Evidence for impaired subunit interaction in chemically deglycosylated human choriogonadotropin

Merz, Wolfgang E.

doi:10.1016/s0006-291x(88)80770-8

Cited by 35 publications

(20 citation statements)

References 29 publications

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“…Degly-hCG by treatment with hydrogen fluoride (HF) becomes homogeneous with regard to pI due to removal of all terminal sialic acid residues [11]together with most of the sugar units. It thus represents an artificial alkaline and markedly smaller variant with a higher pI than naturally occurring pI variants.…”

Section: Discussionmentioning

confidence: 99%

“…1a, b). Intact heterodimeric preg-hCG and degly-hCG were kindly provided by Dr. W. E. Merz, University of Heidelberg, Germany [11]. …”

Section: Methodsmentioning

confidence: 99%

“…In particular, hCG variants from tumor patients are often highly acidic (hCGav), presumably because of being hyperglycosylated and/or oversialylated [9]. In previous investigations, degly-hCG and asialo-hCG [10, 11]displayed higher receptor affinities for the LH/CG-R of rat testis membranes (r-LH/CG-R) [12, 13]than non-modified highly purified urinary preg-hCG [11, 14, 15]. When preg-hCG and asialo-hCG were receptor bound, only 2 of 14 epitopes (i.e.…”

Section: Introductionmentioning

confidence: 99%

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Tumor- and Pregnancy-Derived Isoforms of Human Chorionic Gonadotropin: Biological and Diagnostic Relevance

Lottersberger

Hoermann²,

Mann³

et al. 2003

Horm Res Paediatr

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Objectives: Human chorionic gonadotropin (hCG) and hCG variants are of high clinical importance for the diagnosis of pregnancy, monitoring of abnormal and ectopic pregnancies, testing for Down’s syndrome or monitoring therapy of hCG-secreting malignancies. In serum and urine, hCG appears in microheterogeneous isoforms with respect to protein backbone structure and the extent of glycosylation. The present study reports on the identification, immunological characterization, biological activity of glycosylation isoforms of pregnancy (preg) and tumor-derived (tu) hCG, and the impact of glycosylation on diagnostic immunoassays. Methods: Twenty-two urinary preg- and tu-hCG isoforms were separated by preparative isoelectrofocusing (hCG-pI variants) and characterized by Western blot. Number, topography and accessibility pattern of epitopes on their surface was evaluated by two-site radioimmunoassays using 14 different monoclonal antibodies (mabs). Binding of hCG isoforms to four different LH/CG receptors was investigated in radioreceptor assays, and their biological activity determined by measuring cAMP elevation. Results: All 22 hCG glycosylation variants appeared immunologically intact: each isoform, even when highly acidic, expressed all 14 surface epitopes which were arranged in a topographical manner indistinguishable from crude hCG. hCG isoforms were able to bind to four different receptor variants, with slightly varying affinities, but orientations indistinguishable from each other as shown by identical epitope accessibility patterns. Each of the hCG-pI variants was able to activate the LH/CG-Rs, but with varying reactivities. Conclusions: We conclude that in contrast to deglycosylated hCG, all hCG glycosylation isoforms investigated act as receptor agonists. Moreover, there is no overspecificity of mabs to certain hCG isoforms due to carbohydrate variability that exclude others from diagnostic measurement.

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Section: Discussionmentioning

confidence: 99%

“…1a, b). Intact heterodimeric preg-hCG and degly-hCG were kindly provided by Dr. W. E. Merz, University of Heidelberg, Germany [11]. …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tumor- and Pregnancy-Derived Isoforms of Human Chorionic Gonadotropin: Biological and Diagnostic Relevance

Lottersberger

Hoermann²,

Mann³

et al. 2003

Horm Res Paediatr

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“…Site-directed mutagenesis has revealed that the presence of an oligosaccharide at Am52 is essential for signal transduction . This event might occur in several ways: the oligosaccharide at Asn.52 might bind directly to the IutropidhCG receptor (Calvo and Ryan, 1985;Amano et al, 1989;Kobata, 1993: McFarland et al, 1989;Thotakura et al, 1990); it might maintain the protein in the proper conformation (Keutmann et al, 1985;Merz, 1988;Sairam, 1989;Dighe et al, 1990;Sairam and Jiang, 1992): or it might assure the correct mode of binding of the hormone to the 1utropinlhCG receptor (Ji and Ji, 1990;Schwarz et al, 1991). In contrast, the oligosaccharide at Am78 is not required for hormonal activity , but the stability of the a subunit without glycosylation at Am78 is markedly reduced, as indicated by its rapid degradation in Chinese hamster ovary cells (Matzuk and Boime, 1988).…”

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confidence: 99%

NMR Studies of the Free α Subunit of Human Chorionic Gonadotropin

Beer

Zuylen

Leeflang

et al. 1996

European Journal of Biochemistry

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Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone that is involved in the maintenance of the corpus luteum in early pregnancy. Glycosylation at Am52 of its a subunit (ahCG) is essential for signal transduction, whereas the N-glycan at Am78 stabilizes the structure of the protein.In this study, an almost complete 'H-NMR and a partial "C-NMR spectral assignment for the amino acids and the N-glycans of ahCG and of an enzymatically deglycosylated form, which had a single GlcNAc residue at each of its two glycosylation sites, has been achieved. The secondary structure of ahCG in solution, which was determined based on NOE data, is partially similar to that of the a subunit in the crystal structure of hCG, but large structural differences are found for amino acid residues 33-58.In the crystal structure of hCG, residues 33-37 and 54-58 of the a subunit are part of an intersubunit seven-stranded b-barrel and residues 41 -47 constitute a 3,,,-helix. In contrast, in free ahCG in solution, amino acids 33-58 are part of a large disordered loop, indicating that in intact hCG interactions with the /I subunit of hCG stabilize the conformation of the 0: subunit. The NMR data of ahCG and its deglycosylated counterpart are very similar, indicating that removal of carbohydrate residues other than GlcNAc-1 does not notably affect the conformation of the protein part. However, numerous 'H-NOES between the GlcNAc-1 residue at Am78 and several amino acid residues show that this GlcNAc residue is tightly packed against the protein, being an integral part of the structure of the IX subunit. 'H-NOES across the glycosidic linkages of the glycan, resonance-line widths, and 'H and I3C chemical shifts of the other monosaccharides suggest that the remainder of the glycans at Asn78, and the glycans at Asn.52 are largely extended in solution.

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“…In contrast, the reduction in biopotency in vivo associated with basic isoforms of FSH, in comparison with that of more acidic (sialylated) isoforms, reflects the rapid clearance of non-sialylated glycoproteins from the circulation, and not a reduction in the ability of basic isoforms to initiate a biological response at a cellular level (Ashwell & Harford, 1982;. Differences in glycosylation of members of the gonadotrophin hormone family may also affect solubility (Sairam, 1983), receptor internalization (Niswender et al 1985), secretion (Matzuk & Boime, 1988a,è) and subunit association (Merz, 1988).…”

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confidence: 99%

Expression and characterization of biologically active ovine FSH from mammalian cell lines

Mountford

Brandon²,

Adams³

1994

Journal of Molecular Endocrinology

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Stably transfected cell lines expressing the alpha subunit, beta subunit and alpha/beta heterodimer of ovine (o)FSH have been established following the transfection of Chinese hamster ovary cells with alpha and beta subunit cDNA expression vectors. In the absence of the alpha subunit, FSH beta subunit polypeptides were inefficiently secreted and displayed a short intracellular half-life, while free alpha subunits were readily secreted in the absence of the beta subunit. Cotransfection of oFSH alpha and beta subunit cDNAs led to heterodimer assembly and secretion. While alteration of the nucleotide sequence flanking the beta subunit AUG initiation codon did not appreciably enhance heterodimer biosynthesis and secretion, the replacement of the 5' untranslated and signal peptide-coding regions of the beta subunit cDNA with the corresponding sequences from an oGH cDNA clone was associated with a twofold increase in oFSH heterodimer secretion. The recombinant oFSH had a higher molecular weight than pituitary-derived oFSH, and was more acidic than the native hormone when analysed using isoelectric focusing, suggesting a greater degree of sialylation of the recombinant hormone. A comparison of the activities of the recombinant and native hormones in the porcine testis radioreceptor assay and in the in vitro Sertoli cell bioassay revealed that the recombinant oFSH displayed enhanced biological activity in the Sertoli cell assay when compared with the native hormone.

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Evidence for impaired subunit interaction in chemically deglycosylated human choriogonadotropin

Cited by 35 publications

References 29 publications

Tumor- and Pregnancy-Derived Isoforms of Human Chorionic Gonadotropin: Biological and Diagnostic Relevance

Tumor- and Pregnancy-Derived Isoforms of Human Chorionic Gonadotropin: Biological and Diagnostic Relevance

NMR Studies of the Free α Subunit of Human Chorionic Gonadotropin

Expression and characterization of biologically active ovine FSH from mammalian cell lines

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