Priority paper Partially deglycosylated human choriogonadotropin, stabilized by intersubunit disulfide bonds, shows full bioactivity

Abstract

Several studies indicate that in human choriogonadotropin the N-linked oligosaccharide at position 52 of the A-subunit is important for bioactivity. We have generated choriogonadotropin mutants in which the A52 glycosylation site is removed and the A and β subunits are covalently linked by intersubunit disulfide bonds. These mutants display wild-type receptor binding and bioactivity. Furthermore, we show that removal of the A52 sugar leads to instability of heterodimeric choriogonadotropin. Therefore, we conclude that the A52 oligosaccharide of choriogonadotropin is not involved in signal transduction, but in the stability of the heterodimer.Keywords : human choriogonadotropin; bioactivity ; signal transduction ; protein engineering; glycoprotein.Human choriogonadotropin (CG), luteinizing hormone (LH) position plays a major role in signal transduction in CG [5] and FSH [6, 7]. and follicle-stimulating hormone (FSH) form the family of gonadotropins, which is involved in the regulation of the cellular Recently, we have shown that it is feasible to introduce covalent bonds between the A and β subunits of CG by introducing and endocrine function of the reproductive organs [1]. They consist of A and β subunits that are associated by hydrogen bonds intersubunit disulfide bonds [8]. This class of glycoprotein hormones displays wild-type receptor binding and biological activand hydrophobic contacts. In a given species the A subunit is identical in all glycoprotein hormones, while the β subunits are ity, whereas the thermostability of the hormones is enhanced considerably. Furthermore, these disulfide-bonded mutants were unique and confer receptor specificity on the hormones.A substantial part of the molecular mass of gonadotropins is designed to have overall conformations minimally disturbed relative to that of wild-type CG. Therefore, such molecules provide attributed to complex carbohydrates; in CG approximately 34% of the mass of the glycoprotein is due to complex carbohydrates. a very attractive system to study effects of mutations that in a heterodimeric context could interfere with the stability of the The β subunit of CG has four O-linked carbohydrates at Ser121, Ser127, Ser132 and Ser138. In addition, N-linked oligosaccha-heterodimer. In this paper we analyze the role of the AsnA52 glycosylation site in signal-transducing activities using this class rides are present at Asn52 and Asn78 on the A subunit and at Asn13 and Asn30 on the β subunit. Several studies have ad-of disulfide-bonded CG molecules. We will show that in stable CG, the A52 glycosylation site is not involved in signal transducdressed the role of the oligosaccharide chains on the gonadotropins [2]. It has been shown that the carbohydrate moieties are tion. involved in the folding and secretion of gonadotropins. Furthermore, glycosylation is essential for the plasma half-life of the glycoprotein hormones.MATERIALS AND METHODS The gonadotropins are amongst the most prominent examMaterials. Enzymes for preparing the expression vectors ples in glycobi...

Thyroid-Stimulating Hormone and Thyroid-Stimulating Hormone Receptor Structure-Function Relationships

“…fated in TSH and contains a unique NeuAc ␣2,6 linkage in LH. In contrast to Asn-78, the glycan at Asn-52 in the ␣-subunit L2 loop was found to be highly mobile in the free subunit, but its mobility in the heterodimer is restricted by the seat-belt (80). It is known that the region ␣33-57 is largely involved in signal transduction and significantly altered by subunit dissociation.…”

Towards minimized gonadotropins with full bioactivity

“…The primers were chosen in such a way that the PCR fragments contained appropriate restriction sites 3 H and 5 H of the mutation, so that they could be exchanged with the native a-chain sequence in the expression vector described previously [9,14]. Mutations were confirmed using an automated sequencer (Pharmacia).…”

“…In vitro bioactivity was determined on the human lutropin/ choriogonadotropin receptor as expressed in a stable transfected CHO K1 cell line with a reporter gene that becomes activated specifically after lutropin/choriogonadotropin receptor activation as described previously [14]. Highly purified recombinant human choriogonadotropin was used as a standard.…”

“…The receptor binding of the mutated molecules was quantitated with a radioligand receptor displacement assay on membrane fractions isolated from exponentially growing cells as described earlier [14]. Highly purified recombinant human choriogonadotropin was used as standard.…”

Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins inDictyostelium discoideum

“…In short, CHO cells expressing human HL/CG or FSH receptors also contain a reporter construct with luciferase driven by a cAMP responsive element. On exposure of the cells to the hormone, the reporter gene is activated through the receptor-mediated signal transduction pathway and luciferase activity is measured (5). Highly purified, recombinant gonadotropins produced by CHO cells were used as standards.…”