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.     

Monoclonal antibodies against the beta subunit of human chorionic gonadotropin

The aim of investigation was creation of hybridomas, which produce monoclonal antibodies to the beta-subunit of human chorionic gonadotropin (beta-hCG), characterization of monoclonal antibodies, which necessary for hCG immunoassay in biological fluids, as an immunological methods of detection of early stage of pregnancy and choriocarcinoma. 4 hybridomas, producing monoclonal antibodies to-hCG of IgG1 isotype, were created. On the base of monoclonal antibodies, which produced by D2 hybridoma cell line, test-systems for RIA of hCG in blood serum and urine were elaborated. These test-systems can be used in medical practice for diagnosis of early stages of pregnancy and choriocarcinoma.     

Site-directed mutagenesis defines a domain in the gonadotropin alpha-subunit required for assembly with the chorionic gonadotropin beta-subunit.

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that share a common alpha-subunit, but differ in their hormone-specific beta-subunits. Using site-directed mutagenesis and gene transfer, we studied the region in the common alpha-subunit that has been implicated in the assembly with the beta-subunits. The wild-type or mutated alpha-gene was cotransfected into Chinese hamster ovary cells with the wild-type hCG beta gene. Deletion of the sequence Pro38-Thr39-Pro40 or a change in Tyr37 or Thr39 in the alpha-subunit eliminated or reduced combination with the beta-subunit. Deletion of the sequence Leu41-Arg42-Ser43 had little effect on hCG dimer formation. Disruption of the disulfide bone in the carboxyl end of the subunit did not affect assembly, which suggests that the disulfide bond of Cys59 and Cys87 is not critical for dimer formation. Based on our data and the previously published results from several laboratories, the region encompassed by amino acids 37-40 is a key determinant in initiating and maintaining alpha:beta assembly.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin.

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.     

Localization of human chorionic gonadotropin beta subunit transcripts in ovarian cancer tissue

Recent studies demonstrated that besides placenta and malignant trophoblastic tumors, hCG and especially its beta-subunit is secreted by a varieties of tumors of different origin. The aim of the present investigation was to determine the expression pattern of human chorionic gonadotropin gene in ovarian cancer tissue. The study included 8 patients with epithelial ovarian carcinoma. The expression and distribution of hCGbeta mRNA was assessed by in situ RT-PCR method. The semi-quantitative assessment was performed using computer image analysis. Transformation of the images into the pseudocolour scale showed a clear difference in fluorescence intensity among individual cancer cells. The intensity of ISRT-PCR products corresponding with expression level of hCGbeta demonstrated that its production by individual cancer cells is different. In all studied specimens of the ovarian carcinoma tissue, cancer cells characterized by the presence of active hCGbeta gene were found, whereas noncancerous tissue demonstrated lack of the gene expression. Thus, the study clearly shows that the expression of hCGbeta is the feature of ovarian cancer tissue.     

Domain-dependent protein folding is indicated by the intracellular kinetics of disulfide bond formation of human chorionic gonadotropin beta subunit.

We have measured the intracellular rates of formation of the six disulfide bonds in the human chorionic gonadotropin beta subunit (hCG-beta) to determine whether the folding pathway of this molecule can be described by a simple sequential model. If such a model is correct, the formation of disulfide bonds, which is indicative of tertiary structural changes during protein folding, should occur in a discrete order. The individual rates of disulfide bridging were determined by identifying the extent of disulfide bond formation in hCG-beta intermediates purified from choriocarcinoma cells that had been metabolically labeled for 40 to 120 s and chased for 0 to 25 min. The results of these kinetic studies describe a folding pathway in which the disulfide bonds between cysteines 34-88, 38-57, 9-90 and 23-72 stabilize, in a discrete order, the putative domain(s) involving amino acids 1-90 of hCG-beta. However, the S-S bonds 93-100 and 26-110 begin to form before the complete formation of the disulfide bonds that stabilize the amino acid 1-90 domain(s), and continue to form after complete formation of these disulfide bonds, suggesting that hCG-beta does not fold by a simple sequential pathway. The order of completion of each of the six disulfide bonds of hCG-beta is: 34-88 (t1/2 = 1-2 min), 38-57 (t1/2 = 2-3 min), 9-90 and 23-72, 93-100, and 26-110. Moreover, 60-100% of each of the six disulfide bonds form posttranslationally, and nonnative disulfide bonds do not form in detectable amounts during intracellular folding of hCG-beta.     

Location of major antigenic sites of the beta subunit of human chorionic gonadotropin.

Purified beta subunit of human chorionic gonadotropin (hCG) was partially reduced with beta-mercaptoethanol, carboxymethylated, and digested with chymotrypsin. The peptides were isolated by high-voltage electrophoresis and paper chromatography. Five major disulfide-containing peptides were isolated, and their location in the parent molecule was established by amino acid composition and amino- and carboxy-terminal analyses. All of these peptides inhibited the binding of 125I-labeled hCG by anti-beta hCG serum. The inhibitory effect of these peptides was lost when their disulfide bonds were reduced and alkylated. Synthetic carboxy-terminal peptides were not inhibitory. Based on these data it is concluded that a major antigenic site of hCG resides in the region of residues 21-23 with a disulfide bond connecting cysteine-23 or -26 with the cysteines at positions 72 or 110.     

Cosmid mapping of the human chorionic gonadotropin beta subunit genes by field-inversion gel electrophoresis.

A cosmid clone containing the entire hCG beta gene cluster has been isolated. The restriction map of this clone has been determined by an indirect-end-label FIGE (field inversion gel electrophoresis) method. Analysis of this cosmid clone shows that there are 6 hCG beta genes in human genomic DNA. A previously uncloned portion of the hCG beta cluster, termed the "gap" region, has been shown not to contain any sequences homologous to the hCG beta cDNA. The restriction mapping method employed in this study takes advantage of the superior resolution of FIGE for high molecular weight DNA fragments in the size range 15-50 kb. This method is broadly applicable and permits rapid and accurate restriction mapping for extended regions of genomic DNA that have been cloned into cosmid or lambda vectors.     

Site-directed mutagenesis of the human thyrotropin receptor: role of asparagine-linked oligosaccharides in the expression of a functional receptor

We studied the role of glycosylation in the expression of a functional human TSH receptor. Oligonucleotide-directed mutagenesis was used to replace, separately or together, the Asn codons with Gln in each of the six potential glycosylation sites in the receptor. Recombinant wild-type and mutated TSH receptors were stably expressed in Chinese hamster ovary cells. High affinity TSH binding and the cAMP response to TSH stimulation were abolished in the receptor mutated at Asn77 as well as in the receptor mutated at all six potential glycosylation sites. In the receptor mutated at Asn113, the affinity of TSH binding was markedly decreased (Kd, 2.6 x 10(-8) 3.3 x 10(-10) M in the wild-type receptor). This affinity was too low to permit the transduction of a signal, as measured by an increase in intracellular cAMP generation. Substitution of Asn at positions 99, 177, 198, and 302 did not appreciably affect the affinity of the TSH receptor for TSH binding or its ability to mediate an increase in intracellular cAMP levels. Therefore, either these four potential glycosylation sites are not glycolysated, or alternatively, oligosaccharide chains at these positions do not play a major role in the folding, intracellular trafficking, stability, or expression of a functional receptor on the cell surface. Conversely, our data suggest that N-linked glycosylation of Asn77 and Asn113 does play a role in the expression of a biologically active TSH receptor on the cell surface.     

Different asparagine-linked sugar chains on the two polypeptide chains of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) purified from placenta, like urinary hCG, is shown to have the sialylated forms of three neutral oligosaccharides: Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4(Fucα1→6)GlcNAc (N-1), Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-2) and Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-3). Gel permeation chromatographic analysis of oligosaccharides released from α- and β-subunits of placental hCG has revealed that the α-subunit has one each of sialylated N-2 and N-3, while the β-subunit has one each of sialylated N-1 and N-2.     

Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin beta subunit.

There have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin beta subunit (hCG-beta), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG-beta fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the beta subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects.     

Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction

The role of the human chorionic gonadotropin (hCG) N-linked oligosaccharides in receptor binding and signal transduction was analyzed using site-directed mutagenesis and transfection studies. hCG derivatives with alterations at individual glycosylation sites were expressed in Chinese hamster ovary cells. Receptor binding studies showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on the receptor affinity of the derivatives. Similarly, absence of the N-linked oligosaccharides from the beta subunit or a single oligosaccharide from Asn-78 of alpha had no effect on the production of cAMP or on steroidogenesis. However, the absence of carbohydrate at Asn-52 of alpha decreases both the steroidogenic and cAMP responses. Furthermore, absence of this critical oligosaccharide unit on alpha unmasks differences in the two N-linked oligosaccharides on beta; the beta Asn-13 oligosaccharide but not the beta Asn-30 oligosaccharide plays a more important role in steroidogenesis. Dimers containing deglycosylated beta subunit and an alpha subunit lacking either the Asn-52 oligosaccharide or both oligosaccharides fail to stimulate cAMP or steroid formation. Moreover, these derivatives bind to receptor and behave as competitive antagonists. The use of site-directed mutagenesis was critical in uncovering site-specific functions of the hCG N-linked oligosaccharides in signal transduction and reveals the importance of the Asn-52 oligosaccharide in this process.     

The role of asparagine-linked oligosaccharides in the subunit structure, steroid binding, and secretion of androgen-binding protein.

Testicular androgen-binding protein (ABP) and liver sex hormone-binding globulin are encoded by the same gene. These proteins have the same primary amino acid sequences, but they differ in attached oligosaccharides; the differences are presumably due to cell-specific glycosylation mechanisms. To investigate the role of oligosaccharides in ABP/sex hormone-binding globulin subunit structure, secretion, and steroid binding, mutant rat ABP proteins were constructed that eliminated one or both of the two potential sites of asparagine (Asn)-linked glycosylation. Immunoblot analysis of wild type recombinant ABP yielded the typical heterogeneous banding pattern. Secreted ABP was composed of two protomers of M(r) 46,000 and M(r) 43,000, while cellular ABP yielded three mol wt species (M(r) 43,000, 41,000, and 39,000). Substitution of the Asn residue in either consensus sequence for Asn-linked glycosylation with an Ile residue resulted in increased mobility of the immunoreactive ABP species. These changes are consistent with the loss of an Asn-linked oligosaccharide. Substitution of both Asn residues yielded a single immunoreactive species in the medium and cell extracts that migrated as a M(r) 39,000 protein. These results demonstrate that the mol wt heterogeneity of ABP is due to differential Asn-linked glycosylation of both potential sites. All three mutant forms of ABP were secreted by the COS cells. However, the amount of immunoreactive ABP and [3H]5 alpha-dihydrotestosterone binding in the medium was lower than wild type (100%) in one of the single mutants (65%) and in the double mutant (29%). Unlike the glycosylation mutants, alteration of other residues, not involved in glycosylation, yielded cellular ABP and no detectable medium ABP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure determination of the major N- and O-linked carbohydrate chains of the beta subunit from equine chorionic gonadotropin

The carbohydrate moieties of equine chorionic gonadotropin alpha and beta subunits were released from the protein backbones by successive treatments with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and alkaline borohydride and then fractionated by FPLC and HPLC. The major N- and O-linked glycans of the beta subunit were characterized by 500-MHz 1H-NMR spectroscopy, showing a remarkable structural heterogeneity for the N-glycosidically linked chains, comprising mono-, di-, tri- and tri'-antennary N-acetyllactosamine type of glycans, being partly alpha 1-6 fucosylated at the Asn-bound GlcNAc residue and having alpha 2-6 and alpha 2-3 linked N-acetyl- and N-acetyl-4-O-acetylneuraminic acid residues as sialic acid constituents. Significant differences in this respect were detected for the partially characterized glycans of the alpha subunit. The major part of the O-linked carbohydrate chains, occurring solely in the beta subunit, is formed by tri-, tetra-, penta- and hexa-saccharides. There are indications for the presence of oligo(N-acetyllactosamine) units in both the N- and O-linked glycans of the beta subunit.     

A novel four-amino acid determinant defines conformational freedom within chorionic gonadotropin beta-subunits

On the basis of apparent molecular mass heterogeneity following reducing versus nonreducing SDS-PAGE, we determined that the beta-subunit of macaque (Macaca fascicularis) chorionic gonadotropin (mCG-beta) is more conformationally constrained than the beta-subunit of human chorionic gonadotropin (hCG-beta). The amino acid sequences of these two subunits are 81% identical. To determine the conformational variance source, which was not due to glycosylation differences, we generated a series of hCG-beta-mCG-beta chimeras and identified domains that contributed to CG-beta conformational freedom. We discovered that the CG-beta 54-101 domain contained a small subdomain, residues 74-77, that regulated the conformational freedom of the beta-subunit; i.e., when residues 74-77 were of macaque origin (PGVD), the mutated hCG-beta subunit displayed macaque-like conformational rigidity, and when residues 74-77 were of human origin (RGVN), the mutated mCG-beta subunit displayed human-like conformational freedom and microheterogeneity. Additionally, CG-beta N-terminal domain residues (8, 18, 42, and 46-48) were also found to influence CG-beta conformational freedom when residues 74-77 were of human but not macaque origin. The biological significance of the CG-beta conformational variance was tested using a biological assay that showed that the hCG-alpha-hCG-beta heterodimer facilitated human CG receptor-mediated cAMP-driven luciferase reporter gene activity in HEK cells nearly 1 order of magnitude more effectively than the hCG-alpha-mCG-beta chimera. Together, these data demonstrate that two essential amino acid residues within a four-amino acid subdomain regulated CG-beta conformational freedom and that a conformational difference between hCG-beta and mCG-beta was recapitulated in the context of receptor-mediated CG heterodimer signal transduction activation.