Recombinat hormones: Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon ® )

Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non-covalently associated subunits. We aimed for the expression of a single-chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single-chain hCG is indeed produced by Dictyostelium. Furthermore, this single-chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptorbinding affinity is comparable to single-chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins.Keywords : gonadotropin; single chain; Dictyostelium; human choriogonadotropin; follitropin.The placental human choriogonadotropin (hCG) is involved in the maintenance of pregnancy in the early stages after conception and has important therapeutic applications. This gonadotropin belongs to the family of glycoprotein hormones, which also include follitropin, lutropin and thyrotropin. These hormones are heterodimeric proteins of around 30 kDa formed by a non-covalent association of a common A subunit and a hormone-specific β subunit. Both the A and β subunits of hCG contain two Nlinked oligosaccharide side chains that have an important impact on its conformation and biological activity. A unique feature of the hCG β-subunit is the carboxy-terminal peptide (CTP) which bears four serine-linked oligosaccharides. The major role of the glycosylated CTP seems to be the prolongation of the circulatory half-life of hCG [1].The biosynthesis of the glycoprotein hormones is a highly complex process. In the last decade, it has become clear that folding, assembly and secretion of gonadotropins is assisted by a large set of chaperones and folding enzymes, residing in the endoplasmic reticulum and the Golgi apparatus [2]. Since both the A and the β subunits contain a so-called cystine knot, it can be anticipated that protein disulphide isomerase plays a key role in the facilitation of the folding process [3]. In addition, it has been shown that the N-linked oligosaccharide side chains are required for proper folding, disulphide formation and secretion of hCG [4].The gonadotropins have been expressed in Chinese Hamster Ovary (CHO) cells, and their recombinant derivatives have bioCorrespondence to P. D. J.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum

Heikoop¹,

Grootenhuis

Blaauw³

et al. 1998

“…Plates of cells (100-mm culture dishes) were cotransfected with a selector plasmid [12] and the expression construct using Transfectam reagent as described by the manufacturer (Promega). Cells were selected for neomycin resistance using 0.8 mg´mL 21 G418.…”

Section: Transfection and Selection Of Cho Cellsmentioning

confidence: 99%

Towards minimized gonadotropins with full bioactivity

Heikoop

Winkel

Grootenhuis

1999

Gonadotropins are highly complex glycoprotein hormones consisting of two noncovalently associated subunits, which are heavily glycosylated. Using the X-ray structure of human choriogonadotropin and structure/activity relationships we aimed to design`minimized' gonadotropins of reduced complexity. Our results show that it is possible to reduce the size of natural human choriogonadotropin by one-third of its molecular weight while retaining its wild-type biopotency. To our knowledge, such`mini'-human choriogonadotropins represent the smallest gonadotropins described so far with an lutropin/choriogonadotropin receptor affinity and in vitro biological activity comparable with that of natural human choriogonadotropin. It provides an important step towards the structure/ function-based design of small molecule drugs to the human gonadotropin receptors.Keywords: gonadotropin; human choriogonadotropin; protein minimization; single-chain.Choriogonadotropin, lutropin and follitropin form the family of gonadotropins that play a key role in fertility [1]. They are heterodimeric glycoproteins composed of an a-subunit and a b-subunit, which are noncovalently linked. Both subunits are glycosylated and contain N-linked oligosaccharides, and in the case of the choriogonadotropin b-subunit, O-linked oligosaccharides. Assembly of the common a-subunit with one of the three b-subunits results in the unique properties of each hormone leading to its specific biological activity.The elucidation of the crystal structure of human choriogonadotropin [2,3] has been a major step forward in the understanding of gonadotropin structure/function relationships. However, the mechanism of gonadotropin receptor interactions is poorly understood at present. The reduction of the highly complex heterodimeric glycoprotein hormones to small structured polypeptides or`mini-proteins' that retain their biological activity would be a major step towards achieving the structure/ function-based design of small molecule drugs for the treatment of infertility. Reducing the size and complexity of protein drugs may also open the way for other routes of administration, e.g. nasal rather than by injection. Successful examples of protein minimization projects [4] include the minimization of protein A [5] and atrial natriuretic peptide [6], both to peptides of nearly half the size the original proteins. Even more substantial reductions were recently published by Yanofsky and co-workers [7] who obtained a 15-residue peptide antagonist of the interleukin-1 receptor that may mimic the binding motif of its natural 153-residue antagonist. Wrighton and co-workers [8] reported the discovery of a 20-residue peptide dimer that binds and activates the erythropoietin receptor.In an attempt to reduce the structural complexity of gonadotropins we have engineered a mini-choriogonadotropin in which the two subunits are truncated by a total of 17 amino acid residues and which, in addition, lacks the C-terminal peptide of the b-subunit and its four O-glycosylation sites [9]. Although ...

“…All samples were stored frozen until analysis. ganization was identical to those of pKMS.FSHA g β g [10], except that the DNA encoding the β gene of FSH has been substituted by the cDNA encoding the β gene of CG [11,12].…”

mentioning

confidence: 99%

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

Heikoop

Boogaart

Leeuw

et al. 1998

Self Cite

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...