell line) and in explants of normal first trimester human placental tissue to test the hypothesis that the O-glycosylation of the glycoprotein hormone alpha-subunit at Thr-39 regulates the assembly of the CG alpha beta dimer. This modification of alpha has been shown to ablate its ability to combine in vitro with the beta-subunit of bovine LH and might explain why JAR cells and placental explants secrete uncombined alpha- and beta-subunits in addition to the hCG alpha beta dimer. We have previously detected an O-linked carbohydrate chain at Thr-39 in preparations of secreted free alpha-subunit, but not dimer CG alpha from JAR culture medium. We report here evidence that the O-glycosylation of alpha does not regulate the biosynthetic assembly of the hCG dimer in cultures of JAR choriocarcinoma cells or first trimester placental explants. The intracellular precursor forms of alpha and beta that accumulate in the endoplasmic reticulum and combine in that compartment are not yet modified with O-linked carbohydrate, as determined by measurements of their [3H]galactosamine content after biosynthetic labeling of amino sugars with [3H]glucosamine. Furthermore, only half of the free alpha-subunit secreted by JAR cells and less than 10% of free alpha secreted by 10-week-old placental explants received the O-linked chain. This was shown by determining the ratio of the unglycosylated and glycosylated forms of the tryptic peptide from free alpha that contains the O-glycosylation site (residues 36-42). Based on these findings, we make the following conclusions. 1) O-Glycosylation of alpha-subunit is a late event in the secretory pathway of trophoblasts compared to the rapid combination in the rough endoplasmic reticulum of hCG subunit precursors to form alpha beta dimer. 2) Association of alpha with beta precludes the subsequent addition of the glycan to alpha at Thr-39. 3) The alpha molecules that fail to combine with beta in the endoplasmic reticulum are substrates for the later addition of O-linked carbohydrate, presumably in the Golgi complex, but only a fraction of the free alpha molecules are modified with O-linked carbohydrate.     

Two polyclonal antisera to rat luteal LH/CG receptor with different ligand binding inhibition and immunohistochemical receptor detection capabilities

Polyclonal antisera to a SDS-denatured and partially renatured rat luteal 90 K LH/CG receptor were raised in rabbits, characterized, and their applicability for immunohistochemical location of the receptor examined. The LH/CG receptor was purified by hCG-affinity chromatography and subjected either to a preparative SDS-PAGE or Western blotting. Gel slices containing the SDS-denatured or nitrocellulose strips containing the renatured 90 K LH/CG receptor were used for immunization. The antisera, termed ARS-2 and ARS-3, respectively, possessed similar antibody titres. Both antisera were able to recognize the native, SDS-denatured, and SDS-denatured and reduced forms of the LH/CG receptor on dot blots, but only ARS-3 contained antibodies to the hormone binding site or a region near to it, as it was able to inhibit the hCG binding to the membrane-bound LH/CG receptor in a dilution-dependent manner. Both antisera recognized the receptor-hCG complex, but ARS-2 stained the complex with about 50% less intensity than the free receptor. ARS-3 located the LH/CG receptor distinctly on the luteal cell surfaces in immunohistochemical staining with peroxidase antiperoxidase complex method, but ARS-2, although it possessed similar antibody titre, revealed negligible staining. Thus, the antisera readily recognize the native receptor, but differ in their capability for inhibiting hormone binding. Only ARS-3, produced against the renatured receptor, contains sufficient amounts of antibodies capable of recognizing free and occupied receptors in immunohistochemistry.