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

Matzuk, Martin M.; Boime, Irving

doi:10.1083/jcb.106.4.1049

Cited by 204 publications

(99 citation statements)

References 54 publications

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“…It should be noted that the first prorenin glycosylation site is located at position 69, very close to LysG2 -Arg,,, and therefore it might be responsible of the diminished efficiency of trypsin activation. However, the contribution of the second glycosylation site (or the third, not tested in this work) cannot be discarded, since N-glycosylation at one site may be influenced by events at different sites [22]. The specific influence of an individual glycosylation site on protein secretion or assembly of subunits has been demonstrated for human chorionic gonadotropin a or b expressed in Chinese hamster ovary cells [23,241.…”

Section: Discussionmentioning

confidence: 99%

N‐linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells

Ladenheim

Seidah

Rougeon

1991

European Journal of Biochemistry

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Paris, FranceMost mouse inbred strains carry two renin genes, Ren-I and Ren-2. Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.

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Section: Discussionmentioning

confidence: 99%

N‐linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells

Ladenheim

Seidah

Rougeon

1991

European Journal of Biochemistry

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“…On the molecular level, this can be explained by increased disorder of the protein segments, close to the carbohydrate chain at Asn78, as demonstrated in this study. Moreover, the complete removal of the glycan at Asn78 in the R-subunit by mutagenesis resulted in poor secretion (<20%) of hCG and rapid degradation of the mutant glycoprotein (6). The presence of the -subunit partially stabilizes such mutants and allows about 45% of the subunit to exit the cell in the form of a dimer.…”

Section: Discussionmentioning

confidence: 99%

“…In the R-subunit of hCG, the two glycosylation sites at Asn52 and Asn78, respectively, have remarkably different properties with respect to the stability of the subunit as shown by sitedirected mutagenesis (6). The absence of glycosylation at Asn52 does not affect folding and stability of the R-subunit.…”

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confidence: 99%

Effects of the N-Linked Glycans on the 3D Structure of the Free α-Subunit of Human Chorionic Gonadotropin

et al. 2000

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To gain insight into intramolecular carbohydrate-protein interactions at the molecular level, the solution structure of differently deglycosylated variants of the R-subunit of human chorionic gonadotropin have been studied by NMR spectroscopy. Significant differences in chemical shifts and NOE intensities were observed for amino acid residues close to the carbohydrate chain at Asn78 upon deglycosylation beyond Asn78-bound GlcNAc. As no straightforward strategy is available for the calculation of the NMR structure of intact glycoproteins, a suitable computational protocol had to be developed. To this end, the X-PLOR carbohydrate force field designed for structure refinement was extended and modified. Furthermore, a computational strategy was devised to facilitate successful protein folding in the presence of extended glycans during the simulation. The values for φ and ψ dihedral angles of the glycosidic linkages of the oligosaccharide core fragments GlcNAc2( 1-4)GlcNAc1 and Man3( 1-4)GlcNAc2 are restricted to a limited range of the broad conformational energy minima accessible for free glycans. This demonstrates that the protein core affects the dynamic behavior of the glycan at Asn78 by steric hindrance. Reciprocally, the NMR structures indicate that the glycan at Asn78 affects the stability of the protein core. The backbone angular order parameters and displacement data of the generated conformers display especially for the -turn 20-23 a decreased structural order upon splitting off the glycan beyond the Asn78-bound GlcNAc. In particular, the Asn-bound GlcNAc shields the protein surface from the hydrophilic environment through interaction with predominantly hydrophobic amino acid residues located in both twisted -hairpins consisting of residues 10-28 and 59-84.Human chorionic gonadotropin (hCG) 1 is a glycoprotein hormone involved in the maintenance of the corpus luteum during early pregnancy. It is a heterodimer consisting of noncovalently associated R-and -subunits. Both subunits are glycosylated and contain a cystine knot motif formed by three disulfide bridges as a central structural element (1,2). Besides the role of R-subunit (RhCG or RhCG[glycan 52,78 ]) in the R -dimer, it has been discovered that the noncombined R-subunit of hCG (hCG-R f ), which is produced in significant amounts by the placenta during pregnancy, has an independent biological activity in being able to stimulate prolactin secretion from human decidual cells (3, 4).Glycosylation of proteins has been shown to have many functions, including stabilization of the protein structure (5). In the R-subunit of hCG, the two glycosylation sites at Asn52 and Asn78, respectively, have remarkably different properties with respect to the stability of the subunit as shown by sitedirected mutagenesis (6). The absence of glycosylation at Asn52 does not affect folding and stability of the R-subunit. In contrast, mutant R-subunits lacking glycosylation at Asn78 are poorly secreted and rapidly degraded, although the precise molecular origin of thi...

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“…For construction of the hTSH␤⅐CTP␣ 1ϩ2 single peptide chain, which was deglycosylated only on the ␣-subunit, the vectors pM 2 hTSH␤⅐CTP␣ and pM 2 ␣ 1ϩ2 were used as templates for PCR. The vectors, pM 2 hTSH␤⅐CTP␣ and pM 2 ␣ 1ϩ2 , were prepared in our laboratory as previously described (4,11). The following oligonucleotides were used for the chimeric construction: primer 1, 5Ј-GTGGGAT-CAGGGGGATCCTAGATTTCTGAGTTA-3Ј; primer 2, 5Ј-CACATCAG-GAGCTTGTGGGAGGATCGG-3Ј; primer 3, 5Ј-ATCCTCCCACAAGCT-CCTCATGTGCAG-3Ј; and primer 4, 5Ј-TGAGTCGACATGATAATTCA-GTGATTGAAT-3Ј.…”

Section: Methodsmentioning

confidence: 99%

“…2 ⅐HA is an expression vector that contains the ampicillin (Amp R ) and the neomycin (Neo R ) resistance genes and a strong promoter of the HaMuSV virus, LTR (25,26 (4). Cells were selected for insertion of the plasmid DNA by growth in culture medium containing 0.25 mg/ml of the neomycin analog, G418.…”

Section: Construction Of Expression Vectors-the Eukaryotic Expressionmentioning

confidence: 99%

Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody

Fares¹,

Levi²,

Reznick³

et al. 2001

Journal of Biological Chemistry

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Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, ␣ and ␤. We have recently converted the hTSH heterodimer to a biologically active single chain (hTSH␤⅐CTP␣) by fusing the common ␣-subunit to the C-terminal end of the hTSH ␤-subunit in the presence of a ϳ30-amino acid peptide from hCG␤ (CTP) as a linker. The hTSH␤⅐CTP␣ single chain was used to investigate the role of the Nlinked oligosaccharides of ␣-and ␤-subunits in the secretion and function of hTSH. Using overlapping PCR mutagenesis, two deglycosylated variants were prepared: one lacking both oligosaccharide chains on the ␣-subunit (hTSH␤⅐CTP␣ 1؉2 ) and the other lacking the oligosaccharide chain on the ␤-subunit (hTSH␤⅐CTP␣(deg)). The single chain variants were expressed in CHO cells and were secreted into the medium. hTSH variants lacking the oligosaccharide chains were less potent than hTSH␤⅐CTP␣ wild-type with respect to cAMP formation and thyroid hormone secretion in cultured human thyroid follicles. Both deglycosylated variants competed with hTSH in a dose-dependent manner. The hTSH␤⅐CTP␣ 1؉2 variant blocked cAMP formation and thyroid hormone secretion stimulated by hTSH as well as by the antibody, thyroidstimulating immunoglobulins, responsible for the most common cause of hyperthyroidism, Graves disease. Thus, this variant behaves as a potential antagonist, offering a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH-secreting pituitary adenoma. Thyrotropin (TSH)1 is a member of the glycoprotein hormone family, which includes lutropin (LH), follitropin (FSH), and human chorionic gonadotropin (hCG). These are heterodimers composed of two noncovalent-linked subunits, a common ␣-subunit and a hormone-specific ␤-subunit (1, 2). Assembly of glycoprotein subunits is vital to the function of these hormones. The ␣-and ␤-subunits contain one (TSH␤ and LH␤) or two (␣, FSH␤, and hCG␤) asparagine-linked (N-linked) oligosaccharides (1, 2). These residues have been shown to play a role in determining the biological activity of glycoprotein hormones, including the maintenance of intracellular stability, assembly, secretion, signal transduction, and modulation of plasma halflife (1, 3). Deglycosylation of glycoprotein hormones have been utilized using chemical or enzymatic treatments, but these however cannot discriminate between individual sites, are nonspecific, and provide only completely deglycosylated hormones.Site-directed mutagenesis has become an important tool for studying the structure and function of glycoprotein hormones. However, mutations in either ␣-or ␤-subunits can alter the folding and ultimately inhibit subunit assembly and secretion of the hormone (4 -6). To overcome these limitations, the genes encoding the common ␣-subunit and either the hCG ␤-, FSH ␤-, or TSH ␤-subunits have been genetically fused. The resulting polypeptide chains were efficiently secreted and were biologically active (7-12). The...

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

Cited by 204 publications

References 54 publications

N‐linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells

N‐linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells

Effects of the N-Linked Glycans on the 3D Structure of the Free α-Subunit of Human Chorionic Gonadotropin

Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody

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