The role of the asparagine-linked oligosaccharides of the alpha subunit in the secretion and assembly of human chorionic gonadotrophin

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 subunit. Site-directed mutagenesis of the two asparagine-linked glycosylation sites of hCG alpha was used to study the function of the individual oligosaccharide chains in secretion and subunit assembly. Expression vectors for the alpha genes (wild-type and mutant) and the hCG beta gene were constructed and transfected into Chinese hamster ovary cells. Loss of the oligosaccharide at position 78 causes the mutant subunit to be degraded quickly and less than 20% is secreted. However, the presence of hCG beta stabilizes this mutant and allows approximately 45% of the subunit in the form of a dimer to exit the cell. Absence of carbohydrate at asparagine 52 does not perturb the stability or transport of the alpha subunit but does affect dimer secretion; under conditions where this mutant or hCG beta was in excess, less than 30% is secreted in the form of a dimer. Mutagenesis of both glycosylation sites affects monomer and dimer secretion but at levels intermediate between the single-site mutants. We conclude that there are site-specific functions of the hCG alpha asparagine-linked oligosaccharides with respect to the stability and assembly of hCG.     

steve bAccumulation of nerve growth factor and its receptors in the uterus and dorsal root ganglia in a mouse model of adenomyosis

Background

The pregnancy hormone human chorionic gonadotropin (hCG) and its free subunits (hCG alpha, hCG beta) are produced in the male reproductive tract and found in high concentrations in seminal fluid, in particular hCG alpha. This study aimed to elucidate changes in peptide hormone profiles in patients showing abnormal semen analyses and to determine the genuineness of the highly abundant hCG alpha.

Methods

Seminal plasma was obtained from 45 male patients undergoing semen analysis during infertility workups. Comprehensive peptide hormone profiles were established by a panel of immunofluorometric assays for hCG, hCG alpha, hCG beta and its metabolite hCG beta core fragment, placental lactogen, growth hormone and prolactin in seminal plasma of patients with abnormal semen analysis results (n = 29) versus normozoospermic men (n = 16). The molecular identity of large hyperglycosylated hCG alpha was analyzed by mass-spectrometry and selective deglycosylation.

Results

hCG alpha levels were found to be significantly lower in men with impaired semen quality (1346 +/- 191 vs. 2753 +/- 533 ng/ml, P = 0.022). Moreover, patients with reduced sperm count had reduced intact hCG levels compared with normozoospermic men (0.097 +/- 0.022 vs. 0.203 +/- 0.040 ng/ml, P = 0.028). Using mass-spectrometry, the biochemical identity of hCG alpha purified from seminal plasma was verified. Under non-reducing conditions in SDS-PAGE, hCG alpha isolated from seminal plasma migrated in a manner comparable with large free hCG alpha with an apparent molecular mass (Mr, app) of 24 kDa, while hCG alpha dissociated from pregnancy-derived holo-hCG migrated at approximately 22 kDa. After deglycosylation with PNGase F under denaturing conditions, all hCG alpha variants showed an Mr, app of 15 kDa, indicating identical amino acid backbones.

Conclusions

The findings indicate a pathophysiological relevance of hCG, particularly its free alpha subunit, in spermatogenesis. The alternative glycosylation pattern on the free large hCG alpha in seminal plasma might reflect a modified function of this subunit in the male reproductive tract.     

Site-specific processing of the N-linked oligosaccharides of the human chorionic gonadotropin alpha subunit

Two forms of the gonadotropin alpha subunit are synthesized in placenta and in human chorionic gonadotropin (hCG)-producing tumors: an uncombined (monomer) form and a combined (dimer) form. These forms show differences in their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The slower migration of the monomeric form on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been attributed to a different glycosylation pattern. Previous studies demonstrated different roles of each of the two alpha N-linked glycosylation sites (Asn-52 and Asn-78) in secretion of the uncombined subunit and the biologic activity of hCG dimer. To assess the influence of formation of dimer on the processing pattern at the individual sites, we characterized the N-linked oligosaccharides of monomer and dimer forms of recombinant human choriogonadotropin alpha subunit. Two approaches were employed. First, site-directed mutagenesis was used to alter the two N-linked oligosaccharide attachment sites, thus allowing the expression of alpha subunits containing only one glycosylation site. Second, tryptic glycopeptides of the wild-type subunits were examined. Concanavalin A (ConA) binding and sialic acid content indicated that the oligosaccharides at each glycosylation site of the uncombined alpha subunit are processed differently. Oligosaccharides present at Asn-52 are almost exclusively ConA-unbound and contain three sialic acid residues. The majority of Asn-78-linked oligosaccharides are ConA-bound and disialylated. Both sites are processed independently because no significant differences were observed between the oligosaccharides at the same sites in wild-type and mutant monomeric alpha subunits. By contrast, the majority of the oligosaccharides at both glycosylation sites of the dimer alpha are bound to ConA. Thus, combination primarily affects the processing pattern of the Asn-52-linked species. Because glycosylation at this site is essential for hCG assembly and signal transduction, these data imply a critical link between the site-specific processing and hormone function.     

Mutagenesis and gene transfer define site-specific roles of the gonadotropin oligosaccharides

Human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone and thyroid-stimulating hormone are a family of glycoprotein hormones that share a common alpha subunit but differ in their hormone-specific beta subunits. Using site-directed mutagenesis and gene-transfer, we analyzed the role of the N-linked oligosaccharides of alpha and chorionic gonadotropin (CG)beta in the secretion, assembly, and biologic activity of hCG. Absence of carbohydrate at alpha asparagine (Asn) 52 decreased combination with CG beta but did not alter monomer secretion. Absence of the alpha Asn78 oligosaccharide increased the degradation of the alpha subunit, but the presence of CG beta stabilized this alpha mutant in an efficiently formed dimer complex. Alternatively, absence of both alpha oligosaccharides slowed both secretion and dimer formation but allowed an intermediate level of alpha secreted or dimerized compared to the single-site mutants. Analysis of the CG beta glycosylation mutants revealed that absence of the Asn30 oligosaccharide, but not Asn13, slowed secretion but not assembly, whereas absence of both oligosaccharides slowed both secretion and dimer formation. Analysis of the receptor binding of the hCG glycosylation mutants showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on receptor affinity of the derivatives. However, the absence of alpha Asn52, but not the alpha Asn78 or the CG beta carbohydrate units, reduced the steroidogenic effect, unmasked differences in the beta oligosaccharides, and converted the deglycosylated derivatives into antagonists.     

The polypeptide part of human chorionic gonadotrophin affects the kinetics of alpha 6-sialylation of its N-linked glycans but does not alter the branch specificity of CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase.

Human chorionic gonadotrophin (hCG) is a heterodimeric glycoprotein hormone consisting of an alpha- and a beta-subunit, both containing two N-linked, complex-type glycans. Using this hormone as a model glycoprotein, the influence of its polypeptide part on the activity and specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase (alpha 6-sialyltransferase) was investigated. Initial rates of sialic acid incorporation into the desialylated glycans of hCG alpha and hCG beta in the heterodimer were higher with the alpha-subunit. This appeared to be due to a higher V which, together with a slightly lowered affinity (higher Km), resulted in a higher kinetic efficiency of the sialyltransferase for the glycans of this subunit. By contrast, the kinetic parameters did not differ significantly when the subunits were in the free form, indicating that the differences in the kinetics of sialylation found for the subunits in the heterodimeric state were not caused by the differences in N-linked carbohydrate structures of the subunits. It is proposed that these effects are due to conformational constraints which the polypeptide moieties put on the glycan chains upon dimerization. Furthermore, it was investigated whether the polypeptide of hCG would interfere with the sialyltransferase so as to alter the branch specificity of the enzyme. 1H-NMR spectroscopy (400 MHz) of the glycan chains, alpha 6-sialylated in vitro, showed that the enzyme highly prefers the galactosyl residue at the Gal beta 1----4GlcNAc beta 1----2-Man alpha 1----3Man branch for attachment of the first mol of sialic acid into the diantennary glycans of desialylated hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of residues that confer antibody binding specificity using human chorionic gonadotropin/luteinizing hormone beta subunit chimeras and mutants.

The glycoprotein hormones are a family of conserved heterodimeric proteins which share a common alpha subunit but differ in their hormone-specific beta subunits. We used chimeras of human chorionic gonadotropin (hCG) and luteinizing hormone (hLH) beta subunits to identify residues which enable monoclonal antibodies (mAb) to distinguish the two hormones. The LH beta-CG beta chimeras appeared to fold similar to hCG beta, since they combined with hCG alpha and, depending on their sequences, were recognized by hCG-selective mAbs. Amino acid residues Arg8-Arg10,Gly47-Ala51, and Gln89-Leu92 form a major epitope region and appear to be adjacent to each other on the surface of hCG beta. Gly47-Ala51 and Gln89-Leu92 are recognized by dimer-specific mAbs while Arg8-Arg10 is recognized by mAbs which have highest affinity for the free beta subunit. These observations suggest that the conformation of this region of the beta subunit changes when the alpha and beta subunits combine. Residues which are C-terminal of Asp112 form a second epitope domain. mAbs to the third domain distinguish hCG beta and hLH beta by the presence of Asn77 in hCG beta and can be detected after hCG binds to receptors. These findings were used to develop a model of hCG beta which predicts the locations of these residues and their positions relative to the alpha subunit and receptor interfaces.     

Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction. These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit. The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins. The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants. In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system. A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity. Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active. The preliminary data also suggested that P. pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated. This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.     

Effect of conformation of hCG-beta on generation of hCG-specific antibody

Most antisera generated to isolated highly purified beta subunits of human glycoprotein hormones are not sufficiently sensitive to detect physiologic blood levels of the native hormone. In the dissociated state, beta subunits assume a conformation different from that in the native hormone. Since antisera to alpha subunits have essentially no cross-reactivity between species, highly purified hCG-beta was combined with bTSH-alpha. That hybrid served as immunogen to assess whether sensitive, specific hCG antisera would more likely result than using hCG-beta alone. Of five animals immunized, three developed sufficiently sensitive and specific antisera. The results of these studies strongly suggests that human glycoprotein beta subunits combined with non-human alpha subunit are more likely to yield specific, sensitive antisera than when either isolated beta subunit or the native human glycoprotein hormone, containing common alpha determinants, serves as immunogen.     

Altered glycosylation sites of the delta subunit of the acetylcholine receptor (AChR) reduce alpha delta association and receptor assembly.

We have used mutagenesis to investigate the potential N-glycosylation sites in the delta subunit of the mouse muscle acetylcholine receptor (AChR). Of the three sites, Asn76, Asn143, and Asn169, only the first two were glycosylated when the delta subunit was expressed in COS cells. Because the heterologously expressed delta subunit was similar in its properties to that expressed in C2 muscle cells, the sites of glycosylation are likely to be the same in both cases. In COS cells, mutations of the delta subunit that prevented glycosylation at either of the sites did not change its metabolic stability nor its steady-state level. These results are in contrast to those found previously for the alpha subunit, in which glycosylation at a single site metabolically stabilized the polypeptide (Blount, P., and Merlie, J. P. (1990) J. Cell Biol. 111, 2613-2622). Mutations of the delta subunit that prevented glycosylation, however, decreased its ability to form an alpha delta heterodimer when the alpha and delta subunit were expressed together. When all four subunits of the AChR (alpha, beta, delta, and epsilon) were coexpressed, mutation of the delta subunit to prevent glycosylation resulted in a reduced amount of fully assembled AChR and reduced surface AChR levels, consistent with the role of the heterodimer in the assembly reaction. These results suggest that glycosylation of the delta subunit at both Asn76 and Asn143 is needed for its efficient folding and/or its subsequent interaction with the alpha subunit.     

Existence of associated, non-associated, and oligomeric forms of human chorionic gonadotropin subunits in placental extracts

The molecular sizes of human chorionic gonadotropin (hCG) subunits in the native state in normal first trimester placental extracts were determined by gel filtration on Sephacryl S-300, followed by SDS-polyacrylamide gel electrophoresis, protein blotting, and immunobinding analysis using anti-alpha and - beta antibodies. Mature forms of hCG subunits in the extracts were only found in the same fraction as that which contained standard urinary hCG, indicating an alpha beta dimer. On the other hand, immature forms were detected with a wide range of molecular weights, which were higher than that of standard hCG, suggesting oligomerization of associated or non-associated immature subunits. In order to determine the associated state of these subunits, various forms of associated subunits (hCG alpha beta) in placental extracts were immunoprecipitated with anti-hCG antiserum, which only recognized hCG alpha beta, and Protein A-Sepharose. They were then analyzed by SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions, followed by immunobinding assaying. It has been suggested that there are three kinds of hCG alpha beta S (one mature and two immature). To confirm the above results and to clarify the existence of free subunits, placental extracts were subjected to two-dimensional SDS-polyacrylamide gel electrophoresis. With this technique, high molecular weight forms of immature hCG subunits were found to be present in placental cells as an oligomer of not only the alpha beta dimer but of each subunit as well.     

The glycoprotein hormones: recent studies of structure-function relationships

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.     

Gonadotropin alpha subunit. Differential processing of free and combined forms in human trophoblast and transfected mouse cells

The gonadotropins luteinizing hormone, follicle-stimulating hormone, and human chorionic gonadotropin are composed of two noncovalently linked subunits, alpha and beta. The alpha subunit, identical in all three hormones, is produced in excess over the unique beta subunits by pituitary and placenta, and is secreted as uncombined, or free subunit. Free alpha subunit from both tissues has a larger molecular weight than the dimer form. In bovine pituitary an extra O-linked oligosaccharide is added to free alpha subunit, and this modification has recently been detected at an analogous position (threonine 39) on human alpha subunit secreted by choriocarcinoma cells. To assess the contribution of N-linked and O-linked oligosaccharides to the heterogeneity of human free alpha subunit, we have compared free alpha with human chorionic gonadotropin alpha secreted by explants and cultured cytotrophoblasts of human first trimester placenta. We have also examined the free and combined forms of human alpha subunit expressed in transfected C-127 mouse mammary tumor cells. Processing of the alpha subunit in placental and C-127 cells was similar. Tryptic mapping of placental-derived and transfected alpha subunits indicated that O-glycosylation at threonine 39 was not a major modification. In the presence of the oligosaccharide processing inhibitor swainsonine the difference in size between the free and combined forms of alpha was eliminated in both placental and C-127 cells, indicating that the two forms of alpha differed in their N-linked oligosaccharides. Furthermore, the oligosaccharides of free alpha subunits from placental and transfected cells were resistant to endoglycosidase H, but the combined forms of alpha were partially sensitive to the enzyme. Thus, in human first trimester placenta and mouse C-127 cells, combination of alpha with human chorionic gonadotropin beta alters the processing of N-linked oligosaccharides on alpha subunit.     

N-linked oligosaccharides on free alpha interfere with its ability to combine with human chorionic gonadotropin-beta subunit.

The purpose of the study was to examine the role of N-linked oligosaccharides in preventing combination of free alpha molecules with human chorionic gonadotropin (hCG)-beta subunit to form the intact hormone, hCG. Culture media from JEG cells incubated in the presence or absence of Swainsonine were filtered on Sephadex G-100, and free alpha was identified by radioimmunoassay. Swainsonine interferes with glycosylation by inhibiting alpha-mannosidase II, resulting in formation of hybrid structures. Approximately 50% of the free alpha molecules from Swainsonine-treated cells (Swainsonine pool 2) had an apparent molecular size that was smaller than that of free alpha from control cells. The oligosaccharides of control alpha molecules were resistant to endo-beta-N-acetylglucosaminidase H treatment. In contrast, virtually all of the Swainsonine free alpha molecules contained endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides. Swainsonine free alpha and control free alpha molecules were incubated with an excess of hCG-beta subunit, followed by chromatography on Sephadex G-100. Each fraction was assayed by radioimmunoassay for intact hCG and for alpha. Less than 10% of control free alpha molecules combined with hCG-beta. In contrast, 54% of Swainsonine alpha pool 2 and 40% of Swainsonine alpha pool 1 combined with beta to form hCG. Thus, modulation of N-linked oligosaccharide processing converted free alpha molecules to forms that can combine with hCG-beta. These results indicate that the inability of a substantial portion of control free alpha molecules to combine with hCG-beta is due to the presence of N-linked oligosaccharide structures that interfere with combination.     

Role of carbohydrates in glycoprotein hormone signal transduction

The structure of the polypeptide chains and oligosaccharide moieties of the alpha and beta subunits of pituitary and placental glycoprotein hormones are known. The dimeric polypeptide structure (but not the carbohydrate) is important for binding of the hormone to specific receptors. The N-linked but not O-linked carbohydrates, on the other hand, are required in some manner to activate the effector system. Hormones with depleted carbohydrate content (deglycosylated hormones) interact with receptor but are unable to activate intracellular events. Because of such discordant properties, these forms act as competitive inhibitors of hormone action. Through a combination of chemical deglycosylation procedures and site-directed mutagenesis, the first site of N-glycosylation from the NH2 terminus of the common alpha subunit has been identified to be more critical for glycoprotein hormone signal transduction. Control of glycosylation by the endocrine milieu could contribute to regulation of hormone function by secreting variable forms of agonist/antagonist.     

A single amino acid substitution in an ectopic alpha subunit of a human carcinoma choriogonadotropin

Human choriogonadotropin [hCG] has two dissimilar noncovalently associated subunits, designated alpha and beta. An ectopically secreted hCG alpha subunit that fails to associate with the beta subunit and displays an anomalously high molecular weight on molecular sieve chromatography but not on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been sequenced. A single substitution of Glu56 by Ala56 has been found in the altered subunit. No evidence for conformational differences between normal and ectopic alpha could be found using circular dichroism or intrinsic fluorescence as measures of secondary and tertiary structure, respectively. Hydrophobicity profiles as determined by the method of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132) predicted, however, that the hydrophilic segment, Thr54-Ser55-Glu56-Ser57-Thr58, becomes an extension of the preceding hydrophobic segment when Glu56 is substituted with Ala. This solitary hemoglobin S-like mutation may lead to an altered tertiary structure, self dimerization, or an alteration in glycosylation that could be responsible for the ectopic alpha subunit's failure to associate with the beta subunit.     

An oligosaccharide of the O-linked type distinguishes the free from the combined form of hCG alpha subunit

JAR malignant trophoblast cells produce a free alpha subunit in addition to an alpha combined with beta subunit as hCG. The free alpha is larger by gel chromatography and SDS-PAGE than combined alpha and is unable to associate with beta subunit to form hCG. A tryptic fragment, representing amino acid residues 36-42, derived from free alpha was larger than the corresponding fragment from combined alpha. After neuraminidase treatment, the fragment from free alpha bound peanut lectin agarose, which is specific for Gal beta 1-3GalNAc as found in O-linked oligosaccharides. The fragment also contained Gal and GalNAc (and a lesser amount of GlcNAc) as determined by glycosidase sensitivity and amino sugar analyses. Removal of this tryptic fragment ablated the size difference between free and combined alpha subunits.     

Reciprocal modulation between the alpha and beta 4 subunits of hSlo calcium-dependent potassium channels

Large conductance Ca(2+)-dependent potassium (K(Ca) or maxi K) channels are composed of a pore-forming alpha subunit and an auxiliary beta subunit. We have shown that the brain-specific beta4 subunit modulates the voltage dependence, activation kinetics, and toxin sensitivity of the hSlo channel (Weiger, T. M., Holmqvist, M. H., Levitan, I. B., Clark, F. T., Sprague, S., Huang, W. J., Ge, P., Wang, C., Lawson, D., Jurman, M. E., Glucksmann, M. A., Silos-Santiago, I., DiStefano, P. S., and Curtis, R. (2000) J. Neurosci. 20, 3563-3570). We investigated here the N-linked glycosylation of the beta4 subunit and its effect on the modulation of the hSlo alpha subunit. When expressed alone in HEK293 cells, the beta4 subunit runs as a single molecular weight band on an SDS gel. However, when coexpressed with the hSlo alpha subunit, the beta4 subunit appears as two different molecular weight bands. Enzymatic deglycosylation or mutation of the N-linked glycosylation residues in beta4 converts it to a single lower molecular weight band, even in the presence of the hSlo alpha subunit, suggesting that the beta4 subunit can be present as an immature, core glycosylated form and a mature, highly glycosylated form. Blockage of protein transport from the endoplasmic reticulum to the Golgi compartment with brefeldin A abolishes the mature, highly glycosylated beta4 band. Glycosylation of the beta4 subunit is not required for its binding to the hSlo channel alpha subunit. It also is not necessary for cell membrane targeting of the beta4 subunit, as demonstrated by surface biotinylation experiments. However, the double glycosylation site mutant beta4 (beta4 N53A/N90A) protects the channel less against toxin blockade, as compared with the hSlo channel coexpressed with wild type beta4 subunit. Taken together, these data show that the pore-forming alpha subunit of the hSlo channel promotes N-linked glycosylation of its auxiliary beta4 subunit, and this in turn influences the modulation of the channel by the beta4 subunit.     

Free alpha subunits of glycoprotein hormone with dissimilar carbohydrates produced by pathologically different carcinomas

The two kinds of glycoprotein hormone alpha subunit ectopically produced by an undifferentiated carcinoma of the left femoral region (TM-alpha) and an adenocarcinoma of the right external genitalia (FS-alpha) were examined for amino acid composition, isoelectric focusing, molecular weight, the ability to combine with standard hCG beta and affinity with lectins (Con A, Ricin and PNA). Both TM-alpha and FS-alpha exhibited immunoantigenicity similar to standard hCG alpha. Furthermore, there were no significant differences in the amino acid compositions of TM-alpha, FS-alpha or standard hCG alpha. In isoelectric focusing, while standard hCG alpha exhibited a neutral charge, both TM-alpha and FS-alpha exhibited strong negative charges. FS-alpha was as sensitive to sialidase as standard hCG alpha, whereas most of the TM-alpha exhibited resistance to sialidase. TM-alpha contains sialidase-insensitive peripheral material with a negative charge. The affinity with Ricin-Sepharose indicated that most of the FS-alpha and some of the TM-alpha may contain terminal sialic acid and the penultimate structure, Gal beta 1----4G1cNAc; the affinity with PNA-Sepharose indicated that both may also contain terminal sialic acid and the penultimate structure, Gal beta 1----3GalNAc. These observations suggest that dissimilar glycosylation processes are present in the carcinoma ectopic biosynthesis of glycoprotein hormone alpha subunit.     

A method for assessing the reassembly of a multisubunit glycoprotein by western blotting

The Western blot procedure has been adapted to detect the reassembly of a two-subunit glycoprotein, urinary human chorionic gonadotropin (hCG), directly on the nitrocellulose. This glycoprotein is composed of two nonidentical subunits, alpha and beta. A simple procedure using immunoblotting has been developed to detect reassembly of the monomers to dimer. Three monoclonal antibodies were required for the development of this method: A109, which binds the alpha subunit or hCG; B105, which binds the beta subunit or hCG; and B107, specific for the intact hCG dimer. The alpha subunit and beta subunit of hCG were each electrophoresed and transferred to nitrocellulose, and the transfer was then incubated with the appropriate complementary subunit; reassembly of the dimer was determined by the binding of the monoclonal antibody B107. Evidence that the reassembly occurs directly on the nitrocellulose comes from the fact that B107 immunoreactivity is detected at the molecular weight position of the subunit and not at the dimer molecular weight. A genetically expressed recombinant form of the alpha subunit was also tested for its ability to recombine with the opposite subunit to produce the dimer. The recombinant alpha subunit was determined to have additional carbohydrate which interfered with the binding of the beta subunit. N-Glycanase digestion of the recombinant alpha subunit produced a form which, when incubated with the beta subunit, did recombine on the nitrocellulose and could be recognized by B107.     

Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells

The glycoprotein hormones lutropin (LH) and chorionic gonadotropin (CG) share a common structure consisting of an identical alpha subunit noncovalently linked to a hormone-specific beta subunit. While LH is produced in the anterior pituitary, CG is synthesized in placenta. To compare the assembly, processing, and secretion of human LH and CG in the same cell type, we have expressed their subunits, individually and together, in mouse C-127 mammary tumor cells. Analysis of transfected clones revealed an unexpected difference in the secretion of individually expressed subunits. Whereas alpha and CG beta subunits were rapidly and quantitatively secreted, only 10% of newly synthesized LH beta subunit reached the medium. The remaining subunit was found in an intracellular, endoglycosidase H (endo H)-sensitive pool that had a turnover rate of approximately 8 h. Coexpression with alpha subunit resulted in "rescue" of LH beta subunit by formation of LH dimer, which was efficiently secreted. However, combination of LH beta with alpha was slow, with an overall efficiency of only 50% despite the presence of excess alpha. In contrast, CG beta was rapidly assembled with the alpha subunit after synthesis. The two beta subunits also differed in their influence on the N-linked oligosaccharide processing of combined alpha. The oligosaccharides of LH dimer were endo H resistant, while those of CG dimer remained partially endo H sensitive. Thus, despite a high degree of homology between LH beta and CG beta, the two subunits differ in their secretion as free subunits, their rate of assembly with alpha subunit, and in their effect on the N-linked oligosaccharide processing of combined alpha.